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DarT: The Embryo Test with the ZebrafishDanio rerio - a General Model in Ecotoxicologyand ToxicologyRoland NagelTU Dresden, Institut fur Hydrobiologie, D-Dresden

SummaryThe acute fish test is an animal test whose ecotoxicologicalrelevance is worthy of discussion. The primary aim ofprotection in ecotoxicology is the population and not the indi-vidual. Furthermore the concentration of pollutants in theenvironment is normally not in the lethal range. Therefore theacute fish test covers solely the situation after chemical spills.Nevertheless, acute fish toxicity data still belong to the base setused for the assessment of chemicals. The embryo test with thezebrafisn Danio rerio (DarT) is recommended as a substitutefor the acute fish test. For validation an internationallaboratrycomparison test was carried out. A summary of the results ispresented in this paper. Based on the promising results oftesting chemicals and waste water the test design was validatedby the DIN-working group "7.6 Fischei-Test". A normed testguideline for testing waste water with fish is available. The testduration is short (48 h) and within the test different toxico-logical endpoints can be examined. Endpoints from the embryotest are suitable for QSAR-studies. Besides the use inecotoxicology the introduction as a toxicological model wasinvestigated. Disturbance of pigmentation and effects on thefrequency of heart-beat were examined. A further importantapplication is testing of teratogenic chemicals. Based on theresults DarT could be a screening test within preclinicalstudies.

Zusammenfassung: DarT: Der Embryotest mit dem Zebra-barbling Danio rerio - ein Modell zur Abschatzung okotoxi-kologischer und toxikologischer WirkungenBei dem akuten Fischtest handelt es sicli um einen Tierversuch,dessen okotoxikologische Relevanz diskussionswiirdig ist. Dasprimdre Schutzziel in der Okotoxikologie ist die Population undnicht das Individuum. Aufierdem liegen die Konzentrationenvon Schadstoffen in der Umwelt normalerweise nicht im letalenBereich. Folglich bildet der akute Fischtest nur die Storfall-situation ab. Trotzdem gehort das Ergebnis dieses Tests nachwie VOr zum Grunddatensat; in der Chemikalienbewertung.Der Embryotest mit dem Zebrabiirbling Danio rerio (DarT)wird als Ersatzmethode fur den akuten Fischtest vorgeschlagen.Zur Validierung wurde erfolgreich ein internationaler Labor-vergleichsversucli durchgefiihrt, dessen Ergebnisse vorgestelltwerden. Ausgehend von den positiven Ergebnissen zurChemikalienprufung und zur Abwasserprufung, hat der DIN-Arbeitskreis ,,7.6 Fischei-Test" den Test validiert und eineNormvorschrift fur die Abwasserpriifung vorgelegt. Die Test-dauer ist kur; (48 h) und es konnen verschiedene toxikologischeEndpunkte innerhalb des Tests erfafit werden. Endpunkte desEmbryotestes sind auch eine gute Basisfur die Betrachtung vonquantitativen-Struktur- Wirkungs-Beziehungen (QSAR). Nebendiesen Einsatzmoglichkeiten in der Okotoxikologie wurde auchder Einsat; als ein Modell in der Toxikologie gepruft. Storungenin del' Pigmentierung oder die veranderung del' Herzschlag-frequenz wurden untersucht. Eine weitere wichtige Ein-satrmiiglichkeit ist die Priifung von teratogenen Substanzen.Basierend auf den Ergebnissen konnte DarT ein Screening-Testim Rahmen del' prdklinischen Studien sein.

Keywords: zebra fish, embryo test, replacement, acute fish test, QSAR, teratogenic, heart-beat, pigmentation

1 Introduction

The acute fish test is the initial step intesting chemicals with fish accordingdifferent laws, for example the Germanchemicals act or the plant protection act.Usually, acute toxicity of chemicals isdetermined as a LCso (96 h) value (e.g.,according to OECD guideline 203), i.e.,as the concentration of the test substanceresulting in 50% mortality of the experi-

38

mental fish over a period of 96 hours.There is an extensive data base for acutetoxicity. Different fish species may varyby orders of magnitude with respect totheir sensitivity in acute tests to environ-mental contaminants. Generally, salmonidfish are more susceptible than cyprini-formes or cyprinodontiformes.Death as an endpoint in toxicological

research represents an unambiguousparameter for the individual. However,

the environmental significance of deathof individuals after short-term exposureto high concentrations, except in the caseof accidental spills (e.g. as observed inthe river Rhine in November 1986),is questionable. More frequently, chroniceffects resulting from long-term expo-sure to low concentrations are relevantand should be investigated.Moreover, the ecotoxicological rele-

vance of death is limited by the fact that,

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at least in my opinion, extrapolation ofchronic effects from acute toxicity datais, by principle, impossible (Nagel andIsberncr, 1998). However, it is chronictoxicity which represents the ultimatelyimportant endpoint in ecotoxicology.

In recent years acute toxicity tests withfish have also aroused considerableethical concern. Since acute toxicity tofish is determined in tests with juvenileor adult animals, intact fish are subjectedto considerable pain and suffering, whichis clearly in conflict with current AnimalRights Welfare legislation. Since resultsof LC,o tests are only of minor ecotoxi-cological significance, acute toxicitytests with fish should be replaced interms of the 3R. Possible alternatives tothe acute fish test might be the embryotest with zebrafish Dania rerio (DarT)and cytotoxicity tests with fish cells.

Lange and colleagues (1995) relatedresults of the zebrafish embryo test tothose of cytotoxicity tests with thepermanent cell line RTG-2 derived fromrainbow trout (Oncorhynchus mykiss) for10 selected compounds with differentmodes of action. In most cases thezebrafish embryo was more sensitivethan adult zebrafish and the RTG-2 cells.

In another study for 17 compounds,results of the embryo and the RTG-2cytotoxicity tests were compared toLC,o data from tests with golden ide(Leuciscus idus melanatus) (dataevaluated by F. Moldenhauer and H.Spielmann, personal communication).For either alternative test system, linearregression analysis documentedsatisfactory correlation. The correlationcoefficient for the comparison ofzebrafish embryo test and juvenile

golden ide fish test proved to be some-what better (0.991) than the correlationbetween RTG-2 cytotoxicity andjuvenile golden ide toxicity (0.796). Acomparison of y-axis intercepts revealedthat the zebrafish embryo test (y = -0.13)is more sensitive and reflected the acutetoxicity more accurately than the RTG-2cytotoxicity test (-0.89). These dataindicate that the embryo test (DarT) is aparticularly suitable candidate to replacethe acute fish test at the base level fortesting chemicals with fish.

Furthermore the embryo test has thepotential to be a substitute of fish test inroutine waste water control and it couldbe also a model for testing chemicalsin toxicology. Therefore the method willbe described and an overview of the stateof the art of the zebrafish embryo testis given.

Time (h) Stage

Tab. 1: Stages of embryonic development of the zebrafish Dania rerio at 26±1°C

Characterisation (after Kimmel et aI., 1995)

oo0.75

Fertilisation

Zygote period

Cleavage period

1.25

1.5

2

3

4

5.25

Blastula period

Gastrula period

8

10

zygote

cytoplasm accumulates at the animal pole, one-cell stage

discoidal partial cleavage

1. median vertical division: two-cell-stage

2. vertical division: four-cell-stage

3. vertical and parallel to the plane of the first: eight-cell-stage

4. vertical and parallel to the second plane of division: 16-cell-stage

start of blastula stage

late cleavage; blastodisc contains approximately 256 blastomers

flat interface between blastoderm and yolk

50 % of epibolic movements; blastoderms thins and interface between peribalst andblastoderm become curved

75 % of epibolic movement

epibolic movement ends, blastopore is nearly closed

12

10.5 Segmentation period first somite furrow

20

22

24

30Pharyngula period

3672-96 Hatching period

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somites are developed, undifferentiated mesodermal component of the early trunk,tail segment or metamere

muscular twitches; sacculus; tail well extended

site to side flexures; otoliths

phylotypic stage, spontaneous movement, tail is detached from the yolk; early pigmentation

reduced spontaneous movement; retina pigmented, cellular degeneration of the tail end;circulation in the aortic arch 1

tail pigmentation; strong circulation; single aortic arch pair, early motility; heart beating starts

heart-beat regularly; yolk extension beginning to taper; dorsal and ventral stripes meets at tail;segmental blood vessels: thickened sacculus with two chambers; foregut developmental; neuromasts

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2 Animals, materials and methods

2.1 The zebrafish Danio rerioThe zebrafish Danio rerio (Hamilton-Buchanan, 1922; formerly Brachydaniorerloi is a small cyprinid found intributaries and branches of the GangesRiver in South-East Asia (Eaton andFarley, 1974). This species measures 3-5em as an adult and thrives in both softand hard waters. At 26°C the zebrafishgrows quickly and reaches maturitywithin three months. This species iseasily obtainable, inexpensive, readilymaintainable and, under appropriateconditions, will provide a large numberof non-adherent and transparent eggs(Laale, 1977). One female lays approxi-mately 50·200 eggs per day. Thezebrafish is a r-strategist (Nagel, 1993).The embryonic development was

described in numerous studies (Roosen-Runge, 1938; Hisaoka and Battle, 1958;Laale, 1977; Thomas and Waterman,1978; Kimmel et al., 1988; Kimmel et al.1995) and is the basis for the interpreta-tion of effects caused by environmentalpollutants.The Danio rerio egg is telolecithal, and

cleavage is meroblastic and discoidal.Shortly after fertilisation, cytoplasm ofthe teleost egg accumulates at the animalpole where it surrounds the nucleus ofthe zygote. Only this portion of eggcytoplasm, the so called blastodiscundergoes cleavage, whereas the yolkrich zone is excluded from cleavages. InTable 1 the stages of embryonic develop-ment of zebrafish embryos are sum-marised and will give an impression ofthe complexity of the early development.Selected stages of the embryonic de-

velopment of the zebrafish are shown inFigure 1-4.The zebrafish has been used as a mod-

el in numerous studies in the fields ofmolecular genetics, vertebrate biology aswell as in developmental, neurobiologyand transgenic research (Roosen-Runge,1938; Hisaoka and Battle, 1958; Laale, etal., 1977; Sander and Baumann, 1983;Nagel, 1988; Kimmel et al., 1995;Westerfield, 1995; Lele and Krone, 1996;Goolish et aI., 1999; Wixon, 2000).Furthermore, zebrafish has undoubtelybecome the most important model indevelopmental biology of vertebrates

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(Ekker and Akimenko, 1991; Nusslein-Volhard, 1994; Westerfield, 1995).

2.1.1 Culture, egg production anddifferentiation

A breeding stock of non-treated, maturezebrafish is used for egg production.Females and males are kept at a ratio of1:2 in a glass aquarium filled withcharcoal filtered tap water with anoxygen saturation of more than 80%. Theculture conditions are 26 ± 1°C at a 12hour day/night light regime. Optimal

filtering rates should be adjusted using afilter system. The fish are fed with dryflakes twice per day, and ad libitum withnauplia larvae of Artemia spec. once aday. To ensure optimal water quality re-maining food should be removed daily.To prevent the eggs from cannibalism

by the adult zebrafish the spawn traps arecovered with a stainless steel mesh. Plantimitations made of green glass are usedas spawning substrate. The spawning andfertilisation take place within 30 minutesafter light is turned on in the morning.30-60 minutes after spawning the egg

Fig. 1: Four-cell-stage of an embryoof Danio rerio approximately 1 h afterfertilisation (from Zeller, 1995).

Fig. 3: Normally developed embryo ofDanio rerio after 24 h (from Zeller, 1995).The tail is detached from the yolk andspontaneous movement starts at this time.

Fig. 2: Segmentation phase of an embryoof Danio rerio approximately 12 h afterfertilisation (from Zeller, 1995). The headand tail region as well as the somitesare visible.

Fig. 4: Normally developed embryo ofDanio rerio after 48 h (from Zeller, 1995).Eye and skin pigmentation due tomelanophores, the sacculus containingtwo otoliths as well as the completelydeveloped and well structured spine arevisible. At this stage blood circulation andregular heart beats can be observed.

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traps can be removed and the eggs arecollected in a plastic mesh sieve. A singlemature female lays 50-200 eggs per day.At the culture conditions describedabove, fertilised eggs undergo the firstcleavage after approximately 15 rnin andconsecutive synchronous cleavages form4, 8, 16, and 32 cell blastomeres. At thisstages (4-32 cells) eggs can be identifiedclearly as fertilised and only these shouldbe used for the experiments.

2.2 DarT - The Danio rerio toxicityassay/ Danio rerio teratogenicityassay

The embryo test procedure is describedin Schulte and Nagel (1994) and Nagel(1998) in detail. Following initial range-finding experiments, the toxicity of achemical substance can be determinedby using 24-well multiplates. Afterpreparing a stock solution of the testsubstance, typically five concentrationsare tested.Information about the use of solvents

can be found in Maiwald (1997) and inNagel (1998).40 eggs are transferred to the test

solutions about 60 minutes after light hasbeen turned on to initiate spawning.Fertilised eggs are separated from thenon-fertilised and placed in the multi-plate wells with a pipette using a stereomicroscope. 20 fertilised eggs are placedindividually in 2 mL of the respectivetest solutions to exclude mutualinfluences. The remaining four wells ofeach plate are used as internal controlfilled with dilution water amounting to atotal of 20 controls per test. The dilutionwater corresponded to the reconstitutedwater according to ISO-standard 7346/3,which is diluted 1:5 using deionisedwater. After this procedure the multi-plates are covered with a self-adhesivefoil and incubated at 26°C ± 1°C. Lethal,sublethal and teratogenic endpoints arerecorded using a dissecting microscopewithin 48h. A test is classified as valid,if 90% of the embryos in the controltreatments showed neither sublethal norlethal effects.In Table 2 lethal and sublethal end-

points for both the DarT toxicity assayand the teratogenicity assay are sum-marised.

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3 The embryo test, a model inecotoxicology

laboratories (highest coefficient of varia-tion: 36%). The average value from theinter-laboratory study and LC50 data for16 additional chemicals were comparedto LC,o values resulting from fish acutetoxicity tests with zebrafish (Dania reria)or, if not available, with golden ide(Leuciscus idus melanatus). Regressionanalysis with data for these 37 chemicalsrevealed a good correlation with a slopeof 0,81 and a regression coefficient of0.87 (Schulte et al., 1996).Based on these results an international

inter-laboratory study with 12 laborato-ries, funded by the German Environmen-

3.1 DarT, Danio rerio toxicity test,a substitute of the acute fish testin chemical testing

The method and preliminary results for 6chemicals were published by Schulte andNagel (1994).In three laboratories (University

Mainz, FHG-WCT Schmallenberg, 1GBBerlin), 21 reference chemicals wereinvestigated. Consistency of the resultswas confirmed by the relatively lowcoefficients of variation between the

Tab. 2: Lethal and sublethal endpoints for evaluating the toxicity and teratogeniCityof chemicals on the embryo of Danio rerio (according to Schulte and Nagel, 1994;Nagel, 1998; Bachmann, 2002).

Toxicological endpoints Exposure time (h)

Lethal* 8 24 48 120

Coagulation • • •Tailnot detached • •No somites • •No heart-beat •Sublethal/DevelopmentCompletion of gastrula •Formationof somites •Developmentof eyes • •Spontaneous movement • •Heart beat/blood circulation •PigmentationOedema •TeratogenicMalformationof head • •

sacculi/otoliths • •tail • •heart • •

modifiedstructure of the corda • •scoliosis • •rachischisis • •deformityof yolk • •growth-retardation • •Length of tail** •* After 48 h the four endpoints were assessed to be lethal. Within the teratogenicity test for a bettercomparison with mammalian data only the endpoint "coagulated" is used as a lethal effect.

**There is the option to measure the length of tail after 120 h. In this case the eggs are transfered into waterwithout the test compound after 48 h. After natural hatching the larvae is straightened and the length can bedetermined.

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tal Protection Agency (UBA), was initi-ated (Ful3mann and Nagel, 1997). Threetest substances (EDTA-Na2.2HzO,benzoic acid, 2,4-dinitrophenol) withdifferent toxic potential were repeatedlyblind-tested in twelve laboratories.Lethal and sublethal effects weresurveyed within the first 48 hours ofembryonic development. The twelvelaboratories, which participated in thiscomparative study, performed 61 validtests for the three test substancesaltogether.LCso values for each valid test were

calculated using identical software (datadocumentation and probit analysis inMS-Excel ©FuBmann). Each laboratoryperformed at least one valid test for eachtest substance. One laboratory deliveredthree replicate test results and fivelaboratories two results for EDTA. Tenand nine laboratories delivered tworeplicate test results for benzoic acid and2,4-dinitrophenol, respectively. The toxi-city of the three test substances, asrevealed by the LCso, varied over threeorders of magnitude. Mean LCso (of theaverage value for each laboratory) were727 mgl,", 36.6 mgl,", and 1.13 mgl,"for EDTA-Naz·2H20, benzoic acid, and2,4-dinitrophenol, respectively. Alllaboratories found EDTA-Naz·2HzO tobe the least toxic substance followed bythe other two substances. Varianceamong replicate tests performed by asingle laboratory was always smallcompared to differences betweensubstances. Laboratories 11 and 12,which used different strains of Daniorerio, consistently found higher LCso-values than the other ten laboratoriesfor benzoic acid and 2,4-dinitrophenol(lab 11) or for all three test substances(lab 12).The factor "laboratory" had a signifi-

cant effect on toxicity assessment (LCsu)in this inter-laboratory study, when alltwelve participating laboratories wereconsidered (Friedman's X2=22.2,

X2crit,0<=o.os=19.7,p=0.02). Specificallythe differences of LCso-values betweenlaboratory 1 and 12 (and only betweenthese two) were statistically significant(Wilcoxon-Wilcox slim of ranks differ-ence = 29, critical difference,,=O.05= 28.9).The LC50-va]ucs for the remaining tenlaboratories are no longer significantly

42

different at the 5%-level, if the two labo-ratories were omitted from the analysis,which used different fish strains (for lab1 through lab 10: Friedman's X?=16.2,X2crit.a=oos=16,9,p=O.06).The differencesbetween the LCso values for the threecompounds were about a factor of 10 be-tween the two laboratories.A fairly homogenous body of test

results could be received upon reducingthe data set to the results of the eightintermediate laboratories (for lab 3through lab 10: Friedman's X2=7.1,X2crit,0<=o.o5=14.l,p=0,42).Toxicity estimates derived from the

embryo test are in good accordance withthe acute toxicity test with fish. Togetherwith results of the previous study(Schulte et al., 1996) this study may beconsidered as an important contributionto the validation of the embryo test.Furthermore a draft testguideline wasfromulated based on the results andexperiences of this international inter-laboratory study (Nagel, 1998).By now the database has been extended

to 58 compounds. The correlation isgiven in figure 5. The data corroboratethe fact that toxicity estimates derived

from the emhryo test are in good accor-dance with the acute toxicity test withjuvenile or adult fish. Consequently,from our point of view and in correspon-dence with the animal protection law, theacute fish test has to have substitutedby the zebrafish embryo test (DarT).Although we have no information aboutthe ability to feel pain and passion of theearly developmental stages, we areconvinced this will be lower as forjuvenile or adult fish.

Therefore, and based on a carefulassessment of the data from the varioustests the following test concept with fishis suggested (for details see Nagel andIsberner, 1998):Base level. For ethical reasons, the

conventional acute toxicty test withjuvenile or adult fish should be replacedby the embryo test with zebrafish (DarT).Levell. At level 1 the early life-stage

test (OECD guideline 210) deservespriority over the prolonged fish test ac-cording to OECD guideline 204.Level 2. Only a complete life-cycle

test fulfills the requirements of a chronictoxicity test. It cannot be substituted byan early-life-stage test and extrapolation

8log LC$ acute fish test::: 0.048 + 0.986* log LCm DarT (R2::: 0.84)

e

Fig. 5: Correlation of LC50 acute fish toxicity and LC50 embryo toxicity (DarT).58 data points are from Schulte et al. (1996), Bachmann (1996), Maiwald (1997) andBrust (2001).

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~ct

from ACR data (Acute Chronic Ratio;see also Nagel and Isberner, 1998) is notpossible. Therefore, only a complete life-cycle-test can be recommended.Using zebrafish exclusively is advanta-

geous, because in a tiered approach thesimpler tests can serve as reliable rangefinders for the next study, thus saving an-imals and costs.

3.2 The embryo test, a model inroutine waste water control

The zebrafish embryo test could also bean alternative to fish acute toxicity testsin routine waste water control. Fricciusand coworkers (1995) tested 29 samplesof industrial effluents from 11 differentsewage plants. The embryo toxicitiytest was as or more sensitive than the fishtest according German DIN-Norm 38412,L31.Based on these results and influenced

by the perception that the cytotoxiciy testwith permanent fish cells was no longer acandidate to substitute the fish acutetoxicity test in waste water control the"DIN AK 7.6 Fischei-Test" was estab-lished.27 institutions and 17 laboratories

participated in this working group. Firstthe available draft guideline from the em-bryo test for testing chemical (Nagel,1998) was adapted to the particularconditions testing waste water in differ-

ent dilutions. The same lethal endpointsas in the embryo test (DarT) weredefined as lethal effects (48 b): coagulated,no somite, tail not detached, no heartbeat.Subsequently comparison tests with

reference chemicals and ringtests withwaste water of different toxicities werecarried out. All these studies weresuccessful and therefore a guideline wasformulated which is now available as thenational normed guideline DIN 38415-T6 "Bestimmung der nicht akut giftigenWirkung von Abwasser auf die Ent-wicklung von Fischeiern Iiber Verdun-nungsstufen" .In Germany this test is called the "fish

egg test", The use of this name is notunproblematic. Eggs are exposed but theembryo will be effected and thereforefrom a biological point of view this testis also an embryo test.

3.3 DarT, a model in QSARThe relationship between the biologicalactivity of molecules (e.g. 96-h LCso) totheir chemical structures and correspond-ing chemical and physicochemical prop-erties can be described by mathematicalmodels in quantitative structure-activityrelationships (QSARs) (Lipnick, 1995).The ultimate rationale is the establish-ment of causal relationships betweenfeatures of the chemical structures and

-1 1.....•..

-1...-I-.J + primary0 .• secondaryE:::::L -2 • tertiary•......•..-0L()u -3 -....J-- •~---0) -4 A0

3 5

••• •

log Kow

Fig. 6: Relationship between toxicity (LC50) and lipophilicity (log Kow) of primary,secondary and tertiary aliphatic amines. (from Brust, 2001).

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the observed effects or activities(Nendza, 1998). The effects of toxicantson biota depend on their hydrophobic,polar and electrostatic character and canbe determined by reactive transient inter-actions, hydrogen bonding, covalentbinding and lor steric fit to the interactionsite (Nendza and Russom, 1991).These relationships can be used to

predict the behaviour, accumulationpotentials, and toxicity of chemicals sofar untested, and further to rationalisefuture experiments (Konemann, 1981;Saarikoski and Viluksela, 1982; Nendza,1991).For two groups of aliphatic amines, the

primary and secondary amines, a clearcoherency between lipophilicity andtoxicity can be seen (Brust, 2001). Thetoxicity of aliphatic amines increaseswith increasing lipophilicity (Fig. 6) andcan be described using a simple linearregression model:log(lILCso) = 0.467 •log Kow- 3.429(n = 13; R2 = 0.91)Also for the secondary amines a linear

regression model could be found:10g(lILCso) = 0.278 •log Kow- 3.370(n = 13; R2 = 0.85).Both regressions showed that the rela-

tionship between toxicity and lipophilic-ity of the primary and secondary aliphat-ic amines is significant. However, theslope of the regression model for the sec-ondary amines differs from that of themodel for the primary amines by approx-imately a factor of two.In the case of tertiary amines the

tOXICIty increased with increasinglipophilicity up to a log Kowof approxi-mately 2 and then the toxicity of higherlipophilic tertiary amines decreased.(A more detailed discussion, also onpolar and non polar narcotics, can befound in Brust, 2001) .The possibiltiy to predict the toxicity

of chemicals from homologous seriesbased on their properties - or in generalQSAR-studies-should play a muchgreater role in terms of the 3R. Also in"The European Commission White Paperon the Strategy for a Future ChemicalsPolicy" the reduction of tests withanimals and in this context the use ofQSAR is an important aspect.

43

NAGEL

100 T I .•. :. I 'I' '1', T% effect J. .I. :t z I I t I ~" 1..

90 .•. 1 I"80 .L

1h 70eo5040

g CIl ~ s ~ ~ I.t) ~ ~0 0

~0 gC') .•.. •.. I.t)

0 .•.. C') •..

100 .,. - .,..•. ! '" ~ •• T. .,.. .,.90 J. .L ! r8h 80 !70 T,

I60

I·50 J.

402 .•.. N at CD

~ ~ I.t) 10 0 g 0...:~

N ~ g-- C') .•.. .•.. I.t)0 .•.. C') .•...

1LIn01/l

Fig. 7: Influence of propanolol on the frequence of heart-beat of the zebrafishEmbryo. Exposure time 1 and 8h after 48 h of development. (* significantly reduced;from Zeller, 1995).

4 The embryo test, a model intoxicology

Based on the manifold observationswithin the embryo test we proved theapplications of DarT in toxicology.

4.1 Effect of drugs on heart-beatHeart-beat is an important sublethalendpoint which is routinously measuredwithin DarT. Therefore ist was obviousto test the effects of drugs which are usedto influcene the heart-beat therapeutically.We selected four drugs: Verapamil is ablocker of the calcium channel whichreduces the frequency of heart-beat andthe power of contraction. Propanolol isan antagonist of adrenaline/noradrenaline,at the l3-adrenergen receptors andreduces also the frequency of heart-beatand the power of contraction. Theo-phyllin, an inhibitor of the phosphodi-esterase, and antagonist at receptors ofadenosin, increases the heart-beat andthe power of contraction. This appliesalso to isoprenalin an agonist ofadrenalinlnoradrenalin at the l3-adrener-gen receptors.

44

DarT showed the results which we ex-pected according to the different modesof action (Zeller, 1995). Figure 7 showsthe reduction of heart-beat during expo-sure to propanolol exemplary. The reduc-tion depends on exposure time and con-centration of test substance. Based onthese results we think, DarT could be a

model to detect compounds which influ-ence the cardiac functions of vertebrates.

4.2 Effects of chemicals onpigmentation

Schulte (1997) found a reduced pigmen-tation of zebrafish embryos for severalanilines and phenols. Therefore welooked for compounds which are knownto influence the pigmentation in humans.One candidate was p-tert-butylphenol.Maiwald (1997) observed a concentration-dependent hypopigmentation of all pig-ment cells of the fish embryo (Fig. 8a,b).Hypopigmentation was also observed

in tests with primary, secondary andtertiary amines, except for cyclohexy-larnine and dimethylbutylarnine (Brust,2001).As far as we know there is no toxico-

logical model to detect effects onpigmentation, and therefore DarT couldbe a tool.

4.3 DarT, Danio rerioTeratogenicity Assay

"Spina bifida" was described by Sander(1983). He tested ethanol and colcemideand proposed that this phenomenon canbe caused by teratogenic substanceswithin the most sensitive developmentalphase of ontogenesis. The first sign ofaberrations can be seen in a slower epi-bolic movement and later in a dumb-bellshaped yolk.Zeller (1995) observed this phe-

nomenon in embryos exposed to high

Fig. 8a, b: Influence of p-tert-butylphenol on the pigmentation of the eyes of theembryos of zebrafish. (a: control, b: 1.2 mg/Lj from Maiwald, 1997).

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a) dumb-bell shaped yolk (8 h)b) "spina bifida" (16 h)c) "split embryo" (24 h)

Fig. 9a, b, c: "Spina bifida" one example of malformations which can be seen within DarTj during a exposure to a highconcentration of propanolol (1,000 mrnol/L; from Zeller, 1995).

concentrations of propanolol, a ~-recep-tor blocking drug. Maiwald (1997)found this effect in embryos which wereexposed to acetone and Schulte (1997)observed "Spina bifida" after exposureto malathion. In very few cases, thisphenomenon was also observed inthe toxicity tests performed withhexylamine, diisobutylamine, dibuty-lamine and dimethylbuytlamine (Brust,

2001). The phenomenon is shown inFigure 9.The observation of this malformation

and many other teratogenic effectswithin the many tests carried out in myworking group led to a research projectfunded by BASF AG. The goal was todevelop a screening assay to predict theteratogenic potential of chemicals inmammals (DarT). The tested teratogenic

toxicological endpoints are included intable 2.41 chemicals were selected based

on following criteria: Mammalianteratogens, with or without metabolicactivation. For example diethylene glycoldimethyl ether, N,N-dimethylfromamideand all-trans retinoic acid, Methyl-mercury chloride and valproic acid,respectively. Mammalian non-teratogens

Fig. 10: Scoliosis after exposure tomethylmercury chloride (0.24 mmollL,48 h, from Bachmann, 2002).

Fig. 12: Growth-retardation in larvaeafter exposure to diethylene glycoldimethyl ether (62.2 mmollL, 120 h,* significantally reduced ((p = 0.05)),from Bachmann, 2002).

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Fig. 11: Deformations of tail and head(Rachischisis) after exposure to valproicacid (0.12 mmollL, 24 h, fromBachmann, 2002).

45

_N_AG_E_L i~----~,

as diethyl glycol and glucose. Substancesshowing ambiguous results in mammals.e.g. colcemide and cycloheximide.

All the data and a detailed assessmentof the test will be found inBachmann (2002). in Figure 10, 11 and12 selected effects are shown. DarToffers the possibility to detect effects onthe tail length after 120 h. This extensionof the test is recommended if thestructure of the chorda seems to bemodified within 48 h. One result of thisprocedure is given in Figure 13 fordiethylene glycol dimethyl etherexemplary.

All the effects observed within DarT,lethal and malformations, are evaluatedby probit analysis. An example is givenin Figure 14. The quotient between LC,oand EC,o malformationis an indicator of theteratogenic potential of the compoundand can be calculated as TeratogenicIndex (TI-Value). In the case of all-transretionic acid the LCso is 3.6 urnol/l andthe ECso malformation0.011 urnol/l and theTI-Value consequently 327.3. For thiscompound the teratogenic potential,expressed by the TI -Value, is very clear.But for other compounds the significanceof this index is worthy of discussion (fordetails see Bachmann, 2002).

100

80

20

o0.003

o

malformations lethal

3.3 33.3 3330.03 0.3

concentration [~moI/Ll

Fig. 14: Concentration-effect-relationships of lethality and malformations forall-trans retinoic acid (48 h) (from Bachmann, 2002).

ated up to now the results of 88% ofchemicals are in agreement with findingsin mammals. In 10% (4 compounds)a false positive result was found. Forsalicyclic acid the result was falsenegative in comparison with the effectsin mice and rats. For thalidomide the

For the 41 substances tested and evalu- result was false negative if compared to

1.68

1.64

1.60

E.s 1.56

~c:~ 1.52~ c

1.44 - -........ I ±Std.Dev.

CJ ±Std. Err.1.40 lJ Mean

control 21.5 62.2*

concentration {mmol/lJ

36.6

Fig. 13: Growth-retardation in larvae after exposure to diethylene glycol dimethyl ether(62.2 mmol/l, 120 h, •significantally reduced «p = 0.05)), from Bachmann, 2002).

46

the effects in humans. But the resultcompared with the results of other toxi-cological models, for example with rats,is not false negative but in accordance.

Even for teratogens with metabolicactivation within DarT malformationscould be detected. But the effects werenot as strong as assumed. For examplefor diethyleneglycol dimethyl ether(diglyme) the LOECength of tail was 62.2mmol/l and for metoxyacetic acid theLOEClength of tail was 0.11 mmol/I.Therefore it seems to be meaningfulto investigate the metabolic potential ofthe zebrafish embryo and if necessary tooptimise the test for example by the useof S 9 mammalian liver homogenate.

In summary, the Danio rerio Terato-genicity Assay can be a screening test toinvestigate drugs and other chemicals inan early inventory phase. In terms of the3R DarT is an alternative method toreduce the number of common laboratorytests and replace screening assays withlaboratory animals (Bachmann, 2002).

5 Perspectives

DarT is a rapid, simple and cost-effectivetest. The embryos are available per-manently and timely. Only smallamounts of test substance are required.

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____ i~----------------------------------------------N-A-G--EL~,

Furthermore DarT is an alternative test,because within the test period theembryos will not hatch. According to theGerman Animal Welfare Act, studiesperformed on embryos are classified asnon-animal-test. The study is primarilylimited to the first 48 hours of develop-ment and therefore, in our opinion, canbe justified ethically. Furthermore froma biological point of view we think thatthe use of less far developed stagesof animals is an important issue in termsof3R.

The number of advantages willenormously increase in the near future.The zebrafish is the "Drosophila" ofvertebrates. The genome is or is mostlyknown. There are thousands of mutantsavailable and companies are foundedto use this potential commercially. Theexisting phenotypes can be comparedwhich chemotypes and the phenomenoncan be assessed from a genetic, molecu-lar and developmental point of view.

This development makes greatprogress and could not be foreseen whenwe started ten years ago to substitute theacute fish test by the embryo test withzebrafish.

References

Bachmann, J. (2002). Entwicklung einesTeratogenitats-Screening-Tests mitEmbryonen des Zebrabiirblings Daniorerio. Doctoral thesis, TU Dresden,Germany.

Brust, K. (2001). Toxicity of aliphaticamines on the embryo of the zebrafishDanio rerio - Experimental studiesand QSAR. Doctoral thesis, TUDresden, Germany.

Eaton, R. C. and Farley, R. D. (1974).Spawning cycle and egg production ofzebrafioh, Rrachvdanio rerio, in thelaboratory. Copeia 1,195-209.

Ekker, M. and Akimenko, M. A. (1991).Le poisson zebre (Danio rerio), unmodele en biologie du development.Medicine/sciences 7, 533-560.

Friccius, T. C., Schulte, c., Ensenbach,U. et al. (1995). Der Embryo test mitdem Zebrabarbling - eine neueMoglichkeit zur Priifung und Be-wertung der Toxizitat von Abwasser-proben. VomWasser 84,407-418.

ALTEX 19, SuppJ. 1/02

Fufsmann, O. und Nagel, R. (1997).Laborvergleichsversuch - Embryotestmit dem Zebrabarbling Danio rerio.Federal Environmental ProtectionAgency of Germany, Report No. 10603908.

Goolish, E. M., Okutake, K. and Lesure,S. (1999). Growth and Survivorship oflarval zebrafish Danio rerio on pro-cessed diets. N. Am. 1. Aquacult. 61,189-198.

Hisaoka, H. K. and Battle, H. 1. (1958).The normal developmental stages ofthe zebrafish Brachydanio rerio(Hamilton-Buchanan). 1. Morphol.102,311-326.

Kimmel, C. B., Sepich, D. S. andTrevarrow, B. (1988). Developmentand segmentation in zebrafish.Develop. Suppl. 104, 197-207.

Kimmel, C., Ballard, W., Kimmel, S. etal. (1995). Stages of embryonic devel-opment of the zebrafish. Develop. Dyn.203, 253-310.

Konemann, H. (1981). Quantitativestructure-activity relationships in fishtoxicity studies. 1. Relationship for 50industrial pollutants. Toxicology 19,209-22l.

Laale, H. W. (1977). The biology and useof zebrafish, Brachydanio rerio, infisheries research: A literature review.1. Fish. BioI. 10, 121-173.

Lange, M., Gebauer, W., Markl, J. andNagel, R. (1995). Comparison oftesting acute toxicity of zebrafishBrachydanio rerio and RTG-2 cytotox-icity as possible alternatives to theacute fish test. Chemosphere 30 (11),2087 -2102.

Lele, Z. and Krone, P. H. (1996). Thezebrafish as a model system in devel-opmental, toxicological and transgenicresearch. Biotech. Adv. 14(1),57-72.

Lipnick, R. L. (1995). Structure-activityrelationships. In G. R. Rand (ed.),runaamencals of aquatic toxicologv(609-655). London: Tayler & Francis.

Maiwald, S. (1997). Wirkung vonLosungsvermittlern und lipophilenSubstanzen auf die Embryonalent-wicklung des Zebrabiirblings (Brachy-danio rerio). M. Sc. thesis, TUDresden, Germany.

Nagel, R. (1988). Umweltchemikalienund Fische - Beitriige zu einer Be-wertung. Habilitationschrift. Mainz:

Johannes Gutenbcrg-Universitat.Nagel, R. (1993). Fish and environmen-

tal chemicals - a critical evaluation oftests. In T. Braunbeck, W. Hanke andH. Segner (eds.), Fish-Ecotoxicologyand Ecophysialogy (147-156). Wein-heim: VCH.

Nagel, R. (1998). Fish embryo toxicitytest with the zebrafish Danio rerio.Initial consideration for a OECD DraftGuideline. In W. Heger, S. Jung,S. Martin et al. (eds.), UBA - Texte-band 58/98 (80-93). Berlin: Umwelt-bundesamt.

Nagel, R. and Isberner, I. (1998). Testingof chemicals with fish - a critical eval-uation of tests with special regard tozebrafish. In T. Braunbeck, D. E. Hin-ton and B. Streit (eds.), Fish-Ecotoxi-cology and Ecophysiology (337-352).Basel: Birkhauser Verlag.

Nendza, M. and Russom, C. L. (1991).QSAR modelling of the ERL-D fat-head minnow acute toxicity database.Xenobiotica 21,147-170.

Nendza, M. (1991). QSARs of biocon-centration: validity assessment of logPowlBCF correlations. In R. Nageland R. Loskill (eds.), Bioaccumulationin aquatic systems: Contributions tothe assessment (43-66). Weinheim:VCH.

Nendza, M. (1998). Structure-activity re-lationships in environmental science.London: Chapman & Hall.

Nusslein- Volhard, C. (1994). Of flies andfishes. Science 266 (5185), 572-576.

Roosen-Runge, E. C. (1938). On theearly development - bipolar differenti-ation and cleavage of the zebra fish,Brachydanio rerio. Biol. Bull. 75, 119-133.

Saarikoski, J. and Viluksela, M. (1982).Relation between physicochemicalproperties of phenols and their toxicityand accumulation in fish. Ecotoxicol.Environ. Sa! 6, 501-512.

Sander, K. und Baumann, M. (lOg]).Auslosung von embryonalen Fehlbil-dungen beim Zebrabarbling. Biologiein unserer Zeit 13,87-94.

Schulte, C. and Nagel, R. (1994). Testingacute toxicity in the embryo of ze-brafish, Brachydanio rerio, as an alter-native to the acute fish test: Prelimi-nary Results. ATLA 22,12-19.

Schulte, C., Bachmann, J., Fliedner, A.,

47

_N_A_GE_L i~----~,

Meinelt, T. and Nagel R. (1996). Test-ing acute toxicity in the embryo ofzebrafish (Brachydanio rerio) - an alter-native to the fish acute toxicity test.Proceedings, 2"d World Congress - Al-ternatives & animal use in the life sci-ences. Utrecht, The Netherlands.

Schulte, C. (1997). Entwicklung undValidierung einer Methode zur Er-mittlung der Toxiziuu von Chemikaliengegeniiber Embryonen von Brachy-danio rerio. Doctoral thesis. Mainz:Johannes Gutenberg-Universitat.

Thomas, R. G. and Waterman, R. E.(1978). Gastrulation in the teleost,Brachydanio rerio. Scan. Electr. Mi-croscopy 11,531-540.

Westerfield, M. (1995). The zebrafishbook: a guide for the laboratory use ofzebrafisli (Brachydanio rerio), 3"edition. USA-Eugene: University ofOregon Press, Institute of Neuro-science.

Wixon, J. (2000). Danio rerio, thezebrafish. Yeast 17,225-231.

Zeller, Y. (1995). Einfluj3 van herzwirk-samen Pharmaka auf die Embryona-lentwicklung des Zebrabiirblings,Brachydanio rerio, unter besondererBeriicksichtigung von cardiogenen Ef-fekten. Diplomarbeit. Mainz: JohannesGutenberg -Universi tat.

AcknowledgementsThe investigations were funded byBMBF, Umweltzentrum of the Universi-ty of Mainz, UBA, GDCh and BASF AG.Many thanks to the master students JeanBachmann, Telse Friccius, Thomas Hess,Liane Igel, Monica Lange, Sabine Mai-wald, Christoph Schulte, Peter Stangl,Yvonka Zeller and the Ph.D studentsJean Bachmann, Kristin Brust andChristoph Schulte. All of them have con-tributed and contribute very much to thestate of the art of DarT. Special thanks toKristin Brust and Jean Bachmann, whocontributed parts of their doctoral thesisto this manuscript.It's a great merit of Dr. Pluta from the

German Environmental Protection Agen-cy, that he has organised and led the DINAK 7.6 successfully.

48

Correspondence toProf. Dr. Roland NagelTU DresdenInstitut fur HydrobiologieD-O1062 DresdenE-mail: rnagelwrcs.urz.tu-drcsden.de

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