„Assessing sediments and fish health using a ...s New/SETAC 2009/Hollert SETA… · „Assessing...
Transcript of „Assessing sediments and fish health using a ...s New/SETAC 2009/Hollert SETA… · „Assessing...
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
Bac
kgro
BB
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
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