Assessing sediments and fish health using a weight-of-evidence„Assessing sediments and fish health using a weight-of-evidence approach and effect-directed analyses –

i h f th f fi h d li i th D b i "in search for the causes of fish decline in the Danube river"

H H ll tHenner Hollert

Institute for Environmental Research RWTH Aachen UniversityInstitute for Environmental Research, RWTH Aachen UniversityInstitute for Zoology of the University of Heidelberg

S. Keiter, M. Böttcher, S. Grund, N. Seitz, J. Otte, K. Bluhm & T. Braunbeck (Department of Zoology, University of Heidelberg , Germany)gy, y g , y)

K. Wurm (Gewässerökologisches Labor, Starzach , Germany)

E. Higley, J. Giesy & M. Hecker (University of Saskatchewan and ENTRIX, Canada)

H. Olsman, B. van Bavel & M. Engwall (MTM, Örebro University, Sweden)

G. Reifferscheid & W. Manz (Federal Hydrological Institute, Koblenz , Germany)

L Erdinger (Department of Hygiene University Heidelberg Germany)L. Erdinger (Department of Hygiene, University Heidelberg , Germany)

U. Kammann (Federal Research Centre for Fisheries, Hamburg, Germany)

R. Schönberger & M. Suter (EAWAG, Switzerland) g ( , )

T. Schulze & W. Brack (UFZ Leipzig, Germany)

J. Otte, C. Andersson, A. Abrahamson & B. Brunström (Uppsala University,Sweden)

L.Yang, C. Zinsmeister & U. Strähle (Institute of Toxicology and Genetic, FZK Karlsruhe)

IntroductionIntroductionnn

orm

atio

nor

mat

ion

und

und

info

info

Bac

kgro

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Upper DanubeUpper Danube

IntroductionIntroduction

Fish decline in the upper Danube River

nn

1200SigmaringenRiedlingen Ehingen

tück

] sh

/a]

Karlsruhe

TSCHECHISCHE REPUBLIKRegensburgBaden- Bayernor

mat

ion

orm

atio

n

Nm 200

400

1000

Fang

zahl

en [S

tC

atch

[fis

Karlsruhe

Pforzheim Stuttgart

Reutlingen

REPUBLIKRegensburg

P

Donau

BadenWürttemberg

Bayern

Ingolstadt Bad Abbach

Jochen-steinun

d un

d in

foin

fo ☺1980 1985 1990 1995 2000

mberofcat

0

Reutlingen

ÖSTERREICH

Augsburg

München

Linz

Passau

Tuttlingen

Neckar

Lauc

hert

Riedlingen

Rottenacker

EhingenÖpfingen

Ulm

SchwarzachBac

kgro

Bac

kgro

güte

Sigmaringen Riedlingen Ehingen

III

IV

ality

SCHWEIZ

Friedrichs-hafen

Salzburg

gSigma-ringen

Sigmaringen-dorf

SchwarzachBB

Gew

ässe

rg

I

II

Wat

erqu

a

SCHWEIZ 1970 1975 1980 1985 1990 1995 2000

IntroductionIntroductionnn

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nor

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ion

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und

info

info

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BB

IntroductionIntroduction

Potential impacts? Effects Consequence

n

Structural changes of habitat

orm

atio

n of habitat

Change in temperature

Impairment of health

R d ti f

und

info temperature

Fish removal(Human & animals)

Reduction of food supply

Failing

Decline of fishpopulation

Bac

kgro

(Human & animals)

Ch i l

Failing reproduction

B Chemical contamination Relevance?Relevance?

IntroductionIntroduction

Sediments?

n

• Accumulation of contaminants by adsorption to suspended matter in water phase Sedimentation

orm

atio

n matter in water phase Sedimentation

• Direct exposure of benthic organism and fish offspring, respectively

und

info

p y

• Flood events Remobilisation of sediment-bound contaminants into water phase

Bac

kgro

p

B

IntroductionIntroduction

A pilot study conducted in 2002/03

tudy

grat

ed st

this

inte

gpo

se o

f t „Overall, the ecotoxicological hazard potential shown has indeed to be considered as one potential reason for the decline in fish catches at the upper

Purp Danube River. However, based on the results of this

pilot study, it is not possible to elucidate that chemically induced alterations are responsible for the fish decline“fish decline

Keiter et al. (2006) Environ Sci Pollut Res 13: 308 – 319

IntroductionIntroduction

Objectives?

tudy • Assessment of the ecotoxicological contamination of sediments from

different sites along the pper Dan be Ri er

grat

ed st different sites along the upper Danube River

this

inte

g

• Identification of the relevant hazardous substances and their sources

pose

of t

• Verification of the relevance of sediment contamination for the fish decline

Purp

Conceptal frameworkConceptal frameworkWeight of Evidence –ApproachesTriad Approach according to Chapman (1990)

Line of evidence: community structure Triad-Approach according to Chapman (1990)community structure

Line of evidence:Line of evidence: Biotests

proa

chpr

oach

riad

app

riad

app

… th

e tr

… th

e tr

Line of evidence: Chemical analyses

Conceptal frameworkConceptal framework

… Evaluation of the relevance of

In vitro assays for the fieldceceof

evi

den

of e

vide

n

Chemical analyses Bioassays

al li

nes o

al li

nes o

+Micronucleus Assay

In situ

+Histopathology

In situ

addi

tiona

addi

tiona

Community structure

+ +… a

… a

Chapman & Hollert (2006): Should the Sediment Quality Triad become a Tetrad, a Pentad or Possibly Even a Hexad? J Soils & Sediments

Conceptal frameworkConceptal frameworkcece

of e

vide

nof

evi

den

Chemical analyses Bioassays

+Effect directed

Analyses

al li

nes o

al li

nes o

+

addi

tiona

addi

tiona

Community structure Identification of the contaminants

responsible for the effects

… a

… a

Hecker & Hollert (2009) Effect-directed analysis (EDA) in aquatic ecotoxicology: stateof the art and future challenges, Environ Sci Poll Res, 16:607–613

IntroductionIntroduction

Acute and mechanism-specific endpoints of the in vitro bioassays

•Cytotoxicity – Cell damage/dead?

s

•Embryotoxitiy – Teratogenicity of the sediments?

•Dioxin-like activity – Induction of specific enzymes

Bio

assa

y y p yinvolved in metabolism of xenobiotics (via Ah-receptor)?

•Endocrine activity – Effects to hormonal balance?B

•Gentoxicity – DNA damage?

•Alterations in gene expression patterns (Danio rerio•Alterations in gene expression patterns (Danio reriochip with 20000 genes)

•Immunotoxicity (hIL8 hIL6 and CD54 in Beas2B and•Immunotoxicity (hIL8, hIL6 and CD54 in Beas2B and MM39 cells)

Materials & MethodsMaterials & Methods

Sediment samples

• Sampling periodJanuary-February 2006

mpl

ing

mpl

ing • Sampling sites

1 = Sigmaringen2 L h t (t ib t )

men

t sam

men

t sam 2 = Lauchert (tributary)

3 = Riedlingen4 = Schwarzach (tributary)

Sedi

mSe

dim

( y)5 = Rottenacker6 = Ehingen7 Ö fi

Bavaria (BfG):Jochenstein

7 = Öpfingen

JochensteinBad Abbach

ResultsResults

Genotoxicity of whole sedimentsSediment contact Comet-Assay

using embryos of Danio rerio

in v

itro

oxic

ity in

Gen

ot

Method: Kosmehl et al. 2006, ET&C

Data: Seitz et al. 2007 Mutat. Res.

ResultsResults

Genotoxicity of the sediment extractsMicronucleus assay in vitro with RTL-W1 cells

in v

itro NQOaverage

2 5

3,0

3,5

oxic

ity in

NE

Q [µ

g/g]

1,5

2,0

2,5

Gen

ot

N0,5

1,0NC

Sigmari

ngen

Lauc

hert

Riedlin

gen

Schwarz

ach

Rotten

acke

rEhin

gen

Oepfin

gen

Ingols

tadt

Bad Abb

ach

Joch

enste

in

0,0

River flow directionBöttcher et al. 2009, Keiter et al. 2009

ResultsResults

Genotoxicity in barbels from the field Livern = 5

Micronucleus assay in situ

Erythrocytes from Barbus barbus0,4

*

n = 5 4,6 4,1

2000 cells / sample

in si

tu

[%]

0 2

0,3

*

**

2,1

3,6In vivo! In situ!

toxi

city

i

MN

0,1

0,2

The induction factor

Gen

ot

ngenngen

cker

ngen0,0

NC

(IF) was calculated by dividing the median of each concentration by the

Sigmaring

Riedling

RottenackEhing NC

Flussverlauf

median of the corresponding control group

Flow direction* significant Genotoxicity (χ² Test, p < 0,05)

when compared to negative control (NC) Böttcher et al. (2009); Keiter (2007)

ResultsResults

Dioxin-like activity of the sediment extracts

45000DR CALUX

S h h

EROD, GPC.2D.Luc and DR CALUX assays

] 30000

35000

40000 EROD

2.5

3.0

3.5Procedural control

TCDD

mg-1

*min

-1]

2

3Schwarzach

TCDD

*mg-1

*min

-1]

ctiv

ityct

ivity BioTeqs (pg/g)

TCDD EC25 (pg/ml)

Sample EC25 (g/ml)

Bio-

TEQ

[pg/

g]

20000

25000

1.0

1.5

2.0

EC25: n.b.

Negative control

activ

ity [p

mol

*

1

2

Negative control

activ

ity [p

mol

*

nn--lik

e ac

like

ac

B

5000

10000

15000

0.1 1 10 100 10000.0

0.5

25

[ di t d i ht/ l di ]

ERO

D a

0.01 0.1 1 100

EC25: 0.18 mg/ml

[ di t d i ht/ l di ]

ERO

D a

Dio

xin

Dio

xin

Sigmaringen

Lauchert

Schwarzach

Riedlingen

Rottenacker

Ehingen

Öpfingen

Bad Abbach

Jochenstein0

[mg sediment dry weight/ml media] [mg sediment dry weight/ml media]

Standorte im DonauverlaufGrund et al. (2009)

DiscussionDiscussion

Appraisal of results: dioxin-like activity

• Tested sediments induced AhR-mediated activities in both dioxin-specific bioassays

ctiv

ityct

ivity • Danube River 2006: max. Bio-TEQ 40000 pg/g SEQ (Grund. in prep)

nn--lik

e ac

like

ac

• Danube River 2005: max. Bio-TEQ 5000 pg/g SEQ (Keiter et al. 2008)

• Rhine River: max. Bio-TEQ 1300 pg/g SEQ (Hinger 2003)

Dio

xin

Dio

xin

• Bitterfeld: max. Bio-TEQ 100 000 pg/g SEQ (Brack et al. 2002)

High dioxin-like activities by several sediment extracts

Effects on health of fish in the Danube River cannot be ruled out

Id tifi ti f th b t b EDAIdentification of the substances by EDA

ResultsResults

Multilayer fractionation of the Dioxin-like activityEROD and DR CALUX assays, chemical analysis

ctiv

ityct

ivity

nn--lik

e ac

like

acD

ioxi

nD

ioxi

n

Keiter et al. 2008 Anal. Bioanal. Chem.

ResultsResults

Multilayer fractionation of Dioxin-like activitiesEROD and DR CALUX assays, chemical analysis

ctiv

ityct

ivity

nn--lik

e ac

like

ac

75 % unknown

Dio

xin

Dio

xin

25 % by EPA-PAHs, PCBs, PCDD/Fs

Keiter et al. (2008) Anal. Bioanal. Chem.

ResultsResults

HPLC fractionation of Dioxin-like activities

Lauchert

EROD assay

(Hydroxy-)Quinones, keto-, dinitro-,

104

105

155%

ctiv

ityct

ivity PAHs with 3,4,5 rings

, ,hydroxyl-PAHs, N-Heterocycles

Q [p

g/g]

103

nn--lik

e ac

like

ac

non-ortho-PCBs, PCDD/Fs

Bio-

TE

102

Dio

xin

Dio

xin

x) Ac) RE 18 F1 F2 F3 F4 F5 F6 F7 F8 F9 10 11 12 13 14 15 16 17 18

100

101

RE (Ac:H

x)RE (A

c)

Dial

ys.R

E

Summe F

1-18

F1 F2 F3 F4 F5 F6 F7 F8 F9

F10 F11 F12 F13 F14 F15 F16 F17 F18

Grund et al. (in prep)

ResultsResults

HPLC fractionation of Dioxin-like activitiesEROD assay

c)10000 b)

6% %

a)

ctiv

ityct

ivity

97%

97%

99%

60%18% 99

%10

0% 100%

g/g

SE

Q]

100

1000 86%

84%

98%

% 0%

53%

100%

99%

8%

9694

%

99% 80

% 99%

% 100%

100%

9%

nn--lik

e ac

like

ac

3% 3%

89%

7%9

19%

TEQ

s [n

g

10

100

14%

16%

85%

100

54%

8%4%

4% 6%

100%

9%

Dio

xin

Dio

xin

AMD extra

ctSum

F 9 10 11 13 14 15

AMD extra

ctSum

F 9 10 11 13 14 15

1

AMD extra

ctSum

F 9 10 11 13 14 15

3%

AMAM AM

Bio-TEQ Sum Chem-TEQBenzo(a)anthraceneChrysene

Benzo(a)anthraceneChryseneBenzo[j]fluoranthene Benzo[k]fluoranthene

Benzo[a]pyrene Benzo[g,h,i]perylene Dibenzo[a,h]anthraceneIndeno[1,2,3-cd]pyrene Comparison of the biological response in the EROD assay (Bio-TEQs) and

the calculated PAH toxic equivalents (Chem-TEQs) obtained by chemical

Grund et al. (in prep)

analyses of the dialyzed (AMD) extracts and their fractions of the sediments from the sites at a) Sigmaringen, b) Lauchert and c) Oepfingen.

Materials & MethodsMaterials & Methods

Endocrine activity: H295R bioassay

• NCI-H295R-cell line: human adrenocortical carcinoma cell line

ssay

ssay

• Ability to produce the steroid hormones of each of the three phenotypically distinct zones found in the adult adrenal cortex

95R

bio

a95

R b

ioa

Screening of effects caused by sediment samples of the Danube River on:

adrenal cortex

H29

H29

Screening of effects caused by sediment samples of the Danube River on:• Synthesis of steroid hormones – ELISA • Expression of important genes, involved in steroidogenesis - Real time PCR

MICHIGAN STATE U N I V E R S I T Y

(Hecker et al 2007, Blaha et al. 2006; Gazdar et al. 1990; Hilscherova et al. 2004; Zhang et al. 2005)

U N I V E R S I T Y

DiscussionDiscussion

Appraisal of results: hormone analysis

• Sediment extracts of the sampling sites Riedlingen, Öpfingen and Rottenackercaused alterations (>1,5-fold induction) in production of P, T and E2

alys

isal

ysis • No comparable studies

• First investigation of effects of sediment samples to hormone production in H295R

mon

e m

one

ana

ana

cells

• OECD ring test: Validation of a H295R cell line screening test (Hecker et al. 2007)

Hor

mH

orm

Effects on hormonal balanceEffects on hormonal balance

Impacts on reproduction/sex ratio/several metabolism pathways in vivo cannot be ruled out

ResultsResultsIn co-operation with the ITG-FZK Karsruhe, Prof. Dr. Uwe Strähle

reri

ore

rio))

((Dan

io r

Dan

io r

anal

yses

an

alys

es

A a

rray

aA

arr

ay a

DN

AD

NA

Bluhm et al. (in prep)

ResultsResults

Endocrine activity of sediment extracts

MSO

)3,0

5 mg/ml2 mg/ml0 5 / lM

SO

)3,0

5 mg/ml2 mg/ml

DM

SO)

2 5

3,05 mg/ml2 mg/ml0 5 mg/ml

H295R-Bioassay – Effects on steroid hormoneser

one

solv

ent c

ontro

l, D

M

2,0

2,5 0,5 mg/ml

diol olve

nt c

ontro

l, D

M

2,0

2,5 0,5 mg/ml

tero

neso

lven

t con

trol,

D

2,0

2,5 0,5 mg/ml

alys

isal

ysis

Test

oste

tion

- rel

ativ

e to

s

1,0

1,5

Estr

adtio

n - r

elat

ive

to s

o

1,0

1,5

Prog

est

ctio

n - r

elat

ive

to

1,0

1,5

mon

e an

am

one

ana

MSOBlank

Blankngen

zachngen

genngen

acker

hert

(fold

indu

ct

0,0

0,5

MSOBlank

Blankringen

rzach

ingenngen

ngenacke

rchert

(fold

indu

ct

0,0

0,5

DMSOBlank

Pr Blank

maringen

hwarzach

edlingen

ÖpfingenEhingen

tenacker

Lauchert

(fold

indu

0,0

0,5

Hor

mH

orm

FI =mean value SC

mean value sample *1 FI = 1 for SC

DMS BlanPr. B

lan

Sigmaringe

Schwarza

Riedlinge

ÖpfingeEhinge

RottenackLauche

• Alterations in T concentration: Riedlingen, Öpfingen und Rottenacker• Max induction: Riedlingen 1 8-fold induction

DMSBlan

Pr. Bla

Sigmaring

Schwarza

RiedlingÖpfinge

Ehinge

RottenacLauch

• Alterations in E2 concentration: Riedlingen, Öpfingen, Rottenacker, Sigmaringen, Schwarzach• Max. induktion: Öpfingen > 2-fold induction

D BPr.

SigmarSchwa

RiedlÖpf Eh

Rotten Lau

• Alterations in P concentration: Riedlingen, Öpfingen und Rottenacker• max. induction: Riedlingen 2-fold inductionmean value SCMax. induction: Riedlingen 1,8 fold inductionMax. induktion: Öpfingen > 2 fold inductionmax. induction: Riedlingen 2 fold induction

Conclusion & prospectsConclusion & prospects

•Detection of high genotoxicity in several in vitro bioassays and in the micronucleous assay in situ high relevance of the in vitro results for the field!situ high relevance of the in vitro results for the field!

•Toxic effects on state of health of fish population cannot be ruled out

•Detection of high dioxin-like activities of several sediment extracts in both applied test

onon

systems

• Toxic effects on state of health of fish population cannot be ruled out

• Detection of endocrine disrupting potencies of individual sediment extracts in both applied

oncl

usio

oncl

usio • Detection of endocrine disrupting potencies of individual sediment extracts in both applied

test systems

• Imbalance in the complex network of sensitive regulated steps in the synthesis of steroid

CC hormones

• Effects of endocrine disrupting chemicals in sediments of the Danube River to sex ratio/ reproduction/metabolism of fish population cannot be ruled out

• Identification of “hot spots” along the Danube River

Conclusion: Determined ecotoxicological contamination of the sediments has to be accounted i t t i fl i f t ith t t th d li f fi h l ti i thas an important influencing factor with respect to the decline of fish population in the upper

Danube River.

Conclusion & prospectsConclusion & prospects

Where do we go?

Correlation ??

Exotoxicological potential

Sediment sample

Grund et al. 2009: Di f A i

Grund et al. 2009 ,

ss

Relevance for in situ situation

Bioassays

Diseases of Aquatic Organisms, in press

Higley et al. 2009 in prep.

Pros

pect

Pros

pect

YESSTOPEffect ?

No In situ investigations YES

PP

Chemical AnalysisFractionation

Fractions Identification of relevant

contaminants

Bioassay

AcknowledgmentAcknowledgment

Thanks to: Deutschen Bundesstiftung Umwelt for support

u!ha

nk y

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h