„Assessing
Assessing sediments and fish health using a weight-of-evidence
approach and effect-directed analyses –
i search
in
h ffor the
th causes off fi
fish
h ddecline
li in
i the
th Danube
D
b river"
i "
H
Henner
Hollert
H ll t
Institute for Environmental Research,
Research RWTH Aachen University
Institute for Zoology of the University of Heidelberg
S. Keiter, M. Böttcher, S. Grund, N. Seitz, J. Otte, K. Bluhm & T. Braunbeck (Department of
Zoology,
gy, Universityy of Heidelberg
g , Germany)
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,
L.
Hygiene University Heidelberg , Germany)
U. Kammann (Federal Research Centre for Fisheries, Hamburg, Germany)
R. Schönberger
g & M. Suter ((EAWAG,, Switzerland))
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)
B
Backgro
und info
ormation
n
Introduction
Upper Danube
Introduction
Fish decline in the upper Danube River
Sigmaringen
Riedlingen
Ehingen
1000
400
200
Baden
BadenWürttemberg
Karlsruhe
0
1980
Pforzheim
1985
Stuttgart
1990
Bayern
Bad Abbach
1995
2000Ingolstadt
Reutlingen
Lauchert
Ulm
Rottenacker
Öpfingen
Ehingen
Riedlingen
g
Tuttlingen
Schwarzach
Sigma- Sigmaringenringen dorf
Friedrichshafen
☺
Jochenstein
P
Passau
Donau
Neckar
TSCHECHISCHE
REPUBLIK
Regensburg
IV
Augsburg
Sigmaringen
Riedlingen
Ehingen
Linz
München
Water
qua
ality
Gewässerg
güte
Fangzahlen
[St
tück]
Catch [fis
sh/a]
N m
mber of cat
Background info
B
ormation
n
1200
III
ÖSTERREICH
II
Salzburg
I
SCHWEIZ
1970
1975
1980
1985
1990
1995
2000
B
Backgro
und info
ormation
n
Introduction
Introduction
B
Backgro
und info
ormation
n
Potential impacts?
Structural changes
of habitat
Change in
temperature
Fish removal
(Human & animals)
Chemical
Ch
i l
contamination
Effects
Consequence
Impairment of
health
Reduction
R
d ti off
food supply
Decline of fish
population
Failing
reproduction
Relevance?
Introduction
B
Backgro
und info
ormation
n
Sediments?
• Accumulation of contaminants by adsorption to suspended
matter in water phase Sedimentation
• Direct exposure of benthic organism and fish offspring,
p
y
respectively
• Flood events Remobilisation of sediment-bound
p
contaminants into water phase
Introduction
Purp
pose of tthis integ
grated sttudy
A pilot study conducted in 2002/03
„Overall, the ecotoxicological hazard potential
shown has indeed to be considered as one potential
reason for the decline in fish catches at the upper
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
decline“
Keiter et al. (2006) Environ Sci Pollut Res 13: 308 – 319
Introduction
Purp
pose of tthis integ
grated sttudy
Objectives?
• Assessment of the ecotoxicological contamination of sediments from
different sites along the upper
pper Dan
Danube
be Ri
River
er
• Identification of the relevant hazardous substances and their sources
• Verification of the relevance of sediment contamination for the fish
decline
Line of evidence:
community structure
Conceptal framework
Weight of Evidence –Approaches
Triad Approach according to Chapman (1990)
Triad-Approach
… the trriad app
proach
Line of evidence:
Biotests
Line of evidence:
Chemical analyses
Conceptal framework
… additiona
a
al lines o
of evidence
… Evaluation of the relevance of
In vitro assays for the field
Chemical
analyses
Bioassays
Histopathology
Micronucleus Assay
Community
structure
+
In situ
In situ
+
Chapman & Hollert (2006): Should the Sediment Quality Triad become a Tetrad, a Pentad or
Possibly Even a Hexad? J Soils & Sediments
… additiona
a
al lines o
of evidence
Conceptal framework
Chemical
analyses
Bioassays
+
Community
structure
Effect directed
Analyses
Identification of the contaminants
responsible for the effects
Hecker & Hollert (2009) Effect-directed analysis (EDA) in aquatic ecotoxicology: state
of the art and future challenges, Environ Sci Poll Res, 16:607–613
Introduction
Acute and mechanism-specific endpoints of the in
vitro bioassays
•Cytotoxicity – Cell damage/dead?
B
Bioassay
s
•Embryotoxitiy – Teratogenicity of the sediments?
•Dioxin-like activityy – Induction of specific
p
enzymes
y
involved in metabolism of xenobiotics (via Ah-receptor)?
•Endocrine activity – Effects to hormonal balance?
•Gentoxicity – DNA damage?
•Alterations in gene expression patterns (Danio rerio
chip with 20000 genes)
•Immunotoxicity (hIL8,
(hIL8 hIL6 and CD54 in Beas2B and
MM39 cells)
Materials & Methods
Sediment samples
• Sampling period
Sedim
ment sam
mpling
January-February 2006
• Sampling sites
1 = Sigmaringen
2 = Lauchert
L h t (tributary)
(t ib t )
3 = Riedlingen
4 = Schwarzach (tributary)
(
y)
5 = Rottenacker
6 = Ehingen
7 = Öpfingen
Ö fi
Bavaria (BfG):
Jochenstein
Bad Abbach
Results
Genotoxicity of whole sediments
Sediment contact Comet-Assay
Genotoxicity in vitro
using embryos of Danio rerio
Method: Kosmehl et al. 2006, ET&C
Data: Seitz et al. 2007 Mutat. Res.
Results
Genotoxicity of the sediment extracts
Micronucleus assay in vitro with RTL-W1 cells
Genotoxicity in vitro
3,5
NQOaverage
3,0
NEQ [µg/g]
N
25
2,5
2,0
1,5
1,0
0,5
Si
gm N
ar C
in
La gen
u
R che
ie
d rt
Sc lin
hw ge
n
Ro arz
tte ach
na
c
Eh ker
in
O ge
ep n
fin
In ge
n
g
Ba ols
t
a
d
A dt
Jo bb
ch ac
en h
st
ei
n
0,0
Böttcher et al. 2009, Keiter et al. 2009
River flow direction
Results
Liver
n=5
2000 cells / sample
Genotoxicity in barbels from the field
Micronucleus assay in situ
0,4
n=5
4,6
In vivo!
In situ!
The induction factor
(IF) was calculated
by dividing the
median of each
concentration by the
median of the
corresponding
control group
0,3
MN [%]
Genottoxicity iin situ
Erythrocytes from Barbus barbus
*
3,6
*
*
2,1
02
0,2
4,1
0,1
0,0
a
gm
i
S
* significant Genotoxicity (χ² Test, p < 0,05)
when compared to negative control (NC)
en
ir ng
e
Ri
r
ke
c
a
en
t
t
Ro
en
ng
i
l
d
n
ge
n
i
Eh
NC
FlowFlussverlauf
direction
Böttcher et al. (2009); Keiter (2007)
Results
Dioxin-like activity of the sediment extracts
EROD, GPC.2D.Luc and DR CALUX assays
35000
2.5
30000
1.5
1.0
Bio-TEQ [pg/g]]
B
2.0
0.5
0.0
0.1
-1
BioTeqs (pg/g)
TCDD
25000
Negative
20000 control
EC25 : n.b.
15000
10000
5000
1
3
S h ERODh
Schwarzach
-1
3.0
EROD a
activity [pmol**mg *min ]
3.5
DR CALUX
Procedural
control
40000
-1
Dioxin
n-like acctivity
-1
EROD a
activity [pmol*mg *min ]
45000
10
100
Sample EC25 (g/ml)
2
1
Negative control
EC25 : 0.18 mg/ml
0
1000
[
[mg
sediment
di
t dry
d weight/ml
i ht/ l media]
di ]
0
0.01
Standorte im Donauverlauf
0.1
1
[
[mg
sediment
di
t dry
d weight/ml
i ht/ l media]
di ]
h
n
n
n
er
ch
en
en
ert
ac
tei
ge
ge
ck
za
ng
ng
ch
i
r
a
i
ns
l
bb
r
f in
u
a
n
hin
e
d
p
a
a
A
e
w
E
h
e
t
L
Ö
t
m
d
h
c
Ri
Jo
Ro
Sc
Ba
Sig
Grund et al. (2009)
TCDD EC25 (pg/ml)
TCDD
10
Discussion
Appraisal of results: dioxin-like activity
Dioxin
n-like acctivity
• Tested sediments induced AhR-mediated activities in both dioxin-specific bioassays
• Danube River 2006:
max. Bio-TEQ 40000 pg/g SEQ
(Grund. in prep)
• 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)
• 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 off the
Identification
th substances
bt
by
b EDA
Results
Multilayer fractionation of the Dioxin-like activity
Dioxin
n-like acctivity
EROD and DR CALUX assays, chemical analysis
Keiter et al. 2008 Anal. Bioanal. Chem.
Results
Multilayer fractionation of Dioxin-like activities
Dioxin
n-like acctivity
EROD and DR CALUX assays, chemical analysis
75 % unknown
25 % by EPA-PAHs, PCBs, PCDD/Fs
Keiter et al. (2008) Anal. Bioanal. Chem.
Results
HPLC fractionation of Dioxin-like activities
EROD assay
155%
PAHs with 3,4,5 rings
104
103
non-ortho-PCBs,
PCDD/Fs
102
101
F9
F1
0
F1
1
F1
2
F1
3
F1
4
F1
5
F1
6
F1
7
F1
8
F7
F8
F5
F6
F3
F4
F1
F2
(A
c
R :Hx
E )
(A
c)
Su Dia
m ly
m s. R
e
F1 E
-1
8
100
R
E
Bio-TEQ [pg/g]
Dioxin
n-like acctivity
105
(Hydroxy-)Quinones,
keto-,, dinitro-,,
hydroxyl-PAHs, NHeterocycles
Lauchert
Grund et al. (in prep)
Results
HPLC fractionation of Dioxin-like activities
EROD assay
3%
100%
19%
7%
89%
9
99%
100%
99%
97%
97%
100%
100
0%
99%
%
85%
54%
3% 4% 8%
98%
14%
16%
9%
60%
18%
3%
84%
8%
53%
100%
%
100%
100%
99%
99%
9%
80%
86%
94%
%
100
4%
6%
TEQs [ng
g/g SEQ]
Bio-TEQ
Sum Chem-TEQ
Benzo(a)anthracene
Chrysene
Comparison of the biological response in the EROD assay (Bio-TEQs) and
the calculated PAH toxic equivalents (Chem-TEQs) obtained by chemical
analyses of the dialyzed (AMD) extracts and their fractions of the sediments
from the sites at a) Sigmaringen, b) Lauchert and c) Oepfingen.
9
10
11
13
14
15
ex
tr
Su act
m
F
AM
D
9
10
11
13
14
15
ex
tr
Su act
m
F
AM
D
9
10
11
13
14
15
ex
tr
Su act
m
F
1
AM
D
Dioxin
n-like acctivity
1000
10
c)
b)
a)
96
6%
10000
Benzo(a)anthracene
Chrysene
Benzo[j]fluoranthene
Benzo[k]fluoranthene
Benzo[a]pyrene
Benzo[g,h,i]perylene
Dibenzo[a,h]anthracene
Indeno[1,2,3-cd]pyrene
Grund et al. (in prep)
Materials & Methods
Endocrine activity: H295R bioassay
H29
95R bioassay
• NCI-H295R-cell line: human adrenocortical carcinoma
cell line
• Ability to produce the steroid hormones of each of the
three phenotypically distinct zones found in the adult
adrenal cortex
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
UNIVERSITY
(Hecker et al 2007, Blaha et al. 2006; Gazdar et al. 1990; Hilscherova et al. 2004; Zhang et al. 2005)
Discussion
Appraisal of results: hormone analysis
Horm
mone ana
alysis
• Sediment extracts of the sampling sites Riedlingen, Öpfingen and Rottenacker
caused alterations (>1,5-fold induction) in production of P, T and E2
• No comparable studies
• First investigation of effects of sediment samples to hormone production in H295R
cells
• OECD ring test: Validation of a H295R cell line screening test (Hecker et al. 2007)
Effects on hormonal balance
Impacts on reproduction/sex ratio/several metabolism pathways in vivo
cannot be ruled out
Results
DNA
A array a
analyses (Danio rrerio
rerio))
In co-operation with the ITG-FZK Karsruhe, Prof. Dr. Uwe Strähle
Bluhm et al. (in prep)
Results
Endocrine activity of sediment extracts
H295R-Bioassay – Effects on steroid hormones
(fold induction - relative to solvent control, D
DMSO)
Estrad
diol
Testoste
erone
Progest
terone
(fold
induct
tion
relative
to
olvent control,
MSO
(fold inducttion - relative to so
solvent
s
control, DM
DM
MSO))
Horm
mone ana
alysis
3,0
3,0
3,0
25
2,5
2,5
2,5
mg/ml
555mg/ml
mg/ml
mg/ml
222mg/ml
mg/ml
055mg/ml
0,5
mg/ml
00,5
0,5
/ l
mg/ml
2,0
2,0
2,0
1,5
1,5
1,5
1,0
1,0
1,0
0,5
0,5
0,5
0,0
0,0
0,0
n
n
k
k
er
ch
en
en
SO
gne
n lingeen
lnakkn
lkakn
n
nngen nacckkkeerr
n
nee
cah
rzaach
ngen
OO
i
e
M
i
n
i
e
B
g
e
B
r
a
f
n
n
g
h
S
a
D
g
.
d
n
g
S
g
z
g
r l la
rian
inE ing ottteennaac
acrhwarz edlinRiedlin pfinÖppfing
BBlala
.PBB
DDMM
igam
ari
m
Rottte
w
Eh Eh
i
hwS
PPrr.
Ö Ö
ie
gS
h
m
i
c
R
c
g
R
Ro
SSi
S S
R
ert
chheertrt
u
Laauucch
LLa
value sample
Pmean
concentration:
Riedlingen,
und Sigmaringen,
Rottenacker Schwarzach
• Alterations
in E2 concentration:
Riedlingen,
Öpfingen,Öpfingen
Rottenacker,
• Alterations
in T
*1
FI =
FI = 1 for SC
max. induction:
22-fold
fold
induction
Öpfingen Riedlingen
> 22-fold
fold
• Max. induktion:
Max
1 8-fold
1,8
fold
induction
• Max.
mean
valueinduction
SC
Conclusion & 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!
•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
systems
Conclusio
on
• 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
test systems
• Imbalance in the complex network of sensitive regulated steps in the synthesis of steroid
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
as an important
i
t t influencing
i fl
i factor
f t with
ith respectt to
t the
th decline
d li off fish
fi h population
l ti in
i the
th upper
Danube River.
Conclusion & prospects
Where do we go?
Exotoxicologica
l potential
P
Prospect
s
Grund et al. 2009:
Di
Diseases
off Aquatic
A
i
Organisms, in press
Sediment
sample
Grund et al. 2009 ,
Higley et al. 2009 in prep.
Correlation ??
Relevance for
in situ situation
Bioassays
In situ
investigations
YES
Fractionation
Fractions
Bioassay
Effect ?
No
STOP
YES
Chemical Analysis
Identification of
relevant
contaminants
Acknowledgment
Thanks to:
Th
hank you
u!
Deutschen Bundesstiftung Umwelt for support