SETAC 2008 Annual Meeting
Prof. Giesy and a number of his colleagues, post docs, and students from both Michigan
State University and the University of Saskatchewan attended the 29th annual meeting of
the Society of Environmental Toxicology and Chemistry (SETAC), which was held
November 16-21, Tampa, FL. Prof. Giesy’s group made 22 platform and poster
presentations, the most of any group attending the meeting. The titles of the
presentations are listed below with links to the presentation.
“Assessing Sediments and Fish Health Using a Weight-Of-Evidence Approach and
Effect-Directed Analyses – In Search for the Causes o Fish Decline in the Danube
River.” With H. Hollert, E. Higley, M. Hecker, M. Engwall, G. Reifferscheid, H. Hollert,
S. Grund, T. Braunbeck, S. Keiter, U. Luebcke-Von Varel, T. Schulze, and W. Brack.
“Characterization of Mixed Function Monooxygenase Genes CYP1A1 and CYP1A2 of
Mink (Mustela vison) to Facilitate Study of Dioxin-like Compounds.” With X. Zhang, M.
Hecker, S. Wiseman, P.D. Jones, J.N. Moore, S.J. Bursian, M.J. Zwiernik, M. Hecker,
and J. Newsted.
“Aquatic Toxicology of Perfluorinated Chemicals.” With J. Naile, J. Khim, P.D. Jones
and J.L. Newsted.
“Sensitivity of Chicken and Japanese Quail Embryo Hepatocyte Cultures to Cytochrome
P4501A Induction Upon Exposure to TCDD, PeCDF, and TCDF.” With J.C. Herve, S.W.
Kennedy, S.P. Jones, L.J. Mundy, S.J. Bursian, M.J. Zwiernik and P.D. Jones.
“Molecular Mechanisms Underlying Differences in Sensitivity of Avian Species to
Embryo-toxic Effects of Chlorinated Dioxins and Furans-Recent Advances in the
Characterization of Aryl Hydrocarbon receptor 1 (AHR1) in Birds.” With R. Farmahin,
S.W. Kennedy, D. Crump, S.P. Jones, L. Mundy, S.J. Bursian, M.J. Zwiernik, M.E. Hahn,
and J.A. Head, To: 29th annual meeting, November 16-21, Tampa, FL.
“Application of a Medaka HPG Axis Real Time PCR Array Method to Environmental
Chemical Screening.” With X. Zhang, M. Hecker, A. Tompsett, and P.D. Jones.
“Species-specific Accumulation of Polychlorinated Dibenzo-p-dioxins (PCDDs),
Dibenzofurans (PCDFs), and Coplanar Polychlorinated Biphenyls (PCBs) in Fishes from
the Tittabawassee and Saginaw Rivers (Michigan, USA).” With Y. Wan, P.D. Jones, J.
Khim, R.R. Holem, D.P. Kay, S.A. Roark, and J.L. Newsted.
“Assessment of Upper Danube River Sediments Toxicity Using New Fractionation
Techniques and the Danio rerio Embryo Assay and the Ames Fluctuation Assay.” With
E.B. Higley, T. Seiler, J. Wolz, N. Best, H. Hollert, S. Grund, M. Hecker, U. Lubcke-von
Varel, W. Brack, and T. Schulz.
“Perfluorinated Compounds in Environmental Samples Collected from Inner-Mongolia,
China.” With J. Naile, J. Khim, P.D. Jones, T. Wang, W. Jiao, C. Chen, Y. Lu, and K.
Kannan.
“Perfluorinated Compounds in Sediment and Water from Bohai Bay and its Vicinity,
China.” With J. Khim, J.E. Naile, Y. Wan, P.D. Jones T. Wang, W. Jiao, J. Geng, C.
Chen, and Y. Lu.
“Perfluorooctane Sulfonate and other Fluorochemicals in Soils from Bohai Bay, China.”
With T. Wang, W. Jiao, C. Chen, Y. Lu, L. Wei, W. Guang, L. Jing, J. Khim, J.E. Naile,
Y. Wan, and P.D. Jones.
“Effects of Selected Metals on Early White Sturgeon (Acipenser transmontana) LifeStages.” With D. Vardy, A. Tompsett, M. Hecker, J. Duquette, K. Liber, D. Janz, and M.
Adzic.
“White Sturgeon Growth, Morphology, and Survival after Exposure to Columbia River
Surface Water at two Sites in British Columbia, Canada.” With A.R. Tompsett, D. Vardy,
M. Hecker, S. Wiseman, X. Zhang, K. Liber, and M. Adzic.
“Effects of Polychlorinated Dibenzofurans on Mink.” With D.P. Kay, M. Shotwell, J.
Newsted, M. Zwiernik, S. Bursian, J. Moore, K. Beckett, L. Aylward, and R. Budinsky.
“A Comparison of Methods for Estimating Wildlife Dietary Exposure Concentration
using Measured Concentrations of Dietary Items.” With S.A. Roark, D.P. Kay, S.A.
Newsted, and M.J. Zwiernik.
“An Evaluation of Dibenzo-p-dioxins (PCDDs), Dibenzofurans (PCDFs), and Dioxinlike Polychlorinated Biphenyls (PCBs) in Tissues of Wild Game from the Floodplains of
the Tittabawassee and Saginaw Rivers (Michigan, USA).” With R. Holem, J.J. Matousek,
P.W. Bradley, J.L. Newsted, D.P. Kay, A.L. Blankenship, S.R. Roark, M.S. Shotwell and
A.L. Blankenship.
“An Evaluation of Dibenzo-p-dioxins (PCDDs), Dibenzofurans (PCDFs), and Dioxinlike Polychlorinated Biphenyls (PCBs) in Tissues of Wild Game from the Floodplains of
the Tittabawassee and Saginaw Rivers (Michigan, USA).” With R. Holem, J.J. Matousek,
P.W. Bradley, J.L. Newsted, D.P. Kay, A.L. Blankenship, S.R. Roark, and M.S. Shotwell.
To: 29th annual meeting, November 16-21, Tampa, FL.
“Effects of TCDD, TCDF, and PeCDF Injected into the Air Cell of Japanese Quail
(Coturnix japonica) Prior to Incubation.” With A. Cohen-Barnhouse, S. Bursian, J. Link,
P.D. Jones, Y. Wan, S. Wiseman, S. Kennedy, J. Newsted, and M. Zwiernik.
“Multiple Lines of Evidence Risk Assessment of Great Horned Owls (Bubo virginianus)
Exposed to PCDF/DDs in Midland, MI, USA.” With S.J. Coefield, M.J. Zwiernik, T.B.
Fredricks, R.M. Seston, M.W. Nadeau, D.L. Tazelaar, J.N. Moore, M.S. Shotwell, and
D.P. Kay.
“Enzyme Induction of Several Field-Collected Avian Species as Part of a Site-specific
Risk Assessment on the Tittabawassee River, Midland, MI, USA.” With T.B. Fredricks,
R.M. Seston, P.B. Bradley, S.J. Bursian, M.J. Zwiernik, J.L. Newsted, D.P. Kay, and S.W.
Kennedy.
“Tissue-based Assessment of PCDFs, PCDDs, and PCBs in Great Blue Heron (Ardea
herodias) Residing in the Tittabawassee River Floodplain, MI, USA.” With R.M. Seston,
M.J. Zwiernik, D.L. Tazelaar, T.B. Fredricks, S.J. Coefield, M.W. Nadeau, P.W. Bradley,
and M.S. Shotwell, and D.P. Kay.
“PCDF and PCDD Tissue-based Assessment of American Robins (Turdus migratorius)
of the Tittabawassee River Floodplain, MI, USA.” With D.L. Tazelaar, R.M. Seston, T.B.
Fredricks, S.J. Coefield, M.W. Nadeau, S.J. Bursian, M.J. Zwiernik, M.S. Shotwell, and
D.P. Kay.
“Effects of TCDD, 2,3,7,8-TCDF and 2,3,4,7,8-PeCDF Exposure on CYP1A4 and
CYP1A5 mRNA Abundance in Japanese Quail (Coturnix japonica), Ring-necked
Pheasant (Phasianus colchicus), and Chicken (Gallus gallus) in Ovo.” With S. Wiseman,
Y. Yang, P. Jones, Y. Wan, M. Zwiernik, Zoology S. Bursian, J. Herve, S. Kennedy, and
J. Newsted.
Introduction
Fish decline in the upper Danube River
Fangzahlen
Fa
angzah
hllen
en [Stück]
[St
Stücckk]
Sigmaringen
Riedlingen
Ehingen
in search for the causes of fish decline in the Danube river"
1000
400
200
BadenWürttemberg
Karlsruhe
0
1980
Pforzheim
1985
Stuttgart
1990
Bayern
TSCHECHISCHE
REPUBLIK
Regensburg
Bad Abbach
1995
2000Ingolstadt
Ulm
Öpfingen
Ehingen
Riedlingen
Tuttlingen
Schwarzach
Sigma- Sigmaringenringen dorf
Henner Hollert
IV
Sigmaringen
Riedlingen
Ehingen
Augsburg
Linz
III
Gewäss
Gewässergüte
sse
sergü
se
güt
g
ütte
ü
Rottenacker
-
Jochenstein
Passau
Donau
Reutlingen
Neckar
Lauchert
N mber of cat
Background information
„Assessing sediments and fish health using a weight-of-evidence
approach and effect-directed analyses –
/
1200
München
Institute for Environmental Research, RWTH Aachen University
Institute for Zoology of the University of Heidelberg
ÖSTERREICH
II
Salzburg
Friedrichshafen
I
SCHWEIZ
1970
1975
1980
1985
1990
1995
2000
Introduction
Background information
“
Crisis
r
e
t
a
reshw
F
e
h
„T
S. Keiter, M. Böttcher, S. Grund, N. Seitz, J. Otte, K. Bluhm & T. Braunbeck (Department of
Zoology, University of Heidelberg , Germany)
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)
U. Kammann (Federal Research Centre for Fisheries, Hamburg, Germany)
R. Schönberger & 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)
Introduction
Structural changes
of habitat
Change in
temperature
Fish removal
(Human & animals)
Chemical
contamination
Effects
Consequence
Impairment of
health
Reduction of
food supply
Decline of fish
population
Failing
reproduction
Background information
Background information
Potential impacts?
Introduction
Relevance?
Upper
Upper Danube
Danube
Line of evidence:
community structure
Introduction
Conceptal framework
Weight of Evidence –Approaches
Triad-Approach according to Chapman (1990)
Sediments?
… the triad approach
Background information
Line of evidence:
Biotests
• Accumulation of contaminants by adsorption to suspended
matter in water phase Æ Sedimentation
• Direct exposure of benthic organism and fish offspring,
respectively
• Flood events Æ Remobilisation of sediment-bound
contaminants into water phase
Line of evidence:
Chemical analyses
Introduction
Conceptal framework
… additional lines 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
+
Purpose of this integrated study
A pilot study conducted in 2002/03
Chapman & Hollert (2006): Should the Sediment Quality Triad become a Tetrad, a Pentad or
Possibly Even a Hexad? J Soils & Sediments
„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“
Keiter et al. (2006) Environ Sci Pollut Res 13: 308 – 319
Introduction
Conceptal framework
Chemical
analyses
Bioassays
+
Community
structure
Effect directed
Analyses
Identification of the contaminants
responsible for the effects
Chapman & Hollert (2006): Should the Sediment Quality Triad become a Tetrad, a Pentad or
Possibly Even a Hexad? J Soils & Sediments
Purpose of this integrated study
… additional lines of evidence
Objectives?
• Assessment of the ecotoxicological contamination of sediments from
different sites along the upper Danube River
• Identification of the relevant hazardous substances and their sources
• Verification of the relevance of sediment contamination for the fish
decline
Results
Introduction
Genotoxicity of the sediment extracts
Acute and mechanism-specific endpoints of the in
vitro bioassays
Micronucleus assay in vitro with RTL-W1 cells
•Cytotoxicity – Cell damage/dead?
Genotoxicity in vitro
3,5
NQOaverage
•Embryotoxitiy – Teratogenicity of the sediments?
Bioassays
3,0
NEQ [μg/g]
2,5
2,0
1,5
•Dioxin-like activity – Induction of specific enzymes
involved in metabolism of xenobiotics (via Ah-receptor)?
•Endocrine activity – Effects to hormonal balance?
1,0
•Gentoxicity – DNA damage?
0,5
•Alterations in gene expression patterns (Danio rerio
chip with 20000 genes)
Si
gm N
ar C
in
La gen
u
R che
ie
d rt
Sc lin
hw ge
n
Ro arz
tte ach
na
ck
Eh er
in
O ge
ep n
fin
In ge
n
g
Ba ols
d tad
A
t
Jo bb
ch ac
en h
st
ei
n
0,0
•Immunotoxicity (hIL8, hIL6 and CD54 in Beas2B and
MM39 cells)
River flow direction
Böttcher et al. 2007, Keiter et al. 2007
Results
Liver
n=5
2000 cells / sample
Genotoxicity in barbels from the field
0,4
n=5
In vivo!
In situ!
4,6
0,3
MN [%]
Genotoxicity in situ
Erythrocytes from Barbus barbus
January-February 2006
*
3,6
*
*
2,1
0,2
0,0
a
gm
Si
n
ge
rin
• Sampling period
4,1
0,1
The induction factor
(IF) was calculated
by dividing the
median of each
concentration by the
median of the
corresponding
control group
Sediment samples
Sediment sampling
Micronucleus assay in situ
Materials & Methods
n
er
ge
ck
lin
na
ed
tte
o
Ri
R
in
Eh
n
ge
NC
1 = Sigmaringen
2 = Lauchert (tributary)
3 = Riedlingen
4 = Schwarzach (tributary)
5 = Rottenacker
6 = Ehingen
7 = Öpfingen
Bavaria (BfG):
Jochenstein
Bad Abbach
FlowFlussverlauf
direction
* significant Genotoxicity (² Test, p < 0,05)
when compared to negative control (NC)
• Sampling sites
Böttcher et al. (2007); Keiter (2007)
Results
Results
Dioxin-like activity of the sediment extracts
Genotoxicity of whole sediments
EROD, GPC.2D.Luc and DR CALUX assays
Sediment contact Comet-Assay
using embryos of Danio rerio
2.5
30000
1.0
Bio-TEQ [pg/g]
1.5
0.5
0.0
0.1
-1
25000
Negative
20000 control
EC25 : n.b.
15000
10000
5000
1
3
BioTeqs (pg/g)
TCDD
10
100
Sample EC25 (g/ml)
2
1
TCDD EC25 (pg/ml)
TCDD
Negative control
EC25 : 0.18 mg/ml
0
1000
[mg sediment dry weight/ml media]
0.01
0.1
1
10
Genotoxicity in vitro
35000
Schwarzach
EROD
-1
3.0
2.0
DR CALUX
EROD activity [pmol*mg *min ]
3.5
-1
Dioxin--like activity
Dioxin
-1
EROD activity [pmol*mg *min ]
45000
Procedural
control
40000
[mg sediment dry weight/ml media]
0
h
n
h
n
rt
in
er
en
en
ac
ac
ge
ge
he
ck
ste
ng
ing
bb
fin
rin
uc
arz
na
en
dli
Eh
La
Öp
tte
ma
dA
hw
ch
Rie
Jo
Ro
Sc
Ba
Sig
Method: Kosmehl et al. 2006, ET&C
Standorte im Donauverlauf
Grund et al. (2007) and Grund et al. (in prep)
Data: Seitz et al. 2007 Mutat. Res.
Results
Discussion
HPLC fractionation of Dioxin-like activities
Appraisal of results: dioxin-like activity
EROD assay
Lauchert
155%
PAHs with 3,4,5 rings
104
Bio-TEQ [pg/g]
Dioxin--like activity
Dioxin
105
103
non-ortho-PCBs,
PCDD/Fs
102
Dioxin--like activity
Dioxin
• Tested sediments induced AhR-mediated activities in both dioxin-specific bioassays
(Hydroxy-)Quinones,
keto-, dinitro-,
hydroxyl-PAHs, NHeterocycles
• Danube River 2006:
Æ max. Bio-TEQ 40000 pg/g SEQ
(Grund. in prep)
• Danube River 2005:
Æ max. Bio-TEQ 5000 pg/g SEQ
(Keiter et al. 2008)
• Rhine River:
Æ max. Bio-TEQ 1300 pg/g SEQ
(Hinger 2003)
• Bitterfeld:
Æ max. Bio-TEQ 100 000 pg/g SEQ
(Brack et al. 2002)
High dioxin-like activities by several sediment extracts
101
ÆEffects on health of fish in the Danube River cannot be ruled out
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
ÆIdentification of the substances by EDA
Grund et al. (in prep)
R
E
(A
c
R :Hx
E )
(A
c)
Su Dia
m ly
m s. R
e
F1 E
-1
8
100
Materials & Methods
Results
Multilayer fractionation of the Dioxin-like activity
Endocrine activity: H295R bioassay
EROD and DR CALUX assays, chemical analysis
• 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
Dioxin--like activity
Dioxin
H295R bioassay
• NCI-H295R-cell line: human adrenocortical carcinoma
cell line
MICHIGAN STATE
UNIVERSITY
Keiter et al. 2008 Anal. Bioanal. Chem. in press
(Hecker et al 2007, Blaha et al. 2006; Gazdar et al. 1990; Hilscherova et al. 2004; Zhang et al. 2005)
In co-operation with the ITG-FZK Karsruhe, Prof. Dr. Uwe Strähle
Results
Results
Multilayer fractionation of Dioxin-like activities
Dioxin--like activity
Dioxin
Danio rerio
rerio))
DNA array analyses ((Danio
EROD and DR CALUX assays, chemical analysis
Bluhm et al. (in prep)
75 % unknown
25 % by EPA-PAHs, PCBs, PCDD/Fs
Keiter et al. (2008) Anal. Bioanal. Chem. in press
Acknowledgment
Discussion
Appraisal of results: hormone analysis
Related poster presentations:
Pxx: Eric Higley - Tuesday Poster session
• Sediment extracts of the sampling sites Riedlingen, Öpfingen and Rottenacker
caused alterations (>1,5-fold induction) in production of P, T and E2
Hormone analysis
• 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)
Æ Effects on hormonal balance
Æ Impacts on reproduction/sex ratio/several metabolism pathways in vivo
cannot be ruled out
Conclusion & prospects
•Detection of high genotoxicity in several in vitro bioassays and in the micronucleous assay in
situ Î 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
• Toxic effects on state of health of fish population cannot be ruled out
Conclusion
Deutschen Bundesstiftung Umwelt and the
Federal Hydrological Institut (BfG) for support
• 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 influencing factor with respect to the decline of fish population in the upper
Danube River.
Conclusion & prospects
Where do we go?
Exotoxicological
potential
Sediment
sample
Correlation ??
Relevance for
in situ situation
Prospects
Thank you!
Thanks to:
Bioassays
In situ
investigations
YES
Fractionation
Fractions
Bioassay
Effect ?
No
STOP
YES
Chemical Analysis
Identification of
relevant
contaminants
29th SETAC NA Annual Meeting
AhR-mediated Pathways
Nov 16-20, 2008, Tampa, FL, U.S.A.
Characterization of Mixed Function
Monooxygenase Genes CYP1A1 &
CYP1A2 in Mink (Mustela Vison)
Xiaowei Zhang, Ph.D.
Jeremy N. Moore, John L. Newsted, Matthew J. Zwiernik, Markus
Hecker, Paul D. Jones, Steven J. Bursian, John P. Giesy
University of Saskatchewan
University of Saskatchewan,
Michigan State University, & ENTRIX
University of Saskatchewan,
Michigan State University, & ENTRIX
Objectives
1. Clone and sequence mink AhR, CYP1A1 and CYP1A2 cDNA
2. Develop methods to measure mRNA and protein expression
of mink CYP1A1 and CYP1A2
3. Determine the relationship among CYP1A endpoints (gene
expression, protein levels and enzyme activities) in mink
liver
4. Explore the relationships between CYP1A endpoints and
other toxicological endpoints as well as adipose/liver TCDF
and PeCDF concentrations.
¾
¾
¾
¾
¾
¾
¾
Jeremy N. Moore
John L. Newsted, PhD.
Matthew J. Zwiernik, PhD.
Steven J. Bursian, PhD.
Denise P. Kay, PhD.
Paul D. Jones, PhD
Prof. John P. Giesy, PhD.
Funding
Portions of this work were funded by the Dow Chemical Company
University of Saskatchewan,
Michigan State University, & ENTRIX
University of Saskatchewan,
Michigan State University, & ENTRIX
Methods
Introduction
• Experiment design
– Dose: PeCDF: 100, 390, 1600 ng/kg & TCDF: 500, 2000, 9700 ng/kg
– Time: days 90 and 180
• Biochemical and Molecular methods
–
–
–
–
Acknowledgements
RACE cDNA cloning: AhR, CYP1A1, CYP1A2, -actin
Gene expression: Real time RT PCR method
Western blots: anti-dog CYP1A antibody
EROD & MROD activities
• Chemical methods
– Feed (USEPA methods 8290) and mink tissues (1668)
– TEFs: PeCDF = 0.3 & for TCDF = 0.1 (van den Berg et al. 2006)
University of Saskatchewan,
Michigan State University, & ENTRIX
¾ Mink (Mustela vison) have been proposed as a model sentinel or surrogate
species for assessing the exposure and effects of environmental persistent
organic chemicals
™ polychlorinated dibenzo-p-dioxins (PCDDs), -dibenzofurans (PCDFs) and –
biphenyls (PCBs) that are known to act through the AhR receptor
¾ Cytochrome P450s are up-regulated by aryl-hydrocarbon receptor (AhR)
agonists and have been proposed as indicators or biomarkers for the
exposure of these compounds in ecological risk assessments.
¾ While two CYP enzymes that include 7ethoxy O-deethylase (EROD) and 7methoxy O-demethylase (MROD) have been measured in mink, gene
expression (CYP1A1 and CYP1A2, respectively) associated with these
enzymes had not yet been characterized in mink.
University of Saskatchewan,
Michigan State University, & ENTRIX
Relationships among CYP1As endpoints
Table 5. Spearman rank correlation coefficients (numbers) and probabilities (*) between
expression levels of CYP1A1 mRNA, CYP1A2 mRNA, CYP1As protein, EROD, and
MROD activity in the liver of mink. a
CYP1A1 mRNA
CYP1A2 mRNA
CYP1A2 mRNA
CYP1As protein
cDNA cloning of mink AhR and CYP1As
GenBank ID: AhR:
FJ376816
CYP1A1: EU046493
Table 1. Comparison of the amino acid identities (%) of the mink CYP1A1 and
CYP1A2 to the corresponding isozymes in other species
EROD
Organisms
0.732 ***
0.742 ***
EROD
0.751 ***
0.799 ***
MROD
0.820 ***
a
0.806 ***
Mink CYP1A2
CYP1A2
CYP1A1
CYP1A2
Sea otter
95
74
74
93
Gray seal
91
72
73
84
Harp seal
91
72
73
84
Ribbon seal
90
72
72
85
Dog
89
70
70
81
Cat
86
70
70
80
Human
80
71
69
78
Rat
76
66
64
72
0.757 ***
0.859 ***
Mink CYP1A1
CYP1A1
0.915 ***
CYP1As protein
CYP1A2: EU046494
0.841 ***
Sample size, N=49, *** indicates p < 0.001
University of Saskatchewan,
Michigan State University, & ENTRIX
University of Saskatchewan,
Michigan State University, & ENTRIX
Discussion I
Measurement of mink CYP1A mRNA & Protein
1. The basic mechanism of CYP1A induction via the AhR
mediated pathway is conserved in mink.
2. Predicted protein sequences of CYP1A1 and CYP1A2
indicate that mink have preserved several conserved
traits with other mammalian species and are most
closely related to marine mammals.
3. TCDF and PeCDF behaved as full AhR agonist and
displayed high-intrinsic induction of CYP1A
PCR products on agarose Gel
100pb
200bp
Actin 1A1 1A2
Western blot
Control
Å 58 kDa
+
TCDF &PeCDF
University of Saskatchewan,
Michigan State University, & ENTRIX
Dog
University of Saskatchewan,
Michigan State University, & ENTRIX
Hypothesis
mRNA expression of mink CYP1A1
Ho: Level of CYP1As expression can be used to indicate the
overall exposure to TEQ in mink liver.
Relative hepatic expression level of CYP1A1 mRNA
B: 2,3,4,7,8-PCDF
TCDF_MD
TCDF_HD
Control
Control
University of Saskatchewan,
Michigan State University, & ENTRIX
PeCDF_MD
PeCDF_HD
2.5
***
*
TCDF_LD
PeCDF_LD
PeCDF&TCDF
Fold change
4
6
8
10000
PeCDF_LD
D: Control Groups
0.0 0.5 1.0 1.5 2.0
1000
Fold change
100
Liver TEQ
0
Liver TEQ
10
t=90d
t=180d
2
0.5
1
**
4
6
TCDF_LD
C: Mixture of 2,3,7,8-TCDF & 2,3,4,7,8-PCDF
TCDF vs PeCDF
p-value <0.001
**
**
0
0
2.0
5.0 10.0
**
Fold change
***
*
Control
1.0
Fold change
CYP1As
PeCDF
TCDF
TCDF&PeCDF
***
t=90d
t=180d
2
3
t=90d
t=180d
1
CYP1A2 mRNA v.s. Hepatic TEQ
2
Fold change
4
8
A: 2,3,7,8-TCDF
t=0d
t=90d
t=180d
**
0d
University of Saskatchewan,
Michigan State University, & ENTRIX
90d
180d
Discussion II
4. Positive correlations between adipose TEQ
concentrations and the expression of CYP1A mRNAs
and proteins show that adipose concentrations were
the best predictors of AhR pathway activation.
5. Plots liver/adipose TEQ concentrations to adipose
TEQs along with CYP1A responses indicate that
PeCDF may have been sequestered in the liver unlike
that observed for TCDF.
Dietary and Tissue Concentrations
Daily dietary and tissue concentrations of TCDF and/or
PeCDF in mink
Treatment
Daily dose
(ng TEQ/kg bw/d)
<LODd
0.98
3.8
20
0.62
2.2
9.5
6.9
Control
TCDF
PeCDF
Mixture
bTissue
Adipose
(ng TEQ/kg,
ww) d
<LOD
Liver
(ng TEQ/kg,
ww) d
<LOD
8.9 · 2.8
22 · 4.4
62 · 8.9
74 · 8.2
200 · 21
534 · 104
213 · 22
1.2 · 0.27
2.3 · 0.22
7.1 · 1.1
52 · 18
270 · 25
1600 · 530
360 · 79
Liver/Adipose
ratio
NA
0.15 · 0.06
0.11 · 0.024
0.12 · 0.015
0.70 · 0.23
1.4 · 0.24
3.0 · 0.51
1.6 · 0.30
concentrations are presented as mean · SD.
c LOD = 0.1 ng TEQ/kg, ww
University of Saskatchewan,
Michigan State University, & ENTRIX
University of Saskatchewan,
Michigan State University, & ENTRIX
Discussion III
Sequestration of furans in mink liver
1. The dose of TEQ required for induction of mink
CYP1As, which is AhR-dependent, is lower than that in
other well-examined experimental animal, eg. mouse
and rat.
4
T E Q ratio(Hepatic/Adipose) v.s. adipose T E Q
3
Effects of adipos e TEQ : p < 0.001
2
Effects of c hem ic al: p = 0.2678
0
1. In B6C3F1 mice liver CYP1As was inducible in a range of 400 –
1000 ng TEQ /g tissue as indicated by EROD activity (DeVito et
al., 1997).
2. Wistar (Han) Rat CYP1As was induced in maternal liver at
concentration level of 100 ng TEQ/kg (Bell et al., 2007).
However,
3. The induction of mink CYP1As occurred at as low as liver 1.5
ng TEQ/kg tissue.
1
TEQ Ratio(Hepatic/Adipose)
P eCDF
TCDF
TCDF& P eCDF
5
10
20
50
100
200
500
1000
A dipose TE Q
University of Saskatchewan,
Michigan State University, & ENTRIX
University of Saskatchewan,
Michigan State University, & ENTRIX
Expression of CYP1As vs TEQ
1
1000
5
Fold change
5.0
Fold change
10000
1
10
100
1000
2
20.0
100
TCDF v s PeCDF
p-v alue <0.001
PeCDF
TCDF
TCDF&PeCDF
Chemical ef f ect
p-v alue < 0.005
10000
1
10
100
1000
10000
B: CYP1A1 mRNA v.s. Adipose TEQ
D: CYP1A2 mRNA v.s. Adipose TEQ
F: CYP1As protein v.s. Adipose TEQ
Chemical ef f ect
p-v alue = 0.027
0.5
1
Chemical ef f ect
p-v alue < 0.092
PeCDF
TCDF
TCDF&PeCDF
2
Fold change
2.0
Fold change
5.0
TCDF v s PeCDF
p-v alue <0.222
PeCDF
TCDF
TCDF&PeCDF
1.0
PeCDF
TCDF
TCDF&PeCDF
5
Hepatic TEQ
5.0 10.0
Hepatic TEQ
20.0
Hepatic TEQ
0.5 1.0 2.0
Fold change
10
E: CYP1As protein v.s. Hepatic TEQ
PeCDF
TCDF
TCDF&PeCDF
0.5 1.0 2.0
5.0
TCDF v s PeCDF
p-v alue <0.051
5
10
20
50 100
Adipose TEQ
University of Saskatchewan,
Michigan State University, & ENTRIX
C: CYP1A2 mRNA v.s. Hepatic TEQ
PeCDF
TCDF
TCDF&PeCDF
0.5 1.0 2.0
Fold change
20.0
A: CYP1A1 mRNA v.s. Hepatic TEQ
1. Zhang X. et al. (2008) Sequencing and characterization of mixed
function monooxygenase genes CYP1A1 and CYP1A2 of Mink
(Mustela vison) to facilitate study on dioxin-like compounds.
TAAP (In press)
2. Moore J.N. et al. (2007) Relationships between P450 Enzyme
Induction, Jaw Histology and Tissue Morphology in mink
(Mustela vison) Exposed to Polychlorinated Dibenzofurans
(PCDFs). AECT. (In press)
3. Zwiernik MJ, et al. (2008) Toxicokinetics of 2,3,7,8-TCDF and
2,3,4,7,8-PeCDF in mink (Mustela vison) at ecologically relevant
exposures. Toxicol. Sci. 105(1):33-43.
1
Publications
500
2000
5
10
20
50 100
500
2000
Adipose TEQ
University of Saskatchewan,
Michigan State University, & ENTRIX
5
10
20
50 100
Adipose TEQ
500
2000
Thank you!
Xiaowei Zhang, Ph.D.
Toxicology Centre
University of Saskatchewan
44 Campus Drive,
Saskatoon, SK, S7N5B3, Canada
Tel: 306-966-1204
Fax: 306-966-4796
Email: xiaowei.zhang@usask.ca
Lab Web Site: http://www.usask.ca/toxicology/jgiesy/
University of Saskatchewan,
Michigan State University, & ENTRIX
Background and History
BackgroundandHistory
z
Previousresearchprimarilyfocusedonbrominatedand/or
chlorinatedhalogenatedcompounds
z
Fundamentallydifferentfromtraditionalorganicpollutants
z
Previouslythoughttobechemicallystableandbiologically
inertintheenvironment
z
Globallydistributedinmatricesvaryingfromhumanblood
t
topolarbeartissue
l b
ti
z
Manyuncertaintiesfromanalyticalmethodsfor
quantification to toxicity for wildlife and humans
quantificationtotoxicityforwildlifeandhumans
AquaticToxicologyof
q
gy
Perfluoroctanesulfonate
and Related Chemicals
andRelatedChemicals
SETACNorthAmerica
Tampa FL USA
Tampa,FL,USA
November19th,2008
Physical/Chemical Properties
Physical/ChemicalProperties
Authors
JonathanNaile,JongSeongKhim,
JohnNewstead,PaulJones,andJohnGiesy
• PFOSisafattyacid
analogue
• LogKow isnotuseful
duetoAmphiphilic
properties
• Resistantto
hydrolysis,photolysis,
andbiodegradation
• Preferentially
retained in liver and
retainedinliverand
blood
ToxicologyCentre
UniversityofSaskatchewan
Saskatoon,SK,Canada
Jonathan naile@usask ca
Jonathan.naile@usask.ca
Website:http://www.usask.ca/toxicology
Bioaccumulationandconcentration
tion )
(L b
(Laboratory)
What are they?
Whatarethey?
• BAFfortroutwascalculatedtobe0.32±
f
l l
b
0.05,thereforebasedonlab
h f
b
l b
studiesdietdoesnotappeartobethemajorsourceforPFOS
accumulationinfish
• EnterohepaticrecirculationmaycauseK
Enterohepatic recirculation may cause Kow tounderpredictaccumulation
to under predict accumulation
Apparent
Ku
KineticParameters
b
Kd
Halflife
BCFK
F
F F
F F
F
O
F
Species
Tissue
Bluegill
Edible
Unnedible
Whole
Rainbowtrout Carcass
Blood
Liver
a
a
BCF
484
1124
(L/kg*d)
(1/d)
(L/kg)
(d)
8.9
22
0.0047
0.0052
1866
4312
146
133
856
16
53
240
260
0.0045
0.048
0.057
0 05
0.05
3614
1100
4300
5400
152
15
12
14
ApparentBCFwascalculatedastheconcentrationinfishattheendofthe
exposurephasedividedbytheaveragewaterconcentration
b
BCFKwasestimatedasKu/Kd
O
F F
F
F F
F F
F
Perfluorooctanoate (PFOA)
F
F
F
F F
F F F O
O
S
F
F
F F F F
F F
F
O
Perfluorooctane sulfonate (PFOS)
BioconcentrationandAccumulation
(Fi ld)
(Field)
Chronic Ecotoxicology (Fresh water)
ChronicEcotoxicology(Freshwater)
Basedonthelaboratorytoxicitystudies,PFOSisknowntobe
d
h l b
d
k
b
slightlychronicallytoxictoaquaticorganisms
Trophiclevel
TestSpecies
Test
Duration
Endpoint
NOEC
LOEC
(mg/L)
(mg/L)
EC50/LC50/IC50
(mg/L)
Microorganisms
Microorganismcommunity
96h
Respiratoryinhibition
>870
Schaefer and Flaggs 2000
Microalgae
Selenastrumcapricornutum
96h
Growth(celldensity)
42
68
Drottar and Krueger 2000
96h
Inhibitionofgrowthrate
42
121
Drottar and Krueger 2000
Naviculapelliculosa
Chlorellavulgaris
Macroalgae
Growth(celldensity)
150
96h
Inhibitionofgrowth
206
305
Sutherland and Krueger 2001
96h
Growth(celldensity)
8.2
81.6
263
Boudreau et al. 2003
35d
Communitystructure
Myriophyllumspicatum
42d
Biomass,dw
11.4
12.5
Hanson et al. 2005
42d
Rootlength,cm
11.4
16.7
Hanson et al. 2005
Daphniamagna
Chironomustentans
3
Biomass,dw
2.9
3.4
Hanson et al. 2005
42d
Rootlength,cm
0.3
2.4
Hanson et al. 2005
21d
Adultsurvival
5.3
42.9
10d
Survival
0.05
>0.15
MacDonald et al. 2004
10 d
10d
Growth (chlorophyll a)
Growth(chlorophylla)
0 05
0.05
0 087
0.087
MacDonald et al.
al 2004
Partiallifecycle
Ranapipiens
16wk
Fish
Pimephalespromelas
28d
Microcosm
47d
Earlylifestage
• Reasonsforthedifferenceinclude:interspeciesvariability,
se depe de
sexdependentvariables,dietovertheentirelifespan,and
a ab es, d e o e e e e e spa , a d
precursorsbeingmetabolizedtoPFOS
Boudreau et al. 2003
42d
Amphibians
• Bioaccumulationcalculatedinthefieldrangesgreatly
(6,300to125,000forthecommonshiner),andisoften
much higher than what is predicted in the laboratory
muchhigherthanwhatispredictedinthelaboratory
Sutherland and Krueger 2001
Zooplanktoncommunity
Myriophyllumsibiricum
Invertebrate
96h
• Howeverbigdifferencesexistbetweenlaboratoryandfield
measuredresults
Reference
0.3
Boudreau et al. 2003
3
6.21
Ankley et al. 2004
0.3
3
7.2
Oakes et al. 2005
0.29
0.58
• Moredataisneededtoevaluatebioconcentrationand
bioaccumulationunderenvironmentalconditions
Drottar and Krueger 2000
Chronic Ecotoxicology
ChronicEcotoxicology(Marine)
(Marine)
y
Acute Ecotoxicology
AcuteEcotoxicology(Freshwater)
Y (Fresh water)
Y
Thereislimitedchronicmarinetoxicologicaldataavailable,butin
generalitappearsthatmarinemicroorganismsandinvertebratesbehave
y
similarlytotheirfreshwaterrelatives
Ingeneralbasedonthelaboratorytoxicitystudies,PFOSisknown
tobemoderatelyacutelytoxictoaquaticorganisms
Trophiclevel
hi l l
Macroalgae
Trophiclevel
TestSpecies
Microorganisms Anabaenaflosaquae
Microalgae
Invertebrate
Skeletonemacostatum
Mysidopsisbahia
Test
Duration
96h
Endpoint
NOEC
LOEC
(mg/L) (mg/L)
Growth(celldensity)
93.8
EC50/LC50/IC50
(mg/L)
131
Invertebrate
Inhibitionofgrowthrate
93.8
176
Desjardinsetal.2001
Growth(celldensity)
Growth,#young
produced
>3.2
0.24
>3.2
Desjardinsetal.2001
DrottarandKrueger2000
Test
NOEC
LOEC
LC50
Media
(mg/L)
(mg/L)
(mg/L)
Reference
Daphniamagna
Daphnia
magna
Pimephalespromelas
48h
48
h
96h
FW
FW
886
888
1707
1655
2183
1938
WLI2001
WLI
2001
WLI2001
Lepomismacrochirus
96h
FW
2715
5252
6452
WLI2001
Acute
Water flea
Waterflea
Fatheadminnow
Bluegill
Mysid
a
Selenastrum
Selenastrum
capricornutum
Mysidopsisbahia
96 h
96h
FW
1077
2216
2347
WLI 2001
WLI2001
96h
SW
127
269
372
WLI2001
Chronic
b
Daphnia magna
Daphniamagna
21 d
21d
FW
W t fl
Waterflea
a
Reporteddataarebasedonbiomassmeasurements
b
Reporteddatabasedonreproductionandlengthmeasurements
995
Reference
f
(mg/L)
Desjardinsetal.2001
7d
Frondnumber
15
108
7d
Frondnumber
29.2
59.1
Desjardinsetal.2001
6.6
33.1
31.1
130
d
l
Boudreauetal.2003
Daphniamagna
Biomass
Survival
48h
Immobility
0.8
67.2
Boudreauetal.2003
48h
Survival/immobility
32
61
DrottarandKrueger
48h
2ndgenerationsurvival 12
Growth
Survival
15.6
9.1
PalmerandKrueger
Boudreauetal.2003
DrottarandKrueger
Fish
Pimephalespromelas
96h
Oncorhynchusmykiss
96h
Survival
7.8
Robertson1986
96h
Survival
9.9
Robertson1986
96h
Survival
22
Palmeretal.2002
4.82
3.2
7.97
5.4
6.3
13
DrottarandKrueger
Acute Ecotoxicology
AcuteEcotoxicology(Marine)
(Marine)
AS
Limitedmarinetoxicologydataexists,andtheSheepshead minnow
(Cyprinodon variegatus)studyreportsavalueabovethesolubilityofPFOS
y
insaltwaterbecausetheyadded0.05%methanoltoincreasePFOS
solubility.
Invertebrate
Fish
502
EC50/LC50/IC50
/
/
96h
Trophiclevel
Al
Algae
LOEC
Xenopuslaevis
PFBSwaschosenbecauseitoneofthemainreplacement
chemicalsnowusedinsteadofPFOS
duration
NOEC
(mg/L) (mg/L)
Amphibians
Ecotoxicologyfor
P fl
Perfluorobutanesulfonate(PFBS)
b
lf
(PFBS)
Genus/Species
Endpoint
d i
7d
48h
Desjardinsetal.2001
96h
35d
Test
Duration
Lemnagibba
Reference
96h
Organism
Testorganism/Species
i /
i
Testorganism/Species
Artemiasalina
Test
Duration
Endpoint
NOEC
LOEC
(mg/L)
(mg/L)
EC50/LC50/IC50
Reference
(mg/L)
48h
Survival
9.4
48h
Survival
9.4
48h
Survival
8.9
Robertson1986
Mysidopsisbahia
96h
Survival
3.6
DrottarandKrueger
96h
96h
2ndgenerationsurvival
Crassostreavirginica
Oncorhynchusmykiss
96h
WLI 2001
WLI2001
Cyprinodonvariegatus
Shellgrowth
1.1
0.53
1.8
Robertson1986
Robertson1986
DrottarandKrueger
>3.0
DrottarandKrueger
Survival
13.7
Robertson1986
96h
Survival
13.7
Robertson1986
96h
Survival
>15
Palmeretal.2002
<15
QuantitativeStructureActivity
R l i hi (QSAR)
Relationship(QSAR)
• Shorterthan6or7carbonsdonottendtoaccumulateand
bioconcentrationfactorsareusuallylessthan1.0
• Bioconcentrationtendstogoupbyafactorofabout100
withtheadditionof2carbonsforPFCsC4toC8
• Chainlengthsgreaterthan12appeartohavereduced
toxicity
• LLengthdoesnotappeartobeasimportantforfluorotelomer
th d
t
t b
i
t t f fl
t l
alcohols
QuantitativeStructureActivity
R l i hi (QSAR)
Relationship(QSAR)
Water Quality Criteria for PFOS
WaterQualityCriteriaforPFOS
• Purpose:Toderivewaterqualityvaluesforthose
perfluorinatedcompounds(PFCs)thathavesufficient
andappropriatetoxicitydata
• UsedtheUSEPAGreatLakesInitiativemethodology
b
becauseitprovidedspecificproceduresandmethodsfor
d d
f
d
d
h d f
utilizingtoxicitydatatoderivewaterqualityvalues
p
protectiveofaquaticlife
q
• OVERALLGOAL:Toderivetoxicityreferencevaluesthat
areprotectiveofaquaticlife
Water Quality Criteria (PFCs)
WaterQualityCriteria(PFCs)
LogScale
Chainlengthnotfunctionalgroupmakesthedifference
24mg/LCCC for PFBS
100000
2 9 mg/L CCC for PFOA
2.9mg/LCCC
PFAS
10000
Fish Bioconcentration
Factors
121mg/L CMC for PFBS
25mg/LCMC for PFOA
PFCA
1000
21 ng/LCMC for PFOS
21ng/LCMC
5.1mg/LCCC for PFOS
100
10
47ng/LAWV
47
ng/L AWV for PFOS
17ng/LAWV for PFBS
1
0.1
0
2
4
6
8
10
12
14
Perfluorinated Carbons
Conclusions
• BasedontheGLIaprotectivewaterconcentrationofPFOS
wascalculatedtobe0.46mgPFOS/Lforchronicexposure
and 0 78mg PFOS/L for acute exposure
and0.78mgPFOS/Lforacuteexposure.
• Inmostcaseschainlengthappearstobethemostimportant
factor determining PFC toxicity
factordeterminingPFCtoxicity
• TherearebigdifferencesbetweenBCFcalculatedinthefield
andwhathasbeencalculatedintheLaboratoryy
• Therearestillmanyknowledgegapsandmoreaquatic
toxicitydataisneeded
CMC:criteriamaximumconcentration
CMC
it i
i
t ti
CCC:criteriacontinuousconcentration
AWV:avianwildlifevalue
QuantitativeStructureActivity
R l i hi (QSAR)
Relationship(QSAR)
• LimitedToxicologicaldataavailableformanyPFCs,sothe
useaQuantitativeStructureActivityRelationshipwas
developed to estimate toxicological data where no
developedtoestimatetoxicologicaldatawhereno
measureddataisavailable
Resultsshowthatchain
show that chainlength
lengthisthemostimportantfactor
is the most important factor
• Results
indeterminingtoxicity,althoughfunctionalheadgroupand
theadditionofanamidegroupcanalsobeimportant
ThankYou!
Sensitivity of chicken, ring-necked pheasant and Japanese quail embryo hepatocyte cultures to ethoxyresorufin
J.C. Hervé , S.P. Jones , L. Mundy , M.J. Zwiernik , S. Bursian ,
O-deethylase (EROD) induction upon exposure to TCDD, PeCDF and TCDF
1,2
2
2
3
3
J.P. Giesy4, P.D. Jones4 , Y. Wan4 and S.W. Kennedy1,2
1. University of Ottawa, 2.National Wildlife Research Centre, 3.Michigan State University, 4.University of Saskatchewan
RESULTS
DEFINITIONS
Pheasant
Chicken
EROD activity (pmol/min/mg protein)
• EC50: concentration of a DLC required to induce 50% of the
maximal response.
• ECTCDD-threshold (ECthr): concentration of a DLC required
to induce a response equivalent to 10% of the maximal
response of TCDD.
ABSTRACT
Ethoxyresorufin
O-deethylase
(EROD)inducing
potencies
of
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD),
2,3,4,7,8-pentachlorodibenzofuran (PeCDF)
and 2,3,7,8-tetrachlorodibenzofuran (TCDF)
were determined in domestic chicken, ringnecked pheasant (pheasant) and Japanese
quail (quail) primary hepatocyte cultures. In
pheasant and quail, PeCDF was a more
potent EROD inducer than TCDD. PeCDF
was approximately equipotent at inducing
EROD in hepatocytes in all three avian
species. These findings were surprising
because (a) TCDD has generally been
considered the most potent ‘dioxin-like
compound’ (DLC) and (b) until this study, it
has been assumed that the chicken was
more sensitive to EROD induction by all
DLCs than any other avian species.
fg
• Relative potency (ReP): EROD-inducing potency of a
compound relative to EROD-inducing potency of TCDD in
the same species which can be calculated based on the
EC50 or based on the ECthr: [ EC50 (or ECthr) TCDD) ] ÷
[ EC50 (or ECthr) of compound X)]
• Relative sensitivity (ReS): EROD-inducing potency of a
compound in a species relative to EROD-inducing potency
of the same compound in the chicken, which can be
calculated by comparing the concentration required to cause
equivalent effects. This can be based on any effect level but
is often calculated by comparing EC50 values. Calculation of
the ReS based on the EC50 assumes equal efficacies
(maximal activities) and slopes of the dose response curves.
A less biased estimate of the ReS can be derived from the
threshold concentration for effect, represented in this study
by the ECthr (see Fig. 1 and Table 1).
[(EC50 (or ECthr) of compound X in chicken] ÷
[EC50 (or ECthr) of compound X in species A]
DLCs share the same mechanism of
action, which involves the binding of the
compound to the aryl hydrocarbon receptor
(AHR). However, potencies of different
DLCs range over several orders of
magnitude and sensitivity to these
compounds varies among avian species.
This differential sensitivity to DLCs exists
even among birds within the same order
(e.g. Galliforms (1)). Several studies have
been conducted with DLCs in avian
species but few studies made systematic
comparisons of sensitivity among species.
Data on relative potencies of furans in
birds is also limited. The sensitivity of birds
to induction of EROD is useful for
predicting the sensitivity of avian species
to embrytoxic effects of DLCs (2).
200
200
200
100
100
100
0
0
0
DMSO
10 -4 10 -3 10 -2 10 -1 10 0 10 1 10 2
10 -4 10 -3 10 -2 10 -1 10 0 10 1 10 2
DMSO
Relative potency and relative sensitivity based on EROD
induction
• In chicken hepatocytes, the EROD inducing potencies of
TCDD, PeCDF and TCDF were similar, but in pheasant and
quail hepatocytes, PeCDF was more potent than TCDD (Fig.
2; Tables 2, 3 and 4).
• When exposed to TCDD and TCDF, chicken hepatocytes
were more sensitive to EROD induction than the other two
species, but when exposed to PeCDF, sensitivities were
similar among species (Tables 2, 3 and 4).
10 -4 10 -3 10 -2 10 -1 10 0 10 1 10 2
DMSO
Concentration (nM)
Concentration (nM)
Concentration (nM)
Figure 2: EROD concentration-response curves for chicken, pheasant or quail embryo hepatocytes exposed to PeCDF
(▲), TCDD (○) or TCDF (■) for 24 h. Points represent mean EROD activity caused by a particular concentration on three
replicate cell culture plates; bars represent standard errors and values before the axis break indicate EROD activity
observed for control (DMSO-treated hepatocytes).
PeCDF
TCDD
Pheasant
Quail
Table 1: EC50s, ECthr, ReP values and maximal
activities for TCDD and compound 2 (C2).
Compound
EC50
ReP
EC50
ECthr
ReP
ECthr
Maximal
activity
TCDD
0.03
1
0.001
1
500
C2
0.008
4
0.001
1
300
METHODS
Quail, pheasant and chicken eggs were incubated
until 1 to 3 days prehatch.
CYP1A4 activity (EROD) was measured and EC50,
ECthr, ReP and ReS values were calculated.
Comparison of EC50- and ECthr-based methods
• Both methods indicated that PeCDF was a more potent
EROD inducer than TCDD in pheasant and quail
hepatocytes, but ReP and ReS values differed for EC50 vs.
ECthr approaches – see Fig. 1 for explanation of differences.
• The rank order of potencies and sensitivities were the same
with both methods in all situations (Tables 3 and 4).
Table 2: Maximal EROD responses, EC50 values and ECthr values for EROD data obtained in chicken, pheasant and quail
hepatocyte cultures exposed to TCDD, PeCDF or TCDF for 24 h. Standard errors are shown in brackets.
Figure 1: EROD concentration-response curves for
TCDD and compound 2.
TCDD
PeCDF
300
Max EROD
(pmol/min/mg)
EC50
(nM)
ECthr
(nM)
460 (36)
0.018
(0.0007)
0.00081
(0.00002)
525 (6)
0.085
(0.01)
0.0051
(0.001)
248 (31)
0.19
(0.02)
0.02
(0.001)
TCDF
Max EROD
(pmol/min/mg)
EC50
(nM)
ECthr
(nM)
471 (38)
0.019
(0.0003)
0.0013
(0.0002)
530 (15)
0.025
(0.002)
0.0011
(0.0001)
267 (27)
0.015
(0.0009)
0.00073
(0.0001)
DISCUSSION
Max EROD
(pmol/min/mg)
EC50
(nM)
ECthr
(nM)
443 (17)
0.021
(0.001)
0.0014
(0.0003)
486 (12)
0.11
(0.02)
0.0090
(0.002)
1.6 (0.4)
0.077
(0.01)
285 (24)
PeCDF
TCDD
Species
ECthrbased ReP
EC50based ReP
ECthrbased ReP
EC50based ReP
ECthrbased ReP
Chicken
1,0
1,0
1.0 (0.02)
0.6 (0.1)
0.9 (0.05)
0.6 (0.1)
Pheasant
1,0
1,0
3 (0.3)
5 (0.5)
0.9 (0.2)
0.6 (0.2)
Quail
1,0
1,0
13 (0.7)
30 (5)
0.1 (0.03)
0.3 (0.05)
TCDD
Intercompound comparisons: relative potency as EROD
inducers
The finding that PeCDF is a more potent inducer of EROD
activity than is TCDD in pheasant (3- to 5-fold) and quail (13to 30-fold) is surprising because TCDD is generally
recognized to be the most potent DLC. Possible reasons,
among others, include greater binding affinity of PeCDF to the
AHR and greater resistance to metabolism of PeCDF
compared to TCDD. Current research on embryo-lethal
effects of PeCDF and TCDD in quail (Poster # WP210) is in
general agreement with this finding. A greater potency of
PeCDF compared to TCDD was also reported in the green
frog (3), double-crested cormorant and the Forster’s tern (4).
TCDF
EC50based ReP
B)
Cells were exposed to serial dilutions of TCDD,
PeCDF or TCDF for 24 hours.
TCDF
400
300
A)
Livers were dissected, pooled and digested,
hepatocytes were plated.
CHEMICALS
400
PeCDF
TCDD
TCDF
Table 3: Relative EROD-inducing potencies and sensitivities. A) Relative potency (ReP) values
and B) relative sensitivity (ReS) values. Standard errors are shown in brackets.
OBJECTIVE
To determine the relative sensitivities of
chicken, pheasant and quail hepatocyte
cultures to EROD induction by TCDD,
PeCDF and TCDF.
500
PeCDF
TCDD
TCDF
500
300
Chicken
When maximal activity is lower, as it is for compound 2
(C2), the EC50 is shifted to the left and potency is
overestimated. Relative sensitivity comparisons among
species must also consider differences in maximal EROD
responses.
INTRODUCTION
400
Species
MAXIMAL ACTIVITY (efficacy)
PeCDF
TCDD
TCDF
500
RESULTS
Quail
PeCDF
TCDF
Species
EC50based ReS
ECthrbased ReS
EC50based ReS
ECthrbased ReS
EC50based ReS
ECthrbased ReS
Chicken
1.0
1.0
1.0
1.0
1.0
1.0
Pheasant
0.2 (0.03)
0.2 (0.04)
0.8 (0.07)
1 (0.1)
0.2 (0.05)
0.2 (0.04)
Quail
0.1 (0.01)
0.04 (0.003)
1 (0.07)
2 (0.3)
0.02 (0.004)
0.02 (0.003)
Interspecies comparisons: relative sensitivity to EROD
induction
The fact that all three species showed similar sensitivities to
PeCDF exposure was surprising because the chicken was
thought to be the most sensitive avian species to DLCs.
Moreover, a literature review (1) established that the pheasant
and quail were, respectively, 12 and 218 times less sensitive
than the chicken to the embryolethal effects of DLCs. The
present study with hepatocytes suggests that this order
applies when the hepatocytes are exposed to TCDD and
TCDF, but not to PeCDF. Research on the (AHR), which is
being conducted in our laboratory, will provide valuable
information on molecular mechanisms that could be involved
in the differential sensitivity of birds (Poster # MP36).
Current and Futures Directions
Table 4: Rank order of A) relative potencies and B) relative sensitivities based on EC50 or ECthr. ReP and ReS
values are shown in brackets.
A)
Relative potencies of compounds
B)
Relative sensitivities of species
Chicken
Exposed to TCDD
EC50 based ReP TCDD (1) = PeCDF (1) ≥ TCDF (0.9)
EC50 based ReS
chicken (1) > pheasant (0.2) > quail (0.1)
Ecthr-based ReS
chicken (1) > pheasant (0.2) > quail (0.04)
Ecthr-based ReP
TCDD (1) ≥ PeCDF (0.6) = TCDF (0.6)
Pheasant
Exposed to PeCDF
EC50 based ReP
PeCDF (3) > TCDD (1) ≥ TCDF (0.9)
EC50 based ReS
quail (1) = chicken (1) ≥ pheasant (0.8)
Ecthr-based ReP
PeCDF (5) > TCDD (1) ≥ TCDF (0.6)
Ecthr-based ReS
quail (2) > chicken (1) = pheasant(1)
Quantitative real-time polymerase chain reaction (Q-PCR) assays are
currently being conducted to examine the effects of TCDD, PeCDF and
TCDF on the induction of CYP1A4 and CYP1A5 mRNA in the three avian
species. Porphyrin accumulation and porphyrin patterns will also be
measured in cell cultures exposed to the chemicals.
Acknowledgement
Funded by an unrestricted grant from Dow Chemical Inc., Environment Canada’s
Wildlife Toxicology and Disease Division, Environment Canada’s STAGE (Strategic
Applications of Genomics for the Environment) program and Le Fonds Québécois de
la Recherche sur la Nature et les Technologies.
REFERENCES
1. Head, J. A., M. E. Hahn, and S. W. Kennedy. 2008. Key amino acids in the aryl hydrocarbon receptor
predict dioxin sensitivity in avian species. Environ.Sci.Technol. 42:7535-7541.
Quail
Exposed to TCDF
EC50 based ReP PeCDF (13) > TCDD (1) > TCDF (0.1)
EC50 based ReS
chicken (1) > pheasant (0.2) > quail (0.02)
Ecthr-based ReP
Ecthr-based ReS
chicken (1) > pheasant (0.2) > quail (0.02)
PeCDF (30) > TCDD (1) > TCDF (0.3)
2. Kennedy, S. W., A. Lorenzen, S. P. Jones, M. E. Hahn, and J. J. Stegeman. 1996. Cytochrome
P4501A induction in avian hepatocyte cultures: a promising approach for predicting the sensitivity of
avian species to toxic effects of halogenated aromatic hydrocarbons. Toxicol.Appl.Pharmacol.
141:214-230.
3. Rankouhi, T. R., B. Koomen, J. T. Sanderson, A. T. Bosveld, W. Seinen, and B. M. Van den. 2005.
Induction of ethoxyresorufin O-deethylase activity by halogenated aromatic hydrocarbons and
polycyclic aromatic hydrocarbons in primary hepatocytes of the green frog (Rana esculenta).
Environ.Toxicol.Chem. 24:1428-1435.
4. Sanderson, J. T., S. W. Kennedy, and J. P. Giesy. 1998. In vitro induction of ethoxyresorufin Odeethylase and porphyrins by halogenated aromatic hydrocarbons in avian primary hepatocytes.
Environ.Toxicol.Chem. 17:2006-2018.
Molecular mechanisms underlying differences in sensitivity of avian species to embryotoxic
effects of chlorinated dioxins and furans- Recent advances in the
characterization of aryl hydrocarbon receptor 1 (AHR1) in birds
Reza Farmahin1, 2, Steven J. Bursian3, Doug Crump2, John P. Giesy3, Mark E. Hahn5,
6
2
2
4
1,
2
Jessica A. Head , Stephanie P. Jones , Lukas Mundy , Matthew J. Zwiernik , Sean W. Kennedy
1.Department of Biology, University of Ottawa, Ottawa, ON, Canada.
4. University of Saskatchewan, Saskatchewan, SK, Canada.
2. NWRC, Environment Canada, Ottawa, ON, Canada.
5. Department of Biology, Woods Hole Oceanographic Institution, Woods Hole, MA, USA.
Chlorinated dibenzo-p-dioxins, dibenzofurans, non-ortho substituted polychlorinated biphenyls and other ‘dioxin-like compounds’ (DLCs) are
lipophilic environmental contaminants that are toxic to most vertebrates. Responsiveness to the toxic effects of DLCs varies among species and
strains. For example, some species of birds are 10 to 1000-fold less sensitive to the embryotoxic effects of these chemicals than the highly
sensitive domestic chicken. It has been postulated that differential sensitivity is caused by differences in gene expression that occur subsequent
to the binding of a DLC to the aryl hydrocarbon receptor (AHR). Binding affinity to the AHR is dependent on the confirmation of the ligand-binding
domain (LBD) of AHR1 (there are at least two AHR isoforms in birds; AHR1 and AHR2). The identities of two amino acid residues (aa324 and
aa380) within the AHR LBD contribute to differences in sensitivity to DLCs in chicken and common tern (1). Further research has investigated
whether the identity of these amino acid residues predict embryonic sensitivity to DLCs in eleven avian species (2). Of all the species surveyed,
the chicken (most sensitive) was unique in having the Ile324 and Ser380 genotype. Insensitive species had a Val324/Ala380 genotype; moderately
sensitive species had an Ile324/Ala380 genotype. Interestingly, three species of Galliforms (chicken, ring-necked pheasant and Japanese quail)
are represented by the three genotypes (Fig.1).
The sequences of the LBD of AHR1 have been determined for >70 species of
birds (Kennedy et al., 2009). Among these species, there are a total of six
differences in amino acid residues (aa324, aa380 and four others) within the
LBD. To determine the possible influence of all six amino acids on the
binding affinities of DLCs to AHR1 we will be carrying out site-directed
mutagenesis and other studies similar to the work conducted by Karchner et
al. (1) on all variants of AHR1 LBD in birds. Some of these studies will be
conducted with the three Galliforms mentioned above (chicken, ring-necked
pheasant and Japanese quail) as model organisms. Here we present data on
the full-length sequences of AHR1 for these species and outline some of our
plans for future studies. This research is part of a larger project that includes
egg injection studies (Poster #WP210), hepatocyte culture studies (Poster #
MP33) and field based exposure and response studies (Poster#WP222). The
purpose is to identify the molecular and physiological reasons that underlie
avian species differences and phenotypic responses to DLCs. The ultimate
goal is to develop molecular and cell culture methods that can be used to
predict the sensitivity of wild avian species to 2,3,7,8-tetrachlorodibenzo-pdioxin (TCDD), 2,3,7,8-tetrachlorodibenzofuran (TCDF), 2,3,4,7,8pentachlorodibenzofuran (PeCDF) and other DLCs.
relative
sensitivity
AHR1 genotype
Position :324/380
high sensitivity to
dioxin
IIe/Ser
3. Michigan State University, East Lansing, MI, USA.
6. University of Michigan, Ann Arbor, MI, USA.
Figure 2: Cloning strategy for ring-
Figure 6: Phylogenetic analysis of
necked pheasant and Japanese quail
AHR1 cDNA. Adapter primers (AP) at
two ends of cDNA were paired with
gene specific primers (GSP). 3' and 5'
RACE fragments are shown. The
basic helix loop helix (bHLH) domain
and Per-Arnt-Sim (PAS) domain A and
B repeats are indicated.
selected vertebrate AHR amino acid
sequences. Sequences were aligned using
ClustalW. The tree was constructed using
the minimum evolution criterion in
PAUP*4.0, using the neighbour-joining tree
as the starting tree. Only the N-terminal
half of the protein (~400 amino acids) was
used, because the alignment is less
certain in the C-terminal half. Numbers at
the nodes represent bootstrap values
based on 100 resamplings of the data.
The AHR1 and AHR2 clades are indicated.
Accession numbers are; Black-footed
albatross AHR1 (AB106109), Black-footed
albatross AHR2 (AB10610), Chicken AHR
(NM_204118), Common tern AHR
(AF192503), Pekin duck AHR (AF192501),
Mouse AHR (M94623), Human AHR
(L19872), Beluga whale AHR (AF332999),
Zebrafish AHR2 (AF063446), Killifish AHR1
(AF024591), Killifish AHR2 (U29679),
Atlantic tomcod AHR2 (AF050489), Atlantic
salmon AHR2g (AY052499), Atlantic
salmon AHR2d (AF495590), Drosophila
melanogaster AHR (AF050630).
Name
Length
Name
Length
Percent identity
chicken AHR1
858
ring-necked
pheasant
860
95%
chicken AHR1
858
J.quail AHR1*1
857
95%
chicken AHR1
858
J.quail AHR1*2
859
95%
ring-necked
pheasant AHR1
860
J.quail AHR1*1
857
95%
ring-necked
pheasant AHR1
860
J.quail AHR1*2
859
96%
J.quail AHR1*1
857
J.quail AHR1*2
859
98%
Table 1: The number of amino acid sequences and their similarities in chicken, ring-necked
IIe/Ala
moderate sensitivity
to dioxin
Val/Ala
low sensitivity to
dioxin
pheasant and Japanese quail AHR1 are shown. Two allelic variants of Japanese quail AHR
(J.quail AHR 1*1 and J.quail AHR 1*2) were found (see Figs. 3 and 4).
chicken AHR1
pheasant AHR1
J.quail AHR1*1
J.quail AHR1*2
The four amino acid residue sites within the ligand binding domain of AHR1,
indicated in Fig.5, will be targeted for site-directed mutagenesis
chicken AHR1
pheasant AHR1
J.quail AHR1*1
J.quail AHR1*2
Figure 1: Three broad categories of sensitivity of birds to DLCs have
been proposed based upon the identity of amino acid residues at
positions 324 and 380 within the ligand-binding domain of AHR1 (2).
In vitro translation and transcription (IVTT) assays will be used to synthesize chicken,
ring-necked pheasant, Japanese quail and mutant AHR1 proteins
chicken AHR1
pheasant AHR1
J.quail AHR1*1
J.quail AHR1*2
Binding affinities of TCDD, TCDF and PeCDF to various forms of in vitro expressed
AHR1 proteins will be determined
chicken AHR1
pheasant AHR1
J.quail AHR1*1
J.quail AHR1*2
chicken AHR1
pheasant AHR1
J.quail AHR1*1
J.quail AHR1*2
chicken AHR1
pheasant AHR1
J.quail AHR1*1
J.quail AHR1*2
Binding affinities of TCDD, TCDF and PeCDF to AHR1 for chicken, Japanese quail
and ring-necked pheasant produced in the IVTT studies will be compared to binding
affinities in liver cytosolic fractions from the three avian species
-QQ
chicken AHR1
pheasant AHR1
J.quail AHR1*1
J.quail AHR1*2
1
2
3
Ring-necked pheasant and Japanese quail livers
were flash-frozen
mRNA was isolated and used for synthesizing ss-cDNA
using oligo(dT) primers
AHR ds-cDNA was synthesized using a Marathon™ kit
5
5' and 3' Rapid Amplification of cDNA Ends (RACE) assays
were performed (See Fig. 2 for cloning strategy); products
were run on agarose gels, and bands were excised
6
Excised bands were purified, and then cloned into the
pGEM-T Easy vector
7
Plasmids were transformed into JM 109 competent cells
The efficacies of all in vitro-expressed AHRs to activate transcription of aryl
hydrocarbon responsive elements (AHREs) in transient transfection assay will be
compared
Figure 3: Amino acid sequences of chicken, ring-necked pheasant, and Japanese quail (two
allelic variants; J.quail AHR1*1 and J.quail AHR1*2) AHR1 were aligned using CLUSTALW2.
Amino acids that are identical in two or more sequences are highlighted in blue.
Insertion/deletion (indel) in two allelic variants of Japanese quail AHRs are highlighted in
yellow. The bHLH region and PAS domains are indicated by lines above the alignments. The
ligand binding domain of AHR1 is indicated within brackets.
Results from binding and transfection studies will be used to attempt to understand
the molecular mechanisms that explain differences in sensitivity and response of
chicken, ring-necked pheasant and Japanese quail to ethoxyresorufin O-deethylase
(EROD) induction in cultured hepatocytes (Poster #MP33) and embryotoxicity (Poster#
WP210) when exposed to TCDD, TCDF or PeCDF
J.quail AHR 1*1
J.quail AHR 1*2
Figure 4: Insertion/deletion (Indel) in two allelic variants of Japanese quail AHR1 (J.quail
4
Adaptor was ligated to both ends of the ds-cDNA
8
Plasmid DNA was purified and sequenced
AHR1*1 and J.quail AHR1*2) are shown.
Funded by an unrestricted grant from Dow Chemical Company, Environment Canada’s
Wildlife Toxicology and Disease Division, Environment Canada’s STAGE (Strategic
Applications of Genomics for the Environment) program and a WHOI Sea Grant.
Figure 5: Comparison of
Full-length cDNA sequences for ring-necked pheasant and Japanese quail AHR1 were obtained and alignments with chicken AHR1 (accession #
NM_204118) are shown in Fig. 3
Allelic variants (J.quail AHR1*1 and J.quail AHR1*2) of Japanese quail AHR1 were found (Figs. 3 and 4)
Amino acid residues within the ligand-binding domains of chicken, ring-necked pheasant and Japanese quail AHR1 are compared in Fig. 5
Percent amino acid identities among the ligand-binding domains of chicken, ring-necked pheasant and Japanese quail AHR1 are shown in Table 1
Phylogenetic analysis of selected vertebrate AHR amino acid sequences are shown in Fig. 6
the ligand binding domains
of AHR1 in chicken
(accession number:
NM_204118), ring-necked
pheasant and Japanese
quail. Differences in amino
acids at sites 256,297, 324
and 380 are indicated. Sitedirected mutagenesis at
these sites will be carried
out.
chicken-LBD
pheasant-LBD
J.quail-LBD
chicken-LBD
pheasant-LBD
J.quail-LBD
chicken-LBD
pheasant-LBD
J.quail-LBD
chicken-LBD
pheasant-LBD
J.quail-LBD
1.
Karchner, S. I., Franks, D. G., Kennedy, S. W., and Hahn, M. E. (2006). The molecular basis for
differential dioxin sensitivity in birds: role of the aryl hydrocarbon receptor. Proc.Natl.Acad.Sci
U.S.A 103, 6252-6257.
2.
Head, J. A., Hahn, M. E., and Kennedy, S. W. (2008). Key Amino Acids in the Aryl Hydrocarbon
Receptor Predict Dioxin Sensitivity in Avian Species. Environ.Sci.Technol. 42(19), 7535-7541.
3.
Kennedy, S.W., et al. (2009). The ligand binding domain of aryl hydrocarbon receptor 1 (AHR1) in
seventy-four avian species – A framework for testing hypotheses regarding species sensitivity to
dioxins (in preparation).
Application of a Medaka HPG Axis Real Time PCR Array Method to
Environmental Chemical Testing
Xiaowei Zhang1,2*, Markus Hecker1,3, Amber Tompsett1, Junewoo Park2, Paul D. Jones1,5, John L. Newsted4, John P. Giesy1,5,6
1 Toxicology Centre, University of Saskatchewan, Saskatoon, SK, Canada.
2.Food Safety and Toxicology Center, Center for Integrated Toxicology,
Toxicology, Zoology Department, Michigan State University. East Lansing,
Lansing, MI 48824;
3 ENTRIX Inc. Saskatoon, SK, Canada
4 ENTRIX Inc. Okemos MI, USA
5 Dept.
Dept. Biomedical Veterinary Bioscience, University of Saskatchewan, Saskatoon, Saskatchewan, Canada
6 Dept. Biology & Chemistry,
Chemistry, City University of Hong Kong, Kowloon, Hong Kong, SAR China
ERB_L
VTG.I_L
nnexinM2_L
CHGH_L
VTG.II_L
AR_L
5 0 0 n g /L
5 0 n g /L
3 0 0 u g /L
PRO
3 0 u g /L
3 u g /L
3 0 u g /L
K TC
3 0 u g /L
FAD
1 0 .0 u g /L
3 u g /L
1 0 0 u g /L
1 .0 u g /L
5 0 0 n g /L
EE2
Methods (Cont’)
•SYBR Green technology
•384-well format /ABI system
•3 reference genes
Other endpoints
Endocrine disruptors
Model Chemicals
Fig. 2. Volcano plots of chemically induced changes in gene
expression pattern in males and females. Data are from medaka
exposed to 500 ng EE2/L or 5000 ng TRB/L. Genes plotted farther
from the either the x- or y-axis have larger changes in gene
expression. Thresholds for fold-change (vertical lines, 2-fold) and
significant difference (horizontal line, p < 0.01) were used in this
display.
•17α-ethinylestradiol (EE2)
•17β-trenbolone (TRB)
•Fadrozole (FAD)
•Prochloraz (PCZ)
•Ketoconazole (KTC)
Small fish model
Results
¾ Conservation of basic aspects of the HPG axis across
vertebrates
¾ Small body size & Relatively rapid life-cycle
™ ≤ 4 month generation time from embryos to adults
¾ Genomic sequence (www.ensembl.org)
Fish fecundity
5 0 n g /L
5 n g /L
Real time PCR array
Introduction
Figure 4. Heatmap of the concentration-dependent gene expression profiles in
livers of chemical exposed females. Gene tree was constructed by pearson
correlation metric. Chemical tree was constructed by ‘ToxClust” method, where the
dissimilarity between any two chemicals was calculated by the distance between
the concentration–dependent response curves in the exposure of both chemical.
Figure 5. Relationship between fecundity and gene expression in livers of females. (A)
Fecundity vs hepatic transcript index. The broken line shows the trend of data. (B) Simple
linear regression of log10-transformed fecundity and hepatic transcript index. The
functions describing the relationship are as follows: hepatic transcript index = 0.236 *log10
(ER-α) + 0.326 *log10 (VTG I) + 0.537 *log10 (VTG II) + 0.472 *log10 (CHG L) + 0.343
*log10 (CHG H) + 0.457 *log10 (CHG HM). The formula for the regression model was log10
(fecundity) = 1.616 − 0.4493 *log10(-hepatic transcript index).
Discussion
1.Application of the medaka HPG PCR array facilitated
mechanistic understanding of environmental EDCs
2.Molecular response at mRNA has potential to
quantitatively evaluate chemical induced adverse effects
on reproduction.
3.The medaka HPG axis model has potential to be an
effective ecotoxicological screening tool for EDCs
Methods
References:
Exposure
•Animal: 4 month adult medaka
•Exposure: 5 model chemicals
•Sex: 5 male : 5 female per tank
•Vehicle control: DMSO
•RNA isolation: brain, liver & gonads
HGHminor_L
5 0 0 0 n g /L
TR B
•Fluorescence in situ hybridization (FISH)
•Fecundity (egg production)
•BSI: brain-somatic index
•HSI: hepatic-somatic index
•GSI: gonadal-somatic index
An exogenous agents that interfere with the “synthesis,
secretion, transport, binding, action, or elimination of
natural hormones in the body that are responsible for the
maintenance of homeostasis, reproduction, development,
and/or behavior”
---- Kavlock et al. (1996)
CHGL_L
-4096
-1024
-256
-64
-16
-4
1
4
16
A real time polymerase chain reaction (RT-PCR) array was
developed for studying chemical-induced effects on gene
expression of selected endocrine pathways along the
hypothalamic-pituitary-gonadal (HPG) axis of the small,
oviparous fish, the Japanese medaka (Oryzias latipes).
The Japanese medaka HPG PCR array combines the
quantitative performance of SYBR® Green-based real-time
PCR with the multiple gene profiling capabilities of a
microarray to examine expression profiles of 36 genes
associated with endocrine pathways in brain, liver and
gonad. A pathway-based approach was implemented to
analyze and visualize time -dependent or concentration –
dependent mRNA expression in the HPG axis of Japanese
medaka. The performance of the Japanese medaka HPG
PCR array was evaluated by examining effects of five
model compounds. The organ- gender- and concentration
–specific gene expression profiles derived by the
Japanese medaka HPG axis RT-PCR array provides a
powerful tool to delineate chemical-induced modes of
action. In addition, the medaka real time PCR array
demonstrate potential to quantitatively predict the adverse
effects on reproduction.
ERA_L
F o ld C h a n g e
Abstract
Fig. 1. Cumulative fecundity in medaka exposed to EE2 (A) or TRB (B) in a
7-d test. Data represent the mean cumulative number of eggs per female
collected from 3 replicate tanks, each containing 6 pairs of fish. The
asterisks indicate a significant different (p-value < 0.05) from control group.
Fig. 3. Striped view of concentration-dependent response profile in EE2
exposure of male Japanese medaka. Gene expression data from medaka
treated by 5, 50 and 500 ng EE2/L are shown as striped color sets on the
selected endocrine pathways along the medaka BHG axis. The legend listed in
the upper right corner of the graph describes the order of the three EE2
concentration and the eight colors designating different fold thresholds. LH,
luteinizing hormone; FSH, follicle-stimulating hormone; E2, 17β-estradiol; T,
testosterone; HDL, high-density lipoprotein; LDL, low-density lipoprotein.
1.Zhang, et al., 2008. Aquatic Toxicol. 88, 173–182.
2.Zhang, et al., 2008. Environ Sci Technol 42, 6762-6769.
3.Zhang, et al., 2008. Environ Toxicol Chem. (In press)
4.Park, et al., 2008. Toxicol. Appl. Pharmacol (In press).
5.Tompsett, et al., 2008. Toxicol. Sci. (In press)
Species‐specific accumulation of polychlorinated dibenzo‐p‐dioxins (PCDDs), dibenzofurans (PCDFs), and coplanar polychlorinated biphenyls (PCBs) in fishes from the Tittabawassee and Saginaw Rivers (Michigan, USA)
12
Yi Wan
Yi
Wan1*, Paul D. Jones
Pa l D Jones1, Ryan R. Holem
R an R Holem2, Jong Seong Khim
Jong Seong Khim1, Denise P. Kay
Denise P Ka 2, Shaun A. Roark
Sha n A Roark2, John L. Newsted
John L Ne sted2, and John P. Giesy
and John P Gies 1,2
1Department of Biomedical Veterinary Science and Toxicology Centre, University of Saskatchewan, Saskatoon, SK S7N 5B3, Saskatchewan, Canada
2ENTRIX, East Lansing, MI, USA
ABSTRACT
Factors causing differential accumulation of contaminants among species are a major focus of ecotoxicology and environmental chemistry studies. In this study, polychlorinated dibenzo‐p‐dioxins (PCDDs), dibenzofurans (PCDFs), and non‐ortho‐substituted (co‐planar, dioxin‐like) polychlorinated biphenyl (PCB) cong
eners were analyzed in twelve fish species from the Tittabawassee and Saginaw rivers in Michigan, USA. Based on stable isotope determination the trophic levels for all fishes, excluding migratory walleye and white sucker, ranged from 2.00±0.26 (carp) to 3.26±0.34 (largemouth bass). The greatest PCDD/F conce
eners were analyzed in twelve fish species from the Tittabawassee and Saginaw rivers in Michigan, USA. Based on stable isotope determination the trophic levels for all fishes, excluding migratory walleye and white sucker, ranged from 2.00±0.26 (carp) to 3.26±0.34 (largemouth bass). The greatest PCDD/F conce
ntrations were found in carp (n=50) followed by channel catfish (n=49). ∑PCDD/Fs, ∑PCBs and ∑TEQs in carp were approximately 30‐, 9‐ and 30‐ fold greater than the least concentrations in other fishes. 2,3,7,8‐TCDF, 2,3,4,7,8‐PeCDF, 1,2,3,7,8‐PeCDF and 2.3.7.8‐TCDD were the predominant congeners found in all fi
shes, but relatively small proportions of 2,3,7,8‐TCDF and greater proportions of more‐chlorinated congeners were found in carp and channel catfish. Positive relationships were found between lipid content, body weight and concentrations of ∑PCDD/Fs, ∑PCBs and ∑TEQs, but negative relationships were found bet
ween trophic levels and all the above parameters. Multiple regression analysis demonstrated that lipid content and tropic level were important determining factors for ΣPCBs, but lipid content and body weight were the strongest predictors for ΣPCDD/Fs and ΣTEQs. Furthermore, Biota‐Sediment Accumulation Fact
ors (BSAFs) indicated that differences in bioavailability among chemicals are the main reason for the different patterns of relative concentrations among species.
INTRODUCTION
z
Nine PCDDs, eleven PCDF congeners and twelve non‐ and mono‐ortho PCB congeners in twelve species of fish (314 individuals) collected from the Tittabawassee and Saginaw Rivers were analyzed to study the factors influencing trophic transfer of dioxins in a river ecosystem, and provide site‐specific concentrations for human health risks assessment.
200
100
∑PCDD/Fs (pg/g ww
w)
The Saginaw River and its largest tributary, the Tittabawassee River, have been contaminated by various organic pollutants including PCDD/Fs and dioxin‐like PCBs by historical industrial activities
200
4×105
ΣTEQs (pg/g ww)
z
No research has addressed the factors influencing differences in bioaccumulation of dioxins among species in river ecosystems
300
ΣPCBs (pg/g ww)
z
RESULTS
A number of reports have highlighted the importance of chemical and biological factors in the bioaccumulation and trophic transfer of persistent organic pollutants in aquatic ecosystems ΣPCDD/Fs (pg/g ww)
z
3×105
2×105
100
1000
1000
1000
100
100
100
10
10
y=‐0.49x+2.58
r2=0.2101
p<0.001
1
0.1
1×105
1.0
2.0
10
y=0.82x+0.81
r2=0.3191
p<0.001
1
3.0
0.1
4.0 0.1
1
TL
0
N = 9 12 50 49 20 20 12 58 10 44 15 15
BC BG CA CC FD LB NP SB SU WA WB WS
0
N = 9 12 50 49 20 20 12 58 10 44 15 15
BC BG CA CC FD LB NP SB SU WA WB WS
0
N = 9 12 50 49 20 20 12 58 10 44 15 15
∑PCBs (pg/g ww)
ΣPCDD/Fs in carp were significantly greater than in other species (p<0.01), the next greatest concentrations were found in channel catfish and northern pike.
z
Concentrations in carp were significantly greater than those in other fishes (p<0.01) except channel catfish and white bass.
z
1E+06
100000
1E+05
1E+05
1E+04
1E+04
y=0.62x+4.38
r2=0.2867
p<0.001
10000
y=‐0.44x+5.91
r2=0.2731
p<0.001
1.0
2.0
1E+03
3.0
4.0 0.1
∑TEQs (pg/g ww)
1000
Bay city
20
Saginaw
BG
CA
CC
FD
LB
NP
SB
SU
WA
WB
WS
The δ13C value is an indicator of the origin of nutrients and migratory behaviors, increasing values from freshwater to marine ecosystems 10
The δ13C values of walleye and white sucker were greater than those of other fishes which are probably
greater than those of other fishes, which are probably due to their habitats, since these two fishes live in Saginaw Bay, Lake Huron, and migrate up to the river systems to spawn
δ15N
Other fishes
The fishes studied were those preferentially harvested by anglers and the selection of species was based on information such as creel surveys conducted by state and federal agencies as well as local fisheries experts.
Fish were collected, using standard electro‐fishing equipment (Smith‐Root®) and techniques, from six distinct river reaches, four in the Tittabawassee River and two in the Saginaw River. z
WA
CC
14
The δ15N value is used to calculated the trophic levels of the fishes. TLfish = 2 + ( δ15Nfish – δ15Ncarp ) / 3.4
z
CA
12
The trophic level of carp was the least followed by channel catfish, which is consistent with the bottom feeding behaviors, the top predator among the biological samples in this study is largemouth bass.
z
SU
PCDD/Fs and PCBs were analyzed following EPA methods 1613 and 1668 respectively.
Stable nitrogen isotope analysis was used to quantitatively determine the habitat and
Stable nitrogen isotope analysis was used to quantitatively determine the habitat and trophic levels of these fishes.
z
WA=walleye, WS=white sucker, WB=white bass, SB=smallmouth bass, LB=largemouth bass, CC=channel catfish, CA=carp, FD=freshwater drum, BC=black crappie, NP=northern pike, BG=bluegill, and SU=green sunfish.
z
z
16
z
10
-31
-29
-27
-25
δ13C
-23
100 10
100
1000
10000 100000
Body Weight
1000
y
y=0.62x+0.67
r2=0.1831
p<0.001
100
yy=0.58x‐0.71
r2=0.2024
p<0.001
10
10
10
1
1
1
0.1
0.1
0.1
z
18
z
10000 100000
y=0.30x+3.82
r2=0.0857
0 0857
p=0.006
Lipid
1000
yy=‐0.46x+2.25
r2=0.1872
100
p<0.001
100
1000
1E+03
1
TL
∑PCDD/Fs, ∑PCBs and ∑TEQs in carp in this study were approximately 30‐, 9‐ and 30‐ fold greater than the least concentrations in other fishes, respectively.
, p
y
100
Body Weight
1E+06
z
BC
10
1000000
1000
Concentrations of TEQs in two species (carp and channel catfish) were significantly greater than those in other species (p<0.01).
z
Midland
0.1
100
Lipid
BC BG CA CC FD LB NP SB SU WA WB WS
Species
MATERIALS AND METHODS
10
y=0.79x‐1.08
r2=0.3697
p<0.001
1
-21
1.0
2.0
3.0
TL
4.0 0.1
1
10
Lipid
100 10
100
1000
10000 100000
Body Weight
Statistically significant correlations were observed between tropic level, lipid content, body weight and tissue contaminant concentrations.
z
zMultiple linear regressions were used to further assess the effects of the biological factors on accumulations of dioxins for all local fishes.
zlog(ΣPCBs) = 0.439log(LP)(p<0.001, 28.7%)–0.304TL(p<0.001, 10.4%)+5.304 Lipid content and tropic level were important factors for ΣPCBs , and the percentage contributions were 28.7% and 10.4%, respectively. Negative correlations between concentrations of dioxin‐like PCBs and trophic level were due to the benthivorous fishes occupying lower trophic levels and consequently having an extra uptake pathway, sediment ingestion.
log(ΣPCDD/Fs)=0.634log(LP)(p<0.001,17.7%)+0.646log(BW)(p<0.001,37.0%)–0.976 log (ΣTEQs) = 0.482log(LP)(p=0.001, 10.4%)+0.471log(BW)(p<0.001, 20.2%)–0.633 Lipid content and body weight were strong predictors of concentrations of ΣPCDD/Fs and ΣTEQs, explaining 17.7% and 37.0% of the variance for ΣPCDD/Fs, respectively, and 10.4% and 20.2% of the variance for ΣTEQs, respectively
z
Assessment of Toxicity of Upper Danube River Sediments Using a Combination of Chemical Fractionation,
the Danio rerio Embryo Assay and the Ames Fluctuation Test
Eric Higley1, Stefanie Grund3, Thomas B.-Seiler2, Urte Lubcke-von Varel6 ,Werner Brack6, Tobias Schulz6, Jan Wölz2, Hanno Zielke2, John Giesy1,5, Henner Hollert2, Markus Hecker4
1. University of Saskatchewan, Saskatoon, SK, 2 RWTH, Aachen, Germany, 3 University of Heidelberg, Heidelberg, Germany, 4 ENTRIX, Inc., Saskatoon, SK, 5 City University of Hong Kong, Hong Kong, China, 6 UFZ Leipzig, Germany.
Introduction
Objectives
The world’s river systems provide fresh water to people and support thousands of
species. However, many of the great rivers have been polluted in the past decades.
Possible sources of such pollution include effluents from domestic sewage pants (i.e.
urine and feces, detergents, pharmaceuticals), industry (i.e. PCBs, dioxins, and
metals), agricultural runoff (i.e. pesticides and fertilizers), and storm water runoff from
urban areas (i.e. salts, oil, and antifreeze). Severely contaminated sediments from
many rivers and lakes have been shown to be acutely and chronically toxic to fish and
benthic invertebrate species. For example, sediment samples from the Upper Danube
River that were analyzed in six separate assays were found to have considerable
geno-toxic, cytotoxic, mutagenic, embryo-toxic and estrogenic effects. It has been
hypothesized that decline in fish stocks in the Upper Danube River since the early
1990s may be associated with this pollution. Here, we report on the results of a study
conducted to determine the toxicity of extracts from sediments of the Danube River by
means of the Danio rerio embryo assay, and by assessing lethal and sub lethal
endpoints. In addition, mutagenicity was assessed using the Ames fluctuation assay.
For the sediment samples that revealed toxicity, fractionation of each sample was
performed by separating compounds according to their polarity, planarity, and the size
of the aromatic ring system. 18 fractions for each sediment sample were tested
separately in the Ames fluctuation assay and Danio rerio embryo assay to assess
which group of chemicals within the sediment sample caused the original toxicity.
1. Assess the toxicity of raw sediment extracts from four locations along the Upper
Danube River using the Danio rerio Embryo Assay and Ames Fluctuation Assay
2. Evaluate which groups of chemicals caused the measured toxicities using new
chemical fractionation techniques that separate the raw sediment extracts into 18
different chemical fractions.
3. Analyze all 18 chemical fractions using the Danio rerio Embryo Assay and Ames
Fluctuation Assay.
Results
Figure 1: Map of Germany and sediment sampling locations
Methods
Lauchert reference
Öpfingen
16.0
14.0
12.0
10.0
8.0
6.0
4.0
2.0
0.0
Lauchert
Sigmaringen
***
***
SC
***
* ****
***
***
*
12.5 25.0 50.0 100.0 200.0 400.0
Danio rerio Embryo Assay
16.0
14.0
12.0
10.0
8.0
6.0
4.0
2.0
0.0
TA98 Strain
*
2.0
Sample With or
Location without
1.0
S9
0.0
12.5
25.0
50.0 100.0 200.0 400.0
Sig
PC
Sediment Equivlaent Concentrations (mg/ml) Lauchert reference
Lauchert
Öpfingen
Sigmaringen
TA100 Bacteria Strain -S9
*
*
*
*
Lauchert reference
Lauchert
Öpfingen
Sigmaringen
Lau
**
15.0
Lau
ref
*
*
12.5
25.0 50.0 100.0 200.0 400.0
PC
Sediment Equivalent Concentration (mg/ml) Viable eggs less
than 1 hour old
are collected
Zebrafish are
breed overnight
Add pH
indicator
media
without
histidine
Place histidine deficient bacteria into 384 well
plate without histidine
Incubate at 37 ° C for 48 hours
After 48 hours, any bacteria that have back
mutated and can produce histidine will live and
grow and turn the media from purple to yellow
Count # of wells
that are yellow
+
Zebra fish
egg + ISO
water
0.0
10
XX
11
13
14
X
15
16
X
XXX
X
XXX
X
X
XX
17
XX
XX
X
X
X
X
XXX
Sig
SC
12.5 25.0 50.0 100.0 200.0 400.0
PC
Opf
Sediment Equivalent Concentrations (mg/ml) Lau
Lau ref
Danio rerio embryo assay on whole extracts
XX
Sediment
sample in
DMSO
70
60
50
40
30
20
10
0
Incubate for 48 hours
in 96 well plate
SC
12.5
25
50
+
+
+
+
-
Chemical fractions that showed effects
8
X
10
XX
11
X
XX
X
13
XX
X
X
X
Conclusions
Öpfingen
Sigmaringen
Lauchert
Lauchert Reference
Record
lethal
and sublethal
effects
after 48
hours
9
X
Location without S9
Mortality %
Incubate for 90 minutes with histidine
3
TA100 Strain
Sample With or
5.0
Figure 2. Dose response of four whole sediment extracts ran in the Ames Fluctuation
Assay with and without the liver enzyme S9 mix and on two different bacteria strains
(TA98 and TA100). * indicates significant difference from control.
Sediment
sample in
DMSO
+
+
+
+
-
Chemical fractions that showed effects
10.0
SC
Bacteria culture
Sigmaringen
3.0
SC
M u t a g e n ic E f f e c t ( # o f r e v e r t a n t s )
•Crude sediment extracts and all 18 fractions were analyzed for their toxicity using the
Ames fluctuation assay and Danio rerio egg assay
+
Lauchert
Öpfingen
4.0
PC
TA100 Bacteria Strain +S9
M u t a g e n ic E f f e c t ( # o f r e v e r t a n t s ) •Samples were extracted and fractionated into different chemical groups using a new
technique by Varel et al., 2008 that uses 3 HPLC columns and separates the sample
into 18 fractions according to their polarity, planarity and the size of their aromatic
system
deficient
Lauchert reference
Table 1. Fractions (3 – 17) showing significant increases in the number of mutations
compared to the controls as determined by the Ames Fluctuation Assay. TA98 Bacteria
measures frame shift mutations and TA100 Bacteria measures base pair substitutions.
Sig=Sigmaringen, Opf=Opfingen, Lau=Lauchert, Lau ref=Lauchert Reference.
X = less than a 3-fold increase; XX = 3- to 10-fold increase; XXX = greater than 10 fold
increase.
Opf
•Sediments were sampled (top 5cm) at four locations along the Upper Danube River
using a Van Veenen grabber in January 2006 (Figure 1)
Histidine
5.0
Sediment Equivalent Concentrations (mg/ml) Sampling and extraction
Ames Fluctuation Assay
TA98 Bacteria Strain -S9
M u t a g e n ic E f f e c t ( # o f r e v e r t a n t s )
M u ta g e n ic E f f e c t ( # o f r e v e r ta n t )
TA98 Bacteria Strain +S9
100
Sediment equivalents concentration (mg/ml)
Figure 3. Dose response of four sediment extracts analyzed with the Danio rerio embryo assay
• Mortality of Danio rerio embryos increased in a dose-dependent manner when
exposed to whole sediments collected at Öpfingen and Sigmaringen, but none of the
fractionated samples were toxic. These results indicate that the observed toxicity was
likely due to the combination of groups of chemicals in the whole sediment samples.
• Toxicity was observed for whole sediments from Sigmaringen, Öpfingen and Lauchert
in the Ames Fluctuation Assay only when TA98 bacteria with S9 were tested. Toxicity
was also found in the fractionated samples in both bacterial strains, although the
pattern was inconsistent.
• However, toxicity was measured in fractions 10 and 15 of every sediment sample
except Lauchert reference. Previous work has found that fraction 10 can contain sixringed PAHs (i.e. benzo(a)pyrene or benzo(k)fluoranthene) and fraction 15 can
contain more non-polar chemicals like benzocarbazole and benzanthrone. Further
work using other analytical techniques may identify which chemicals caused the
observed toxicity.
Reference:
Varel U, Streck G, Brack W. 2008. Automated fractionation procedure for polycyclic aromatic compounds in sediment
extracts on three coupled normal-phase high-performance liquid chromatography columns. Journal of
Chromatography A. 1185:31-42
Perfluorinated Compounds in Environmental Samples Collected from Inner‐Mongolia, China
Jonathan Naile1,*, Jong‐Seong Khim1, Tieyu Wang2, Wentao Jiao2, Chunli Chen2, Yonglong Lu2, Matt Zwiernik3, Kurnthachalam Kannan4, Paul D. Jones1, and John P. Giesy1,2,5
1Department of Veterinary Biomedical Sciences & Toxicology Centre, University of Saskatchewan, Saskatoon, SK, Canada
2Research Center for Eco‐environmental Sciences, Chinese Academy of Science, Beijing, China 3Department of Zoology, National Food Safety and Toxicology Center, Center for Integrative Toxicology, Michigan State University,
East Lansing, MI, USA
Environmental Health Sciences, School of Public Health, State University of New York at Albany, Albany, NY
5Department of Biology and Chemistry, City University of Hong Kong, Kowloon, Hong Kong, SAR China
4Wadsworth Center, New York State Department of Health and Department of
ABSTRACT
Inner‐Mongolia is a region of China which historically has seen little development and industry. Sediment (n=7), water (n=8), and biological (n=12) samples were collected from Inner‐Mongolia to determine the extent of perfluorinated compound (PFC) pollution in a less industrialized region and to shed light on their long‐range transport and ultimate fate of these compounds. Our results indicate that PFCs are only moderately concentrated in sediments and water samples. Some biological samples contained detectable concentrations of several PFCs including PFOS (0.48‐1.1 ng/g) and PFOA (0.09‐1.2 ng/g), however, their concentrations were mostly less than the detection limit. There is currently some debate as to whether soil and sediment are the ultimate sink for PFCs as they are for many neutral organic compounds. PFCs detected in these environmental samples from Inner‐Mongolia likely represent background globally distributed concentrations in China. Overall, the detection of PFCs and their precursors in various environmental matrices from remote regions suggest their long range transport and distribution. BACKGROUND
RESULTS
CONCLUSIONS
z
z
Fundamentally different from traditional organic pollutants
z
Difficult to study due to unique chemical properties
z
Thought to be chemically stable and biologically inert in the environment
140.0
z
Large scale production from the 1950’s to 2000
120.0
z
Wide range of applications from surfactants to pharmaceuticals
100.0
z
First found in the environment in 1997
z
3M Company and US EPA announce voluntary phase out of PFOS manufacturing
Globally distributed in matrices varying form human blood to wildlife tissue
z
Many uncertainties from analytical methods for quantification to toxic effect thresholds
Concentration (ng/L)
z
z
z
z
z
China is experiencing a time of rapid growth and development and areas that were once pristine are now becoming more urbanized to meet the growing demand for resources
Inner Mongolia is the Mongol autonomous region of Northern China
Samples collected from Inner Mongolia should help shed light on the effects of urbanization and PFCs distribution on a remote region of the world
To date most PFC research in China has focused on the southern mainland or Hong Kong areas
z
131.0
z
80.0
z
PFOS
60.0
50.0
PFOA
40.0
z
18.8
20.0
1.8
INTRODUCTION
z
Average Concentrations of PFOS and PFOA in Surface Water Samples Collected from China 7.9
0.0
4.9 7.8
0.1 0.3
6.9
1.3 3.1
7.0
Xiaoqing South Costal Costal Pearl River 3 4
Yangtze Dalian
Inner River 1 China Sea 2 2 2 3 China Hong Kong
River
Mongolia
Average calculated from Zhao et al. 20071, Yamashita et al. 20052, So et al. 20073, and Ju
Samples were collected during August of 2006 and were stored frozen until analysis
Samples were extracted using modified Solid Phase Extraction (SPE) methods to optimize recovery and minimize contamination1,2,3
z
Recoveries for all 8 compounds were greater than 70% thus concentrations were not corrected
Negative ESI‐HPLC‐MS/MS operated in MRM was used for data analysis quantification
z
z
z
The use of Teflon related materials were avoided during all steps of sample collection and analysis A second column was inserted directly upstream of the HPLC injector port to separate any possible contamination coming from the eluents or instrument Sediment and biological samples collected from Inner Mongolia, China do not appear to be heavily contaminated with PFCs PFCs found in these samples likely represent a background level contamination as result of global distribution Concentrations are consistent with other remote environments
Monitoring and Assessment of Exposure and Potential Biological Effects of Perfluorinated Compounds in the Yellow Sea Region of China and Korea
z Find environmental levels of target persistent and toxic contaminants and if appropriate determine loadings and sources
z Determine distribution and source characteristics (and possibly fate, transport, or food web) PFCs in Sediment (ng/g dry wt.)
Shilawusu River
Xiaohei River
Dahei River
Xiaohei River
Xiaohei River
Dahei River
Yellow River
PFOS
PFOA
TDHA
PFNA
PFDA
C11
C12
PFHS
<0.1
<0.1
<0.1
<0.1
<0.1
<0.1
<0.1
<0.5
<0.5
<0.5
<0.5
<0.5
<0.5
<0.5
<0.1
<0.1
<0.1
<0.1
<0.1
<0.1
0.13
<0.1
<0.1
<0.1
<0.1
<0.1
<0.1
<0.1
<0.1
<0.1
<0.1
<0.1
<0.1
<0.1
<0.1
<0.1
0.13
0.30
0.26
0.25
0.24
0.36
0.78
<0.5
<0.5
<0.5
<0.5
<0.5
<0.5
<0.5
<0.5
<0.5
<0.5
<0.5
<0.5
<0.5
Toad, Dahei River
Toad, Shilawusu River
Toad, Confluence of Xiaohei and Dahei Rivers
Toad, Dahei River
Frog, Xiaohei River
Carp, Yellow River
Frog, Xiaohei River
Grasshopper, Yellow River Grasshopper, Shilawusu River
Grasshopper, Confluence of Xiaohei and Dahei Rivers
Grasshopper, Dahei River
Praying Mantis, Xiaohe River
of target contaminants
z Address potential biological effects associated with samples
z Identify potential toxic chemicals based on a TIE and mass balance approach
z Establish background monitoring data for target contaminants and possibly develop site‐specific environmental quality guidelines
z Prioritize the site‐specific contaminants of concern in the study area
REFERENCES
PFCs in Biological Samples (ng/g wet wt.)
z
Water concentration of PFCs found in inner Mongolia are similar or lower than most other regions of china
et al. 20084
METHODS and QA/QC
z
PFOA was found in all water samples and was consistently found at the highest concentrations relative to the other PFCs monitored for ONGOING and FUTURE WORK
0.6 1.3
1.2
PFOS was found in all but one of the water samples PFOS
PFOA
TDHA
PFNA
PFDA
C11
C12
PFHS
<0.3
<0.3
<1.5
<1.5
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
<1.5
<1.5
<1.5
<1.5
<1.5
<1.5
<0.3
<1.5
<0.3
<0.3
<0.3
<1.5
<1.5
<1.5
<0.3
<0.3
0.48
1.10
<0.3
<0.3
<1.5
<1.5
<1.5
<1.5
<1.5
<1.5
<0.3
<0.3
<0.3
0.87
<0.3
<0.3
0.93
<0.3
<0.3
0.36
<0.3
<0.3
<0.3
<0.3
<0.3
0.51
<0.3
<0.3
<1.5
<1.5
<1.5
<1.5
<1.5
<1.5
<1.5
<1.5
<1.5
<1.5
<1.5
<1.5
<1.5
<1.5
<1.5
<1.5
<1.5
<1.5
<0.3
<1.5
<0.3
<0.3
<0.3
<1.5
<1.5
<1.5
<0.3
<0.3
<1.5
<1.5
<0.3
<0.3
<0.3
2.36
<0.3
<0.3
<1.5
<1.5
<1.5
<1.5
<1.5
<1.5
1 Higgins, Christopher P., et al. "Quantitative Determination of Perfluorochemicals in Sediments and Domestic Sludge." Environmental Science & Technology 39.11 (2005): 3946‐56.
2 So, M. K., et al. "Alkaline Digestion and Solid Phase Extraction Method for Perfluorinated Compounds in Mussels and Oysters from South China and Japan." Archives of Environmental Contamination and Toxicology 50.2 (2006): 240‐48.
3 Yamashita, Nobuyoshi, et al. "Analysis of Perfluorinated Acids at Parts‐Per‐Quadrillion Levels in Seawater Using Liquid Chromatography‐Tandem Mass Spectrometry." Environmental Science and Technology 38.21 (2004): 5522‐28.
Perfluorinated Compounds in Sediment and Water from Bohai Bay and Its Vicinity, China
Jong Seong Khim1*, Tieyu Wang2, Wentao Jiao2, Jonathan E. Naile1, Jing Geng2, Chunli Chen2, Yonglong Lu2, Yi Wan1, Paul D. Jones1, John P. Giesy1,3,4
1
3
Department of Biomedical Veterinary Sciences and Toxicology Centre, University of Saskatchewan, Saskatoon, SK S7N 5B3, Saskatchewan, Canada
2 Research Center for Eco-environmental Sciences, Chinese Academy of Science, Beijing, China
Zoology Department, Center for Integrative Toxicology, National Food Safety and Toxicology Center, Michigan State University, East Lansing, MI 48824, USA
4 Department of Biology and Chemistry, City University of Hong Kong, Kowloon, Hong Kong, SAR China
INTRODUCTION
2. Instrumental Analysis
2. Spatial Distribution of PFCs
z Bohai Bay and its vicinity (north coast of China) contains several industrial
complexes and a large commercial harbor.
z PFCs were concentrated from sediment and water by use of solid phase extraction.
z PFOS was detected in sediment from nearly all locations in Bohai Bay, while
PFOS was detectable in only two locations from Guanting Reservoir.
z Despite the fact that perfluorinated compounds (PFCs), are known to have been
used extensively in the region with potential for intentional and accidental release,
little was known regarding the current status of PFCs concentrations in this region.
z The present study was one of the first efforts to examine the concentrations,
distribution, and potential ecological effect of PFCs in this area of China.
z PFCs were identified and quantified by liquid chromatography interfaced with a
triple quadrapole tandem mass spectrometer (LC-MS/MS).
I. Collection
II. Extraction
III. Clean-up
z PFOA was detected in water from all locations from both Bohai Bay and Guanting
Reservoir, which suggests widespread distribution of PFOA.
z Relatively greater PFOS concentrations were observed in water from Bohai Bay
than Guanting Reservoir, which could be explained by industrial activities in
Bohay Bay and nearby Tianjin.
z We present the results of instrumental analyses on the distribution of 17 PFCs
including perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) in
sediment and water from Bohai Bay and Guanting Reservoir.
1950s
z PFOA in sediments from Bohai Bay and Guanting Reservoir were found to be
similar, indicating widespread contamination of PFOA throughout the study area.
3. Comparison to Environmental Quality Criteria
Large scale production of PFCs begins
z Concentrations of neither PFOS nor PFOA in water samples exceeded
concentrations thought to be protective of aquatic life both.
1997
PFCs first reported
in soil, water, air, and wildlife
z Overall, the PFCs detected in environmental samples from these areas were
relatively low to moderate compared to other studies in Asia and likely represent
background globally distributed concentrations of these compounds.
2000
3M and US EPA announced voluntary
phase out of PFOS manufacturing
Log Scale
2001present
Global distribution including remote areas
reported but little is known for PFCs in China
Fig. 4. Sample preparation and extraction sequence (e.g. sediment samples)
25 mg/L-CMC for PFOA
2.9 mg/L-CCC for PFOA
RESULTS & DISCUSSIONS
21 ug/L-CMC for PFOS
Fig. 1. Historical review for PFCs study
1. Occurrence & Concentrations of PFCs
z Of 17 PFCs measured, PFOS and PFOA were found to be the predominant
compounds in both sediment and water.
MATERIALS & METHODS
1. Study Area
z Sediment and water samples were collected from Bohai Bay and its vicinity city of
Tianjin (Fig. 2) and from Guanting Reservoir (Fig. 3).
z Soil samples were also collected, but soil PFCs data presented elsewhere.
Sediment & Water
Soil
z Concentrations of PFOS and PFOA in sediment were as great as 2.15 ng/g DW
(mean=0.46, n=15) and 0.74 ng/g DW (mean=0.31, n=19), respectively.
z Concentrations of PFOS (mean=1.79 ng/L, n=15) and PFOA (mean=4.18 ng/L,
n=15) in water samples were generally three orders of magnitude less than
corresponding sedimentary concentrations.
z There is currently some debate as to whether soil and sediment are the ultimate
sink for PFCs as they are for many other organic compounds.
Table 1. Concentrations of PFOS and PFOA in sediment (ng/g DW) and
water (ng/L) samples from Bohai Bay and Guanting Reservoir, China
Location
Samples
PFOS
PFOA
Bohay Bay
n=8, Aug 2007
Sediment
0.01-2.15
(mean=0.56)
0.05-0.74
(mean=0.31)
Water
0.10-10.5
(mean=2.55)
3.00-12.0
(mean=6.80)
Sediment
0.09-0.15
(mean=0.12)
0.06-0.57
(mean=0.30)
Water
0.13-0.52
(mean=0.29)
0.55-2.26
(mean=1.19)
Guanting Reservoir
n=7, Aug 2007
Fig. 2. Map of the Bohai Bay
showing sampling locations
(n=8, Aug 2007)
Fig. 3. Map of the Guanting Reservoir
showing sampling locations
(n=7, Aug 2007)
SETAC 29th Annual Meeting
November 16-20, 2008
Tampa, FL, USA
5.1 ug/L-CCC for PFOS
10.5 ng/L-PFOS-max
12.0 ng/L-PFOA-max
in Bohai Bay, China
47 ng/L-AWV for PFOS
CMC: criteria maximum concentration
CCC: criteria continuous concentration
AWV: avian wildlife value
Fig. 5. PFCs in Bohai Bay vs. Water Quality Criteria
Perfluorooctane Sulfonate and other Fluorochemicals in Soils from Bohai Bay, China
Tieyu Wang1, Jonathan E. Naile2, Jong Seong Khim2, Wentao Jiao1, Jing Geng1, Chunli Chen1, Yonglong Lu1, Yi Wan1, Paul D. Jones2, John P. Giesy2,3,4
1 Research Center for Eco‐environmental Sciences, Chinese Academy of Science, Beijing, China Department of Biomedical Veterinary Sciences and Toxicology Centre, University of Saskatchewan, Saskatoon, SK S7N 5B3, Saskatchewan, Canada
3 Zoology Department, Center for Integrative Toxicology, National Food Safety and Toxicology Center, Michigan State University, East Lansing, MI 48824, USA
4 Department of Biology and Chemistry, City University of Hong Kong, Kowloon, Hong Kong, SAR China
12
ABSTRACT
Perfluorinated compounds (PFCs), such as perfluorooctanesulfonate (PFOS) and related compounds, have recently been identified in the environment and have become the subject of increasingly intense environmental research. Despite their detection both in biota and in aqueous media, little attention has been paid to their possible presence in soils. The limited available data indicates that some PFCs such as PFOS and perfluorooctanoic acid (PFOA) may strongly sorb to solids, thus soil may be suspected to be an important sink for PFCs as they are for many other neutral organic compounds. In the present study, the concentrations and distribution of 5 PFCs were quantified in soil samples (n=18) collected from the Beijing and Tianjin regions of China; the latter being the biggest industrialized coastal city of Bohai Bay. Among the PFCs measured, PFOS and PFOA were found to be the most predominant compounds with the greatest concentrations. PFOS and PFOA concentrations in soil ranged from 0.01 to 4.69 ng/g and from 0.01 to 2.77 ng/g, on a dry weight basis, respectively. Other PFCs showed relatively lower concentrations compared to PFOS and PFOA and most were below the detection limits. PFCs concentrations detected in this study were not likely sufficient to induce ecological or human health effects, however, the present data does provide some insight into the potential sources of PFCs in Chinese industrialized costal areas. Further studies are needed to elucidate the occurrence, exposure and possible sources of PFCs in different environmental media in these areas.
METHODS and QA/QC
BACKGROUND
z
Fundamentally different from traditional organic pollutants
z
z
Difficult to study due to unique chemical properties
z
z
Thought to be chemically stable and biologically inert in the environment
z
Large scale production from the 1950’s to 2000
z
Wide range of applications from surfactants to pharmaceuticals
z
First found in the environment in 1997
z
3M Company and US EPA announce voluntary phase out of PFOS manufacturing
z
Globally distributed in matrices varying form human blood to wildlife tissue
z
Many uncertainties from analytical methods for quantification to toxic effect thresholds
z
z
z
z
z
Recoveries for all 8 compounds were greater than 70% thus concentrations were not corrected
z
Negative ESI‐HPLC‐MS/MS operated in MRM was used for data analysis
The use of Teflon related materials were avoided during all steps of sample collection and analysis
z
A second column was inserted directly upstream of the HPLC injector port to separate any possible contamination coming from the eluents or instrument z
0.6
Concentration (ng/g DW)
z We present the results of instrumental analyses on the distribution of 5 PFCs including perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) in sediment and water from Bohai Bay and Guanting
Reservoir.
Soil samples collected from the Bohai Bay region of China do not appear to be heavily contaminated with PFCs PFOS was found at concentrations above the limit of detection (0.1 ng/g) only 50% of the time
The soils around Bohai Bay and Guanting reservoir do not appear to be a substantial sink for PFCs and may suggest that sediment, water, or biota are the ultimate sink Concentrations of PFCs found in the soil are not great enough that toxicological effects would be expected
Overall, the PFC concentrations detected in soil samples from this area were relatively low to moderate when compared to the other few studies that have looked at soil concentrations of these compounds.
RESULTS
z Bohai Bay and its vicinity (north coast of China) contains several industrial complexes and a large commercial harbor. z The present study was one of the first efforts to examine the concentrations, distribution, and potential ecological effect of PFCs in this area of China. z
Samples were extracted using a modified Solid Phase Extraction (SPE) method to optimize recovery and minimize contamination1
INTRODUCTION
z Despite the fact that perfluorinated compounds (PFCs), are known to have been manufactured and used extensively in the region little was known regarding the current status of PFCs concentrations in this region. CONCLUSIONS
Samples were collected during August of 2007 and were stored frozen until analysis
ONGOING and FUTURE WORK
Average Concentration of PFCs Detected in Soil Samples from Northeastern China
Monitoring and Assessment of Exposure and Potential Biological Effects of Perfluorinated Compounds in the Yellow Sea Region of China and Korea
z Find environmental levels of target persistent and toxic contaminants and if appropriate 0.5
determine loadings and sources
0.4
z Determine distribution and source characteristics (and possibly fate, transport, or food web) 0.3
Bohai Bay, Tianjin
Guanting, Beijing
0.2
of target contaminants
z Address potential biological effects associated with samples
z Identify potential toxic chemicals based on the TIE and mass balance approach
0.1
z Establish background monitoring data for target contaminants and possibly develop the 0
site‐specific environmental quality guidelines
PFOS PFOA TDHA
PFNA PFDA z Prioritize the site‐specific contaminants of concern in the study area
REFERENCES
Occurrence & Concentrations of PFCs
z Of the 17 PFCs measured 5 were routinely found about the limit of detection
Sediment & Water
Soil
z PFOS and PFOA were found to be the predominant compounds present in the soils
around Bohai Bay and Guanting reservoir respectively
z Concentrations of PFOS in soil were as great as 4.7 ng/g DW (mean=0.88, n=8) and as
low as 0.09 ng/g DW
Fig. 1. Map of the Bohai Bay showing sampling locations
(n=10 Aug 2007)
z There is currently some debate as to whether soil and sediment are the ultimate sink
for PFCs as they are for many other organic compounds.
Fig. 2. Map of the Guanting Reservoir
showing sampling locations
(n=8, Aug 2007)
SETAC 29th Annual Meeting
November 16‐20, 2008
Tampa, FL, USA
1 Higgins, Christopher P., et al. "Quantitative Determination of Perfluorochemicals in Sediments and Domestic Sludge." Environmental Science & Technology 39.11 (2005): 3946‐56.
PFOS
F
F
F
C
C
F
F
F F
C
C
F F
F F
C
C
FF
F
C
C
FF
O
S
F O
O
Effects of Selected Metals on Early Life-Stages of White Sturgeon (Acipenser transmontanus)
D. Vardy1, A. Tompsett1, J. Duquette1, K. Liber1, D. Janz1, M. Adzic3, M. Hecker1,2, J.P. Giesy1
1 Department
of Biomedical Sciences and Toxicology Centre, University of Saskatchewan, Saskatoon, SK, Canada.
2ENTRIX,
Inc., Saskatoon, SK, Canada
3Teck,
Spokane, WA, USA
Abstract
Results
• 100% mortality occurred between hatch and day 10 for the two highest doses of Cu (Fig.2)
and the highest dose of Cd and Zn (Fig.3,4).
• Cd 4 (10.24 µgL) treatment experienced greater mortality near the end of the exposure
period (day 40) compared to other treatments.
100
75
75
75
Cu 7.2 ug/L
Cu 43.2 ug/L
25
Cd 0.02 ug/L
Cd 0.16 ug/L
Cd 1.28 ug/L
Cd 10.24 ug/L
25
Cu 259.2 ug/L
Cd 81.92 ug/L
Control
20
30
40
50
10
Objectives
1. Develop a species-specific dose-response relationship for Cu, Cd and Zn that will be
used to establish metal toxicity threshold values for white sturgeon.
2. Collect information that will be used along with metal speciation models to predict
thresholds for effects of these metals on eggs and larvae of white sturgeon under field
conditions
Methods
• Continuous flow-through exposure systems were designed and used to test 5
different exposure concentrations per metal based upon environmentally relevant
concentrations found in the Columbia River and concentrations expected to produce
toxic effects (Fig.1):
Cu 0.2 µg/L (ppb)—260 µg/L
Cd 0.02 µg/L (ppb)—82 µg/L
Zn 1 µg/L (ppb)—1300 µg/L
• Fertilized white sturgeon eggs were obtained from the Kootenay Trout Hatchery, Fort
Steele, B.C.
• Embryos, larvae and juveniles were exposed for 65 days and the surviving juveniles
were euthanized, measured, weighed and fixed in formalin.
• 96hr static renewal LC50 tests were conducted with 8-day old larvae.
• Further morphological analyses are currently being conducted at the University of
Saskatchewan Toxicology Centre.
B
30
40
50
60
0
10
20
Days
30
40
50
60
Days
Fig.3 Cadmium cumulative mortalities
100
Cu In Situ
75
50
• 96hr LC50 values for Cu, Cd and Zn were 74.3 µg/L, 15.3 µg/L and 156 µg/L, respectively.
• Water effects ratios (WER) indicate a 4 fold factor for Cd and Zn and a 0.5 fold factor for
Cu between Columbia River water and standard laboratory water for early life-stages of white
sturgeon (Table 1).
Discussion
• Copper affects sodium regulation across the gills and appears to affect early life-stages
of white sturgeon during initial exposure, especially at the higher doses (Fig.2,5).
• Cadmium is known to disrupt calcium uptake but has also been found to bioaccumulate
within the kidneys and liver. In the present study, cadmium appears to have a
pronounced acute effect at the highest dose at an early stage and a more chronic effect
in the second to highest dose towards the end of the exposure period (Fig.3).
• Zinc is an essential nutrient and most fish can tolerate relatively high concentrations.
In this study, only the highest dose of zinc (1296 µg/L) had a pronounced effect early in
the experiment. A slight increase in mortality was experienced in the second to highest
does near the end of the exposure period (Fig.4).
• A sensitive transition period from yolk sac to exogenous feeding (~day 20-35) was
discovered within the controls and all treatment groups (except the high metal doses
where 100% mortality occurred prior to feeding) that promoted fish mortality (Fig.2,3,4).
• The drastic increase in mortalities across all groups during the transition feeding stage
has raised the question of whether it may be more appropriate to test early life-stages
of white sturgeon at independent time intervals, thereby excluding this period of time
that is characterized by a naturally greater mortality.
• A significant dose-response relationship is apparent in the copper treatment when
examining day 1-20, the period prior to the sensitive transition feeding stage (Fig.5).
100
Cd In Situ
75
50
Zn In Situ
75
50
25
0
1
10
100
1000
ug/L
WQG (2.6 µg/L)
25
0
0.01
0
0.1
WQG (0.028 µg/L)
1
10
100
1000
ug/L
1
WQG (7.5 µg/L)
10
100
ug/L
100
75
50
25
0
Table 1. Sensitivity of early life-stages of white sturgeon exposed to
Cu, Cd and Zn in laboratory and Columbia River water
96hr LC50
Lab
In Situ
WER
Cu
74.3
38.7
0.52
2.6
30 - 210
Cd
Zn
15.8
156
62.5
646
3.96
4.14
0.028
7.5
4.1 - 5.3
38 - 257
(µg/L)
Env. Can. Rainbow
WQG
Trout *
* LC50 values for Cu, Cd and Zn were obtained from Kamo, 2008.,
Besser, 2007., and Hansen, 2002., respectively
***
*** ***
***
**
*
***
CT
RL
p < 0.05
p < 0.01
Metal analyses
Histology and bio-energetic analyses
Development of a metal speciation model
Proposed field experiments in 2009
References
*
***
Doses
p < 0.001
Fig.5 Cumulative mortalities for Cu, Cd and Zn during the first 20 days
10000
• Regulatory decisions are often based upon the most sensitive species within an ecosystem and
the present study helps to characterize white sturgeon sensitivity to metals.
• Rainbow trout are relatively sensitive to metals, and for comparison, a range of LC50 values for
Cu, Cd and Zn are displayed in Table 1.
• The results of this study suggest that early life-stages of white sturgeon are relatively sensitive
to Cu in comparison to rainbow trout, and yet more tolerant to Cd and Zn.
•
•
•
•
Swim Up Phase Mortality (Day 1-20)
1000
Fig.6 96hr acute LC50 tests for Columbia River water and standard laboratory water (conducted through a
parallel study with A.Tompsett)
Future and upcoming work
*
**
***
Besser et al., 2007. Sensitivity of mottled sculpins (Cottus bairdi) and rainbow trout
(Onchorhynchus mykiss) to acute and chronic toxicity of cadmium, copper and zinc.
Env. Tox. and Chem. Vol 26, No.8, pp. 1657-1665.
Golder Associates Ltd. 2007. White sturgeon spawning at Waneta, 2007 investigations.
Report prepared for Teck Cominco Metals Ltd. Trail Operations. Golder Report No. 071480-0031F, 28p.
Hansen, 2002. Relative sensitivity of bull trout (salvelinus confluentus) and rainbow trout
(oncorhynchus mykiss) to acute exposures of cadmium and zinc. Env. Tox. and Chem.
Vol 21, pp. 67-75.
Kamo et al., 2008. An application of the biotic ligand model to predict the toxic effects of
metal mixtures. Env. Tox. and Chem., Vol 27, No.7, pp 1479-1487.
Fig.1 A: Flow-through exposure system; B: Exposure chamber design
Acknowledgments:
Zn Lab
Fig.4 Zinc cumulative mortalities
Cu
1
Cu
2
Cu
3
Cu
4
Cu
5
Cd
1
Cd
2
Cd
3
Cd
4
Cd
5
Zn
1
Zn
2
Zn
3
Zn
4
Zn
5
A
20
100
Zn 1296 ug/L
Cd Lab
25
Mortality (%)
It has been reported, however, that year old juveniles released into the Columbia River as
part of a recovery initiative exhibit good survival, growth rates and body condition. Habitat
alteration, varying flow regime, poor nutrition, genetic bottlenecks, predation and pollution
have all been suggested as possible explanations. Presently, little toxicity data exist
characterizing the sensitivity of white sturgeon to metals such as Cu, Cd, Zn.
Zn 216 ug/L
0
0
60
Fig.2 Copper cumulative mortalities
There is evidence that adult white sturgeons are spawning and depositing viable eggs in
certain areas of the Canadian reach of the Columbia River, especially at Waneta Eddy
located just north of the U.S.-Canada border, but only limited numbers of young of the year
(YOY) have been found in habitats considered suitable for this life stage (Golder Associates
Ltd., 2007).
Zn 36 ug/L
Control
0
10
Cu Lab
Zn 6 ug/L
25
125
125
125
Zn 1.0 ug/L
Control
0
0
50
% Mortality
Cu 1.2 ug/L
50
% Mortality after 96hr - Zn
% Mortality after 96hr - Cd
% Mortality after 96hr - Cu
% Mortality
Cu 0.2 ug/L
• Early-life stages of white sturgeon appear to be less sensitive to Cd and Zn in Columbia River
water compared to standard laboratory water and relatively more sensitive to Cu (Fig.6).
• Complexation of metals with organic materials decreases bioavailability and in turn toxicity to
fish and could explain the lower toxicity of Cd and Zn in river water.
• The decrease in Cu toxicity in laboratory water compared to river water is surprising and merits
further investigation.
• Environment Canada’s water quality guidelines (WQG) for Cu, Cd and Zn in the Columbia River
are displayed in Fig.6 and Table 1. The LC50 values for the metals of concern for early lifestages of white sturgeon are well above the set water quality guidelines.
% Mortality
50
% Mortality
100
% Mortality
100
Days
Introduction
Cumulative Zinc Mortality
Cumulative Cadmium Mortality
Cumulative Copper Mortality
% Mortality
Poor recruitment of white sturgeon Acipenser transmontanus in the Columbia River has
been documented since the 1970s. There are many possible causes for this phenomenon,
including water pollution (e.g., waterborne metals released by a metallurgical facility and
other industrial and municipal facilities). In general, little is known about the potential toxicity
of metals such as Cu, Cd, and Zn to white sturgeon and their potential influence on survival
of eggs and/or juveniles. The purpose of this study was to establish baseline laboratory
toxicity data for the exposure of early life-stages of white sturgeon to Cu, Cd, and Zn that
can be used in risk assessments, and, in combination with field experiments conducted in a
parallel study (see A. Tompsett et al.; White sturgeon hatch and survival after exposure to
Columbia River surface water at two sites in British Columbia, Canada; SETAC), to assess
the potential toxicity of these metals in waters of the Columbia River. Embryos, larvae and
fry were exposed to increasing concentrations of dissolved Cu, Cd, and Zn for 65 days using
laboratory based flow-through exposure systems. In addition, 96hr LC50 static toxicity tests
were conducted for each metal in order to gather information to calculate water effect ratios
(WER) between laboratory and separate concurrent field studies (see above). Preliminary
results indicate that early life-stages of white sturgeon are more sensitive to Cu and Zn
during the first 20 days post hatch compared to Cd which had a greater impact during
prolonged exposure.
Funding for this project was provided by Teck American Incorporated. Thanks to the Kootenay Trout Hatchery, Dr. Liber’s Lab, Eric Higley, Jonathan Naile, the UofS undergraduate team and the US-EPA for their advise during the planning stage of the studies.
White sturgeon hatch and survival after exposure to Columbia River surface water at two sites in British Columbia, Canada
AR Tompsett1, D Vardy1, M Hecker1,2, M Adzic3, M Allan1, JH Smith1, X Zhang1, K Liber1, DM Janz1, and JP Giesy1,4
1Dept. of Veterinary
Biomedical Sciences & Toxicology Centre, University of Saskatchewan, Saskatoon, SK, Canada
2ENTRIX, Inc., Saskatoon, SK, Canada
3Teck, Spokane, WA, USA
4Department of Biology and Chemistry, City University of Hong Kong, Kowloon, China
Days 12-27 and Days 38-60
Abstract
• Corrected for mortality on days 28-37, there were no significant treatment differences in cumulative
mortality (Figures 6 & 7)
The subpopulation of white sturgeon (Acipenser transmontanus) that resides in the Columbia River between
the Hugh L. Keenleyside dam in British Columbia, Canada and the Grand Coulee dam in Washington state,
USA has suffered nearly 30 consecutive years of poor recruitment. Factors such as altered flow regime due to
damming, loss of critical habitat, predation, and pollution have been suggested as causes for the lack of
recruitment, but none has been convincingly linked with the disappearance of young-of-the-year sturgeon. In
the current study, surface water toxicity up- and downstream of a large metal smelter was examined as a
possible contributor to the life-stage specific bottleneck in the white sturgeon population. Hatchery fertilized
eggs from wild brood stock were exposed to Columbia River surface water from 8 hr to 60 d post-fertilization
at two sites, one upstream and one downstream from the smelter effluent outflows. A filtered city water
control group was also examined to characterize any effects of inputs upstream of the study area not related
to the smelter. The exposures took place in mobile laboratories outfitted with flow-through exposure
chambers that allowed the white sturgeon to be exposed to the river water in real-time, a close
representation of the natural exposure scenario. Preliminary data suggests that neither hatch nor survival
through 60 d was affected by river water exposure. Evaluation of growth rates and histological endpoints in
larvae are ongoing.
River
Intake
Figure 6: Cumulative Mortality – 12-27 d
16
14
Overflow
to River
85L
Reservoir
Control
20
Upstream
15
Downstream
10
% Mortalit
% Mortality
Fully Replace Every
6hr (205 L)
% Mortalit
% Mortality
25
12
Control
10
Upstream
8
Downstream
6
4
Recirculating
System (205 L)
5
2
0
0
12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27
38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61
Day of Exposure
Day of Exposure
Figures 6 & 7: Both figures exhibit similar mortality curves for all treatments during both time periods.
Figures 6 & 7 coincide with the yolk-sac and exogenous feeding stages of sturgeon larvae, respectively
Survival to 60 d Post-Fertilization
40 L Streams
• The density of fish initially in each chamber
explains most of the ‘treatment differences’ in
mortality (R2=0.983, data not shown)
• Number of fish surviving to 60 d postfertilization may be a better indication of
treatment effects
• No significant differences in number of fish
surviving per treatment at 60 d (Figure 8)
Figure 3: Experimental setup
• Poor recruitment of white sturgeon in the trans-boundary
region of the Columbia River (Figure 1)
Flow-through systems with continuous river water renewal. Water in the system was completely
renewed every 6 hr. To maintain appropriate flow-rates and water quality parameters, water was also
recirculated within the system.
• Adult sturgeon spawn and lay fertilized eggs that successfully
hatch
• However, few young-of-the-year have been found in habitats
considered suitable for this life stage
Results
• Hatchery reared juveniles released to the river exhibit good
survival and growth
• No significant treatment differences (Figure 4)
• Hatch rates ranged between 76 – 82%
• Lower hatch rates in river water treatments
were due to fungal growth
Survival to Hatch
90
Figure 8: Number of fish surviving to 60 d
Percent Hatch
Figure 1. Overview of the Columbia River
The boxed area indicates the trans-boundary
region, which contains the study area.
Project Objectives
18
30
Introduction
• Effluent inputs from a metal smelter in Trail, BC, Canada
have been suggested as a contributor to reproductive failure
Figure 7: Cumulative Mortality – 38-60 d
35
post-fertilization. No significant differences in
survival to 60 d in control (C), downstream (D), and
upstream (U) treatments
80
• Expose early life-stages of white sturgeon to Columbia River
surface water at 2 sites (Figure 2)
• Filtered city water control also evaluated
• Evaluation of hatch, survival, growth, and morphology at each site
70
Figure 4: White Sturgeon Hatch Rates
Discussion
Hatch rates were not significantly different between control
(C), downstream (D), and upstream (U) treatments.
60
C
D
U
Treatment
Smelter Site
Cumulative Mortality of White Sturgeon Larvae
Days 12-60
• Percent mortality of control larvae was significantly greater than river water treatments at exposure termination
• However, cumulative mortality curves show similar trends across treatments (Figure 5)
-except from 28-37 d
-magnitude of mortality rate greater in controls
• Period of greatest mortality (28-37 d) coincides with transition to exogenous feeding
Columbia
River
• River water from downstream of the metal smelter had no adverse effects on white sturgeon hatch rate.
• Most larval mortality occurred from 28-37 d post-fertilization, which coincides with transition to exogenous
feeding. This period is generally considered to be very sensitive for white sturgeon.
• Outside of the transition period, cumulative mortality curves were similar across treatments.
• Number of mortalities was highly dependent upon initial stocking density
-all densities were below ASTM recommendations
-future studies with white sturgeon should use lower stocking densities
• Number of fish surviving to 60 d post-fertilization did not differ by treatment.
• River water from downstream of the metal smelter probably had no adverse effect on survival to 60 d, but
more advanced statistical analysis to correct for stocking density is required to confirm this conclusion.
Future Work
•
•
•
•
Pen d’Orielle
River
Cumulative Mortality
Analysis of survival, growth, and morphology data.
Analysis of water samples for trace metals.
Histological and molecular analysis of selected samples.
Expansion of project in 2009 to includes sites in both Canada and USA.
90
80
70
40
Control
Upstream
Downstream
30
20
10
Day of Exposure
60
57
54
51
48
45
42
39
36
33
30
27
24
21
0
18
Experiments performed riverside in retrofitted commercial trailers
Flow-through systems with continuous renewal and recirculation (Figure 3)
4 replicate systems per treatment
Sturgeon eggs from wild brood stock introduced into systems 8 hr post-fertilization
Eggs hatched and larvae grown to 60 d post-fertilization
Dead eggs and larvae collected, counted, and preserved daily
Bi-weekly water samples for metal analysis
Larvae euthanized, weighed, measured and preserved for subsequent analyses at exposure termination
Plot of cumulative larval mortality for the
duration of the exposure
50
15
•
•
•
•
•
•
•
•
Figure 5: Cumulative Mortality,
Days 12-60
60
12
Methods
% Dead
% Mortality
Figure 2. Study sites on the Columbia River
The reference site was located just upstream of the smelter site in Trail, BC. The downstream
site was located above the confluence of the Columbia and Pen d’Orielle Rivers at the USA/CAN
border.
Acknowledgements
This research was funded by an unrestricted grant from Teck. The authors would like to thank A. Jonas, S. Sedgwick, K. Smyth, E.
Higley, J. Naile, and J. Duquette for laboratory assistance. R. Ek and the Kootenay Trout Hatchery provided fertilized white
sturgeon eggs and invaluable guidance. We would also like to thank the City of Trail, Selkirk College, USEPA, and Teck ( B.
Duncan and R. Brown).
SETAC North America Meeting, November 16-20, 2008, Tampa, Florida, USA
EFFECTS OF POLYCHLORINATED DIBENZOFURANS ON MINK
Denise Kay∞, Matthew Zwiernik§, Steven Bursian‡, Kerrie Beckett†, Lesa AylwardΩ, Robert Budinsky*, Melissa Shotwell∞, Jeremy Moore§,
John Newsted∞, and John Giesy§#
∞ENTRIX Inc., Okemos, MI, USA. §Department of Zoology, National Food Safety & Toxicology Center, Michigan State University (MSU), East Lansing, MI, USA. ‡Department of Animal Science,
Center for Integrative Toxicology, MSU, East Lansing, MI, USA. †Stantec Consulting Services Inc., Topsham, ME, USA. ΩSummit Toxicology L.L.P., Falls Church, VA, USA. *The Dow Chemical
Company, Midland, MI, USA. #Department of Biomedical Veterinary Sciences & Toxicology Centre, University of Saskatchewan, Saskatoon, Saskatchewan, CA.
INTRODUCTION
Mink are often predicted to have the greatest potential for adverse effects in multi-species risk
calculations for sites with a substantial aquatic habitat where polychlorinated dibenzo-p-dioxins
(PCDDs), dibenzofurans (PCDFs), polychlorinated biphenyls (PCBs) and other dioxin-like
compounds are the contaminants of concern (COC)1. This is because mink:
™ are apical carnivores
™ consume a great amount of food relative to their body mass
™ are among the mammals that are more sensitive to aryl hydrocarbon receptor (AhR)mediated effects
Thus, remedial criteria are often derived for mink in situations where risks are predicted to occur
due to AhR-active compounds2. Hence it is important that exposure concentrations at which
adverse effects are predicted to occur be as accurate as possible to appropriately protect wildlife
from adverse effects due to chemical exposure but also to protect from habitat destruction due to
remediation based on misunderstanding of critical effect concentrations.
Considerable toxicological information is available on the effects of PCBs and PCDDs on mink,
but limited toxicological information is available for PCDFs. This report compares the toxic
effects reported for laboratory and field studies on mink with both mixed and single dioxin-like
congener exposures and demonstrates that exposure concentrations at which adverse effects
occur cannot be determined reliably for complex mixtures in which PCDFs dominate the total
calculated TEQ values, thereby suggesting that the values of the mammalian-specific TEFs
suggested by the WHO may overestimate the toxic potency of PCDFs to mink.
Table 1. Estimated average first-order elimination rate constants, based on data from both 90and 180-d time points, for 2,3,7,8-TCDF and 4-PeCDF by dose group. N=6 except where noted.
kg-1
d-1)
Daily dose TEQ (ng
2,3,7,8-TCDF
0.98
3.8
20
Mixture: 4.1 TCDF and 2.8 PeCDF (n=5)
2,3,4,7,8-PeCDF
0.62
2.2
9.5
Mixture: 2.8 PeCDF and 4.1 TCDF (n=5)
First order rate
constant, d-1
Mean (S.D.)
Estimated
half-life, d
Mean
1.6 (0.6)
2.6 (0.7)
4.1 (0.6)
4.3 (0.7)
0.43
0.27
0.17
0.16
0.086 (0.012)
0.095 (0.008)
0.087 (0.019)
0.094 (0.008)
8.1
7.3
8.0
7.4
DISCUSSION
In both the Tittabawassee River Field Study and the Laboratory Chronic Exposure to 2,3,7,8TCDF, concentrations of TEQ2006-WHO-mammal to which the mink were exposed exceeded those at
which adverse effects, based on studies with PCDD or PCB congeners, would have been
expected. Yet in both instances where PCDF congeners were the sole or predominant source of
the TEQ2006-WHO-mammal, predicted adverse effects were not observed. The apparent discrepancy
between predicted and observed relative potency for 2,3,7,8-TCDF and mixtures containing
2,3,7,8-TCDF as compared to TCDD- and PCB 126-containing mixtures may be in part due to
dissimilar metabolic transformation and elimination.
Uncertainties associated with the relative potencies of individual components which would
differentially affect mixtures of varying composition can be demonstrated by comparing the data
collected from the 2,3,7,8-TCDF study reported herein to a parallel study, conducted at the same
facility (MSU Experimental Fur Farm) and using the same methodologies, of 3,3’,4,4’,5pentachlorobiphenyl (PCB 126)5 (Table 2).
Table 2. Reproductive outcomes resulting from mink dietary exposure to PCB 126 and 2,3,7,8,TCDF.
Chemical
studied
PCB 1265
2,3,7,8,-TCDF4
Table 3. Effect levels for mink dietary exposure to dioxin-like compounds in ng TEQ2006-WHO-mammal/kg diet, ww.
“It is important that exposure concentrations
at which adverse effects are predicted to occur
be as accurate as possible to appropriately
protect wildlife from adverse effects due to
chemical exposure but also to protect from
habitat destruction due to remediation based
on misunderstanding of critical effect
concentrations.”
METHODS AND MATERIALS
Study
Housatonic River fish lab study8
PCB 126 lab study5
Saginaw River fish lab study9
Saginaw River fish lab study9
Housatonic River fish lab study8
Saginaw River fish lab study9
Housatonic River fish lab study8
Housatonic River fish lab study9
Tittabawassee River wild mink3
2,3,7,8-TCDF lab study4
Three primary studies discussed herein include a three-year field study of mink chronically
exposed to a mixture of PCDFs and PCDDs under natural conditions, a laboratory chronic
exposure study in which mink were exposed to 2,3,7,8-tetrachlorodibenzofuran (2,3,7,8-TCDF)
through diet, and a laboratory evaluation of the toxicokinetics of 2,3,7,8-TCDF and 2,3,4,7,8pentachlorodibenzofuran (4-PeCDF).
Tittabawassee River Field Study3
Forty-eight wild mink, 22 from the study area and 26 from reference areas, were collected
throughout the Tittabawassee River, Midland, Michigan, USA drainage basin during the winters of
2003-2005.
™ Concentrations of dioxin, furan, and dioxin-like PCB congeners were measured in the
dietary items and livers of mink
™ Estimates of the daily dose were created from site-specific dietary composition and
measured dietary item residue concentrations
™ Necropsies included gross and histological examinations, including jaw examination for the
presence of squamous epithelial cell proliferation as described in Beckett et al.6
Laboratory chronic exposure to 2,3,7,8-TCDF4
This laboratory study was designed to determine the toxic effects threshold for mink exposed to
2,3,7,8-TCDF through the diet.
™ Thirty 10-m old adult female mink (P0) were fed diets containing 0.0 (Control), 240, or 2400
ng 2,3,7,8-TCDF/kg feed on a wet-weight (ww) basis (0, 26, and 240 ng TEQ2006-WHO-mammal
/kg, respectively)5
™ Dietary exposure was started 3 wk prior to the initiation of breeding
™ Adults and kits were examined for sub-lethal effects including kit growth, organ masses,
and tissue histology
™ Necropsies included gross and histological examinations, including jaw examination for the
presence of squamous epithelial cell proliferation
Laboratory toxicokinetic evaluation of 2,3,7,8-TCDF and 4-PeCDF7
A controlled laboratory feeding study was performed to determine the toxicokinetics of 2,3,7,8TCDF and 2,3,4,7,8-PeCDF using mink as a mammalian model.
™ Mink were exposed to three concentrations each of the congeners and to a binary mixture
of the two congeners through the diet (Table 1)
™ Three animals from each of the dose groups were sampled on day 90 and 180
™ 2,3,7,8-TCDF and 4-PeCDF residues were measured in liver, adipose, and scat
™ Necropsies included gross and histological examinations, including jaw examination for the
presence of squamous epithelial cell proliferation
™ CYP1A1 and CYP1A2 enzyme activities were measured
Sum
TEQs
50
24
57
36
12
22
6.8
4.3
31
240
PCB
126
TEQs
41
24
19
11
9.8
7.2
5.4
3.2
2.4
0
2,3,7,8
TCDF
TEQs
0.3
0
2.1
0.9
0.1
0.7
0.1
0.1
8.8
240
% Jaw
lesions
100% (6/6)
80% (12/15)
75% (6/8)
57% (4/7)
33% (2/6)
0% (0/8)
17% (1/6)
0% (0/6)
0% (0/22)
0% (0/8)
Dioxin
TEQs
0.9
0
20
14
0.3
11
0.3
0.3
4.4
0
Furan
TEQs
1.9
0
14
8.8
0.5
3.0
0.3
0.3
22
240
RESULTS
Study3
Tittabawassee River Field
A mink hazard assessment based on concentrations of furans, dioxins, and PCBs in site-specific
dietary items from the Tittabawassee River, and toxicity reference values (TRVs) derived from
mixtures of other Ah-R active compounds resulted in values of hazard quotients (HQ) that were
greater than 1.0, which suggested potential adverse effects for mink3. However, there were
no statistically significant differences in any of the measured parameters between mink
exposed to a median estimated dietary dose of 31 ng TEQ2006-WHO-mammal/kg ww, and mink from an
upstream reference area where they had a median dietary exposure of 0.68 ng TEQ2006-WHOmammal/kg ww. Surveys of the conditions of individual mink, and the mink population, including track
surveys, trapping, age distributions and sex ratios indicated that the mink population was not being
adversely impacted. 75% of the 31 ng TEQ2006-WHO-mammal/kg, ww in the mink diet were due to
PCDFs with a majority of that originating from TCDF (31%) and 4-PeCDF (37%)3.
Laboratory chronic exposure to 2,3,7,8-TCDF4
Similarly, chronic exposure of mink to TCDF concentrations as great as 2400 ng TCDF/kg ww feed
(240 ngTEQ2006-WHO-mammal/kg ww feed) exhibited transient decreases in body masses of kits
relative to the controls as the only statistically significant effect observed.
Laboratory toxicokinetic evaluation of 2,3,7,8-TCDF and 4-PeCDF7
The laboratory study of the toxicokinetics of 2,3,7,8-TCDF and 4-PeCDF in mink demonstrated that
2,3,7,8-TCDF is quickly metabolized relative to 4-PeCDF7(Table 1).
“The apparent discrepancy between
predicted and observed relative potency
for 2,3,7,8-TCDF and mixtures containing
2,3,7,8-TCDF as compared to TCDD- and
PCB 126-containing mixtures may be in
part due to dissimilar metabolic
transformation and elimination.”
Non-ortho PCB
TEQs
44
24
20
12
10
7.2
5.8
3.4
2.5
0
Concentration in
diet
Reproductive outcome
240 TEQ/kg diet
Complete reproductive failure
240 TEQ/kg diet Whelping rate not different from
control (80%)
The most comprehensive comparison of mixture and congener toxicological
potency can be made by comparing all of the available dose response
relationships between concentrations of TEQ and occurrence of squamous
epithelial cell proliferation or jaw lesions. Jaw lesions are a sensitive response
of mink to 2,3,7,8-TCDD, PCB 126, and mixtures of dioxin-like compounds.
The environmental mixtures that resulted in jaw lesions had great proportions
of non-ortho PCBs, specifically, PCB 126 (Table 3). There was no clear
relationship between the presence or frequency of jaw lesions and the total
concentration of TEQ2006-WHO-mammal, contributed by PCDD or PCDF, 2,3,7,8TCDF or mono-ortho PCBs. This does not mean that there is not a dose
response for these compounds but rather the data set is limiting.
CONCLUSION
The results of these studies suggest that the values of the mammalian-specific TEFs suggested
by the WHO overestimate the toxic potency of PCDFs to mink. Therefore, hazard cannot be
accurately predicted by making comparisons to TRVs derived from exposure studies conducted
with PCBs or PCDDs in situations where mink are exposed to TEQ mixtures dominated by
PCDFs.
ACKNOWLEDGEMENTS
Funding for the field study described herein was provided through an unrestricted grant from The
Dow Chemical Company to Michigan State University. The laboratory study of chronic exposure
to 2,3,7,8-TCDF was funded in part by a grant from the Michigan Great Lakes Protection Fund.
The toxicokinetic study was funded and supported by The Dow Chemical Company.
REFERENCES
1. Basu N., Scheuhammer A.M., Bursian S.J., Elliott J., Rouvinen-Watt K. and Chan H.M.
Environ Res 2007; 103:130-144.
2. Kannan K., Blankenship A.L., Jones P.D. and Giesy J.P. Hum Ecol Risk Assess 2000; 6:181201.
3. Zwiernik M.J., Kay D.P., Moore J., Beckett K.J., Khim J.S., Newsted J.L., Roark S. and Giesy
J.P. Environ Toxicol Chem 2008; 27: 2076-2087.
4. Zwiernik M.J., Beckett K.J., Bursian S., Kay D.P., Holem R.R., Moore J., Yamini B. and Giesy
J.P. Integrated Environ Assess Manag Accepted.
5. Beckett K.J., Yamini B. and Bursian S.J. Arch Environ Contam Toxicol 2008; 54:123-129.
6. Beckett K.J., Millsap S.D., Blankenship A.L., Zwiernik M.J., Giesy J.P. and Bursian S.J.
Environ Toxicol Chem 2005; 24:674-677.
7. Zwiernik M.J., Bursian S., Alyward L., Kay D.P., Moore J.N., Rowlands C., Woodburn K.,
Shotwell M., Khim J.S., Giesy J.P. and Budinsky R.A. Toxicol Sci 2008; 105: 33-43.
8. Bursian S.J., Sharma C., Aulerich R.J., Yamini B., Mitchell R.R., Beckett K.J., Orazio C.E.,
Moore D., Svirsky S. and Tillitt D.E. Environ Toxicol Chem 2006; 25:1541-1550.
9. Bursian S.J., Beckett K.J., Yamini B., Martin P.A., Kannan K., Shields K.L. and Mohr F.C.
Arch Environ Contam Toxicol 2006; 50:614-623.
A comparison of methods for estimating wildlife dietary exposure concentration
using measured concentrations of dietary items
Shaun A. Roark1, Denise P. Kay1, John. L. Newsted1, Matthew J. Zwiernik2, and John P. Giesy2,3
1Entrix,
Inc, Okemos, MI, USA, 2Michigan State University, East Lansing, MI., USA., 3University of Saskatchewan, Saskatoon, Saskatchewan, Canada.
Approach 1: Descriptive statistics for each category
ng TEQ/kg ww
1e+02
Median of each category
Concentrationdiet Equation
25 ng TEQ/kg wet wt food
Mean of each category
Concentrationdiet Equation
31 ng TEQ/kg wet wt food
1e+00
95 UCL of mean of each category
Concentrationdiet Equation
34 ng TEQ/kg wet wt food
1e-01
95th centile of each category
Concentrationdiet Equation
71 ng TEQ/kg wet wt food
Max of each category
Concentrationdiet Equation
104 ng TEQ/kg wet wt food
1e+01
Crayfish
Fish
Frog
Muskrat
Plant
Shrew Sm. Mammal
1. Summary statistics calculated independently for each category of dietary item
2. Each summary statistic input (mean, median, etc.) into dietary concentration equation as Cdiet(i)
3. Result from equation for each summary statistic used to represent dietary concentration
6
4
Median of each category
Concentrationdiet Equation
2
Mean of each category
Concentrationdiet Equation
0
95 UCL of mean of each category
Concentrationdiet Equation
95th centile of each category
Concentrationdiet Equation
Max of each category
Concentrationdiet Equation
-2
Crayfish
Fish
Frog
Muskrat
Plant
Shrew Sm. Mammal
METHOD
Compare several approaches to characterize dietary TEQ concentration using
measured dietary item TEQ concentration data and dietary proportions
⎛ N
⎞
Concentrationdiet = ⎜ ∑ (C diet(i) x Pi )⎟
⎝ i =1
⎠
Fraction of mink diet
Number of samples
Crayfish
0.08
15
Forage fish (composites)
0.52
7
Frog
0.04
106
0.19
13
Plant
0.09
25
Shrew
0.0048
35
Small Mammal
0.0752
38 ng TEQ/kg wet wt food
68 ng TEQ/kg wet wt food
104 ng TEQ/kg wet wt food
Approach 3: Iteratively sampled measured values
1. One measured value randomly sampled from each dietary category (i.e., fish, frog, etc.)
2. Each value weighted by dietary proportion (percentage of diet)
3. Weighted values summed to estimate one possible dietary concentration
4. Random sampling repeated ~10,000 times to generate the distribution of potential dietary
concentrations
128
Dietary items were sampled from multiple locations on the Tittabawasse River, MI, USA. Dietary
proportions were based on site-specific data for the mink on the Tittabawassee River.
Approach
Median
Mean
95%UCL
(of mean)
95th centile
Maximum
1
2
3
4
25
25
29
29
31
21
31
31
45
38
31
31
71
68
54
54
104
104
82
119
1e+02
1e+01
1e+00
1e-01
Crayfish
1500
RESULTS
• Approaches 1 and 2 yielded similar estimates for quantiles (median and 95th centile) of
data, but had lesser agreement for estimates based on mean and variation
• Approaches 3 and 4 resulted in nearly identical descriptions of dietary concentration
except for the the maximum
• Approach 3 did not reach the “true” maximum based on measured data even using
10,000 iterations
• Approach 4, which used unbounded distributions for 5 of 7 categories, somewhat
overestimated the “true” maximum based on measured data.
Approaches 1 and 2
• Very easy to implement, conceptually simple, commonly used
• Summary statistics require assumptions about distributions of data
• No valid approach to estimating variation around dietary concentration estimates
• Equation results based on summary statistics for each category has unclear relationship
to “true” dietary concentration
1e+03
Histogram of concentration in diet (Approach 3)
Fish
Frog
Muskrat
Plant
Shrew
Sm. Mammal
1000
Approach 3
• Requires no assumptions about distribution of data in each category
• Results in readily described and visualized distribution of potential dietary concentration
• Accurately estimates dietary concentrations if sample size is sufficient in all categories
• More difficult to implement; may require software such as Crystal Ball, R, or SAS
Median: 29 ng TEQ/kg wet wt food
Approach 4
• More complicated to implement than Approach 3 due to distribution-fitting
• Distribution-fitting may not be possible for some data (small N or bimodal distribution)
• Use of unbounded distribution has potential for a large overestimate – athough this did
not occur here with 10,000 randomly drawn samples from each fitted distribution.
Mean: 31 ng TEQ/kg wet wt food
500
95th centile: 54 ng TEQ/kg wet wt food
Maximum: 82 ng TEQ/kg wet wt food
0
40
60
80
Dioxin & Furan TEQ, ng/kg ww food
Histogram of bootstrapped medians
Bootstrapping used to estimate confidence on median and mean
• 5000 replicates of the dietary distribution were generated
• Quantiles were estimated (2.5% and 97.5%) for the median and mean of the dietary
concentration
• All data were used in each bootstrap replicate
Histogram of bootstrapped means
800
1000
800
600
400
200
0
600
400
200
0
28.0
28.4
28.8
29.2
30.4
Median TEQ, ng/kg ww food
30.6
30.8
31.0
31.2
Mean TEQ, ng/kg ww food
1e+03
ng TEQ/kg ww
1. Distribution (i.e., gamma, lognormal) was fitted to data in each category with ≥ 15 samples
• Best-fit distribution selected with Crystal Ball for categories
• Crayfish and frog: lognormal distribution.
• Plant, shrew, and small mammal: gamma distribution
• Komolgorov-Smirnov tests using R indicated raw data were not different from fitted
distribution (P>0.05)
2. Randomly sampled one item from each category distribution (i.e., fish, frog, shrew, etc.)
3. Weighted each concentration by dietary proportion (percentage of mink diet)
4. Summed weighted concentrations to estimate the possible dietary concentration
5. Repeated random sampling with replacement 10,000 times to generate distribution of potential
dietary concentrations
1e+00
1e-03
1e-06
Crayfish
Fish
Frog
Muskrat
Plant
Shrew Small Mammal
Histogram of concentration in diet (Approach 4)
Number of occurances
Mink dietary proportions and sample size for each dietary category
Muskrat
21 ng TEQ/kg wet wt food
Approach 4: Distributions fit to categories and iteratively sampled
C = chemical concentration (soil, sediment, diet)
Cdiet = chemical concentration in each dietary item (i)
Pi = proportion of dietary item (i) in the diet
Dietary Item Category
25 ng TEQ/kg wet wt food
back-transform
log(ng TEQ/kg ww)
8
20
OBJECTIVE
Find best method to estimate dietary concentration using a robust set of
measured concentrations in dietary items from multiple dietary categories
Summary of results from each approach (ng TEQ/kg ww food).
Approach 2: Same as approach 1, but with log-transformed data
ng TEQ/kg ww
INTRODUCTION
• Use of measured concentrations of dietary items can reduce uncertainty in
dietary exposure estimates compared with estimates modeled from soil and
sediment
• U.S. EPA Guidance on exposure characterization (USEPA 1992, 1997)
recommends using exposure estimates based on central tendency (CT) and
reasonable maximum (RM) exposure
• Identifying the most accurate approach to incorporate measured data into a
complex diet, such as that of the American mink, is challenging because:
• Sample size varies among categories of dietary items
• Multiple species included in some categories of dietary items
• Statistical distribution of data in categories of dietary items is difficult to
characterize
1. Summary statistics calculated independently for each category of dietary item
2. Each summary statistic input (mean, median, etc.) into dietary concentration equation as Cdiet(i)
3. Result from equation for each summary statistic used to represent dietary concentration
1e+03
Number of occurances
ABSTRACT
In ecological risk assessment, US EPA guidance recommends characterizing exposure with
measures of central tendency (CT) and reasonable maximum (RM). However, the choice of
parameters to represent these measures differs depending on specific guidance and on
characteristics of the data. To address this issue, methods were compared for estimating parameters
to describe dietary concentration based on measured concentrations of dioxin and furan TEQ (WHO
2006) in dietary items for mink on the Tittabawassee River (Michigan, USA). The first approach was
to estimate each parameter (median, mean, 95% upper confidence limit of the mean (95%UCL), 95th
centile, and maximum) for each dietary category, then weight by dietary proportion, and finally sum
each parameter across categories. The second approach was similar to the first, but raw data were
log-transformed. The third approach was to iteratively sample (with replacement) one item from each
category, weight by dietary proportion, and sum. This method generates the distribution of possible
dietary concentrations without assumptions about the distribution of the data in each category. The
fourth approach, a variation of the third, was to fit a distribution to the data in each dietary category
with n≥15, then randomly sample from each distribution, weight by dietary proportion, and sum.
Where n<15 in a category (two occurrences here), the data were sampled iteratively, as in the third
approach. Using the first and second approaches, the median, mean, 95%UCL, 95th centile, and
maximum dietary concentrations were 25, 31, 45, 71, and 104 ng TEQ/kg and 25, 21, 38, 68, and
104 ng TEQ/kg, respectively. Using the third and fourth approaches, the median, mean, 95%UCL,
95th centile, and maximum were 29, 31, 31, 54, 82 ng TEQ/kg and 29, 31, 31, 54, and 119 ng
TEQ/kg, respectively. The results for mean and 95%UCL of the first and second approaches were
strongly influenced by the log-transformation. The results of the third and fourth approaches were
similar, but use of unbounded distributions in fourth approach overestimated the maximum. The
iterative sampling approaches do not require making assumptions about the distribution of the data in
each dietary category, and unlike the first two approaches, the resulting distribution of the dietary
concentration can be readily described and presented graphically.
Median:
29 ng TEQ/kg wet wt food
Mean:
31 ng TEQ/kg wet wt food
95th centile:
54 ng TEQ/kg wet wt food
Maximum:
119 ng TEQ/kg wet wt food
1500
1000
500
Histogram of bootstrapped medians
0
20
40
60
80
100
Dioxin & Furan TEQ, ng/kg ww food
1000
800
600
400
200
0
600
400
200
0
28.5
Bootstrapping was used to estimate confidence on median and mean
• 5000 replicates of the dietary distribution were generated as described above in Approach 3
Histogram of bootstrapped means
800
29.0
29.5
Median TEQ, ng/kg ww food
30.6
30.8
31.0
31.2
31.4
Mean TEQ, ng/kg ww food
DISCUSSION
• To estimate exposure using measured concentrations in dietary items, the choice of
the best approach to describe CT and RM exposure can be unclear, yet may influence
conclusions regarding risk.
• It is difficult to accurately characterize variation in the dietary concentration estimate
based on summary statistics for each dietary category (Approaches 1 & 2).
• The use of a resampling procedure (Approach 3) avoids assumptions about the
distribution of the data, and therefore may provide the best characterization dietary
concentration based on measured tissue concentrations in dietary items.
• Use of unbounded Monte Carlo procedures (Approach 4) can overestimate the
maximum but the 95th centile is representative of reasonable maximum.
• Use of resampling procedures produces a concentration distribution that is easy to
describe and present graphically.
QUESTIONS FOR FURTHER STUDY
• Concentrations for fish used here were based on composite samples – would the use
of individual samples change the results?
• The data set used here is robust – what is the effect of reduced sample size on
accuracy and uncertainty of these approaches?
• Is the gain in apparent accuracy of the resampling approach outweighed by the
uncertainty in other parts of the model (e.g. dietary proportions, groupings of species
in dietary catgories)?
REFERENCES
1. US EPA. 1997. Ecological risk assessment guidance for superfund: process for designing and
conducting ecological risk assessments - interim final. EPA 540-R-97-006.
2. US EPA. 1992. Framework for ecological risk assessment. EPA/630/R-92/001.
3. Crystal Ball 7 software. http://www.oracle.com/crystalball/index.html
4. R: A Language and Environment for Statistical Computing. http://www.R-project.org
5. Zwiernik, M.J., D.P. Kay, J. Moore, K.J. Beckett, J.S. Khim, J.L. Newsted, S.A. Roark, and J.P.
Giesy. 2008. Exposure and effects assessment of resident mink (Mustela vison) exposed to
polychlorinated dibenzofurans and other dioxin-like compounds in the Tittabawassee River Basin,
Midland, Michigan, USA. Environmental Toxicology and Chemistry 27(10):2076-2087.
ACKNOWLEDGEMENTS
AJ Bailer provided an initial statistical consultation and samples of R code for resampling data.
SETAC 29th Annual Meeting, November 16-20, 2008, Tampa, FL, USA
An evaluation of 2,3,7,8-tetrachlorodibenzo-p-dioxin equivalents in
tissues of wild game from the floodplains of the Tittabawassee and
Saginaw Rivers (MI, USA)
Ryan R. Holem1, John J. Matousek1, Patrick W. Bradley1, John L. Newsted1, Denise P. Kay1, Alan L. Blankenship1,2, Shaun A. Roark1, Melissa S. Shotwell1, and John P. Giesy3
Inc., Okemos, MI, USA; 2Univ. of Michigan, Ann Arbor, MI, USA; 3Dept. Biomedical Sciences and Toxicology Centre, Univ. of Saskatchewan, Saskatoon, Saskatchewan, CA.
Abstract
The Tittabawassee River is located in central Michigan and flows southeast
through Midland and into the Saginaw River and eventually to the Saginaw Bay
of Lake Huron. Previous studies have reported elevated soil, sediment, and
fish concentrations of dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs)
downstream of Midland. In addition, elevated polychlorinated biphenyl (PCB)
concentrations have been found in soils, sediments, and fish from the Saginaw
River. It was suspected that wild game residing in this area may contain
detectable concentrations of these contaminants. To evaluate this, whitetailed deer, wild turkey, fox squirrel, cottontail rabbit, Canada goose, and wood
duck were collected from several locations along the Tittabawassee and
Saginaw Rivers (Figure 1). Edible tissues from these animals were analyzed
for PCDDs and PCDFs, and some were also analyzed for dioxin-like PCBs.
Results based on concentrations of 2378-tetrachlorodibenzo-p-dioxin
equivalents (2006 WHO TEQs) have been summarized and are reported in
this poster.
Figure 3. Total TEQ in edible tissues of birds: skin-on vs. skin-off
ng TEQ/kg, wet weight (ND = 0, 2006 WHO TEFs)
1ENTRIX,
Table 1. Wild game collection summary - 237 animals
Species
SF
SC
IPA
SNWR
CISGA
White-tailed deer
Wild turkey
Cottontail rabbit
Fox squirrel
Wood duck
Canada geese
13
12
12
-
10
11
10
-
10
12
11
10
-
11
8
10
12
12
12
11
2
12
12
12
12
Rationale
•
•
N
•
(Lake Huron)
SF
Saginaw River
•
IPA
2
SC
TEQ, ND=1/2 DL) were observed in muscle tissue from deer, rabbit, and squirrel
TEQ concentrations were greatest in livers of white-tailed deer and skin-on tissues
of wild turkey and wood duck (range of means: 0.2 – 43 ng/kg ww D/F TEQ,
ND=1/2DL)
In birds, TEQ concentrations in skin-off samples were less than in skin-on samples
(Figure 3)
The greatest TEQ concentrations were observed in tissues of animals from the
central sampling location, IPA (Figure 2, 4)
D/F TEQ values for deer muscle and liver, squirrel, and turkey were comparable
(mean TEQ values ≤ 0.5 ng TEQ/kg, ND=1/2 DL) in animals from the upstream
reference (SF) and downstream (CISGA) sampling locations (Figure 2, 4)
PCB contribution to total TEQ was minimal with the exception of wood duck tissues
in which PCB contribution to total TEQ ranged from 30-50% (Figure 3, ND=0)
CISGA
5
ng TEQ/kg, wet weight (ND = ½ DL, 2006 WHO TEFs)
•
1
5
4
3
2
1
skin-off
skin-on
skin-off
skin-on
skin-off
skin-on
Wild turkey
Canada goose
Wood duck
(Locations 2-5)
(Locations 4&5)
(Locations 4&5)
Results
• Overall, the least TEQ concentrations (range of means: 0.1–0.9 ng/kg ww D/F
and PCBs had not been evaluated
Saginaw Bay
6
Figure 4. PCDD/PCDF TEQ in edible tissues of rabbit and squirrel
the Tittabawassee and Saginaw River floodplains
Figure 1. Sampling locations along the Tittabawassee and
Saginaw Rivers
PCB TEQ
Dioxin and Furan TEQ
7
0
• Wild game species commonly pursued by hunters can be found throughout
• The degree of exposure of floodplain-residing wild game to PCDDs, PCDFs,
8
3
Cottontail rabbit
Fox squirrel
2
1
0
SF*
SC*
IPA
SNWR
Upstream
CISGA
Downstream
*Cottontail rabbits not targeted at this location
3
Tittabawassee River
4
SNWR
Discussion & Conclusions
• Average total TEQ concentrations in wild game tissues were less than
Figure 2. PCDD/PCDF TEQ in white-tailed deer livers and skin-on
wild turkey tissue
•
•
•
•
(SF)-Sanford –upstream reference area
2
(SC)- Smith’s Crossing
3
(IPA)- Imerman Park Area
4
(SNWR)- Shiawassee National Wildlife Refuge
5
(CISGA)- Crow Island State Game Area
Methods
Animals were collected from locations upstream and downstream of
Midland (Figure 1) by use of traps and firearms in 2003 and 2007
Whole animals were removed from collection locations (i.e., entrails
removed at laboratory) to prevent contamination from other media such as
floodplain soils
Tissues commonly consumed by humans were removed from each animal
and freeze-fractured (i.e., cryogenic homogenization)
Tissue samples were analyzed for 17 PCDD/PCDFs; some also for 12
dioxin-like PCBs
TEQ concentrations (2006 WHO TEFs) were compared between species,
locations, and tissue types
100.0
ng TEQ/kg, wet weight (ND=1/2 DL, 2006 WHO TEFs)
•
1
O
O
50.0
Deer liver
Turkey skin-on
O
O
O
O
O
O
O
10.0
O
O
O
O
O
O
O
O
O
O
O
5.0
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
1.0
O
O
O
O
O
0.5
O
O
O
O
O
O
O
0.1
O
O
References
O
O
O
O
Hilscherova,K., Kannan,K., Nakata,H., Yamashita,N., Bradley,P., McCabe,J., Taylor,A.B., and Giesy,J.P. 2003.
Environmental Science and Technology 37: 468-474
O
O
O
O
O
O
MDEQ. 2002. Baseline Chemical Characterization of Saginaw Bay Watershed Sediments. August 29, 2002
O
MDEQ. 2003. Final report - Phase II Tittabawassee/Saginaw River dioxin flood plain sampling study. June 2003
O
O
O
O
O
SF
Upstream
O
O
O
O
O
O
average total TEQ in fillets of many species of fish collected from the
Tittabawassee and Saginaw Rivers
• Wood ducks and wild turkeys commonly feed on invertebrates, which likely
increases exposure to PCDDs, PCDFs, and PCBs through ingestion of
sediment/soils
• Greater concentrations observed in skin-on bird tissues compared to skinoff due to the high lipid content of skin
• Age, diet, and site fidelity likely key factors contributing to exposure of wild
game to PCDDs, PCDFs, and PCBs
O
SC
IPA
SNWR
Van den Berg,M.; Birnbaum,L.S.; Denison,M.; De Vito,M.; Farland,W.; Feeley,M.; Fiedler,H.; Hakansson,H.; Hanberg,A.;
Haws,L.; Rose,M.; Safe,S.; Schrenk,D.; Tohyama,C.; Tritscher,A.; Tuomisto,J.; Tysklind,M.; Walker,N.; Peterson,R.E. 2006.
Toxicological Sciences 92(2), 223-241.
CISGA
Downstream
Acknowledgements
This work was funded by The Dow Chemical Company
Effects of TCDD, TCDF, and PeCDF Injected Into the Air Cell of Japanese Quail (Coturnix japonica) Prior to Incubation
A. M. CohenCohen-Barnhouse1, S. J. Bursian1, J. E. Link1, J. P. Giesy2, P. D. Jones2, Y. Wan2, S. Wiseman2, S. W. Kennedy3, J. Newsted4, M. J. Zwiernik5
of Animal Science, Michigan State University, East Lansing,
Lansing, MI, USA. 2Department of Veterinary Biomedical Sciences and Toxicology Centre,
Centre, University of Saskatchewan, Saskatoon, SK, Canada.
3National Wildlife Research Centre, Environment Canada, Ottawa, ON,
ON, Canada. 4Entrix, Okemos, MI, USA.
o Egg injection studies were conducted to confirm proposed avian sensitivity
classification using TCDD, 2,3,7,8-tetrachlorodibenzofuran (TCDF) and
2,3,4,7,8-pentachlorodibenzofuran (PeCDF).
Objective
16
7.26
0.888
10
TCDF = 3.31
(n
0
< 4 g/
g
0.312
eg
g)
3.8
20
LD-50
PeCDF = 0.85
4‐7 7‐10
10‐14 14 <
Age of em
bryo (d)
0
1.808
3.8
3.84
7.26
7.58
B
91.00
8.92
12.00
37.00
0.92
1.82
7.58
3.80
3.84
7.26
0.14
0.89
3.56
egg)
Number observed
Limb Deformities
C
3
egg)
7.13
8.13
F
9.13
4.13
5.13
Dose (ng
/g
6.13
1.13
0.13
12.00
37.00
0.92
0
2.13
2
0
egg)
D
4
3.13
Number observed
5
1
Dose (ng
/g
TCDF
TCDD
PeCDF
6
Dose
Group
40
0
35
0.128
30
0.192
25
0.488
G
o There were no consistent changes in body mass or relative organ mass in TCDD-, PeCDF- and TCDFexposed quail when compared to vehicle control
Conclusions
o The relative potencies (RePs) of TCDF and PeCDF compared to TCDD were 3.4 and 13.3, respectively
0.888
20
9.42
4.6
2.42
15
10
0.888
5
0.192
0
< 4 Figure 3: Deformities observed in quail embryos exposed to 2,3,7,8-TCDD, 2,3,4,7,8PeCDF, or 2,3,7,8-TCDF. Cranial deformities (top left) included exencephaly (A) and
microphthalmos (B) or anophthalmos (C); bill deformities (top right) included incomplete
(D) or lack of upper or lower beak development (E) and crossbill (F); trunk deformities
(bottom left) included gastroschisis; and limb deformities (bottom right) included club foot
and curled toes (G).
16
TCDF Embryo
Mortality
TCDD = 11.25
E
1
0.888
40
30
Results
2
1
91.00
50
2
8.92
0.624
3
1.82
0.312
60
4
3.56
0.144
70
5
0.16
0
80
TCDD
PeCDF
TCDF
6
0.24
91
Dose
Group
90
Tissue weighing
3
16.00
eg
g)
37
Do
se
Chick necropsy
o 12-d-old chicks
ƒ Weighed
ƒ Tissues removed and weighed
• Liver
- CYP1A 4 and CYP1A 5 mRNA
abundance (Wiseman et al.,
WP229)
• Brain, heart, bursa and spleen
4
Dose (ng
/g
Trunk Deformities
12
14 <
PeCDF Embryo
Mortality
Incidence
Tagged 12-d-old chick
egg)
A
8.92
o Hatchlings transferred to battery
Eggs in incubator tray
Dose (ng
/g
TCDD
PeCDF
TCDF
5
0
0.40
0
1.824
Number observed
4‐7 7‐10
10‐14
Age of em
bryo (d)
g/
g
0.16
0
< 4 1
3.56
(n
Sealing injection site with
melted paraffin
5
Do
se
Injection with positive
displacement pipettor
0.4
2
Bill Deformities
6
0
0.92
8.92
1.824
10
3
0.62
15
o Unhatched eggs (d 20) opened to
determine:
ƒ Age of embryo at death
ƒ Presence and type of deformities
Number observed
0.4
37
4
1.81
20
5
0.07
Incidence
0.24
PeCDF
TCDD
TCDF
6
0.31
0.16
25
4‐7 7‐10
10‐14 14 <
Age of em
bryo (d)
0
eg
g)
o Avian species classification model based on three TCDD-sensitivity
categories:
ƒ Most sensitive (Chicken)
ƒ Moderately sensitive (Ringneck pheasant)
ƒ Least sensitive (Japanese quail)
0.072
30
1.472
2.42
o The ReP of PeCDF based on hatchability of Japanese quail eggs is similar to an ReP value of 13-30
based on induction of CYP1A activity in cultured Japanese quail hepatocytes determined in a
complimentary study (Herve et al., MP33)
2.62
g/
g
o Differences have been attributed to amino acid substitutions in the ligandbinding domain of the aryl hydrocarbon receptor (AhR; Farmahin et al., MP36).
o Eggs incubated for 19 d (37.6°C, 52%
humidity)
0
35
(n
o A molecular basis for differences in sensitivity to 2,3,7,8- tetrachlorodibenzop-dioxin (TCDD)-like compounds among avian species has been suggested.
Injection site over air cell
Cranial Deformities
Dose
Group
Do
se
Introduction
Drilling shell using Dremel® tool
o Injection of 0.1 µl/g egg into the air cell of
Japanese quail eggs with:
ƒ Triolein
ƒ TCDD – 11 doses (0.072 to 91 ng/g)
ƒ PeCDF – 10 doses (0.144 to 16 ng/g)
ƒ TCDF – 10 doses (0.128 to 9.42 ng/g)
Results
TCDD Embryo
Mortality
0.07
0.16
0.24
0.40
Methods
Abstract
Amino acid substitutions in the ligand-binding domain of the aryl hydrocarbon
receptor (AhR) have been proposed to determine, as the molecular basis for
differential sensitivity of birds to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-like
compounds (Farmahin et al., (MP36). The results of recent studies have
suggested that birds can be classified into one of three TCDD-sensitivity
categories: very sensitive (chicken), moderately sensitive (ringneck pheasant),
and sensitive (Japanese quail). A series of egg injection studies are being
conducted to confirm the proposed avian sensitivity classification. The effect of
TCDD,
2,3,7,8-tetrachlorodibenzofuran
(TCDF)
and
2,3,4,7,8pentachlorodibenzofuran (PeCDF) on hatchability of Japanese quail eggs and
growth and survival of hatchlings is reported here. Doses ranging from 0.07 to
91 ng/g, ww egg were injected into the air cell prior to incubation. Hatchlings
were maintained for 12 d to assess growth and survivability. A sample of the
chicks was weighed, euthanized, and necropsies conducted on the day of
hatching and at 12-days of age. Selected tissues from 12-day-old chicks were
removed, weighed, and processed for histological assessment. Subsamples of
liver were processed for determination of concentrations of the target
compounds as well as induction of cytochrome P4501A activity. LD-50 values
based on hatchability data were determined to be 11.25 (0 – ∞; 95% confidence
intervals), 3.31 (2.02 – 5.75) and 0.85 (0.27 – 1.53) ng/g, ww egg, for TCDD, TCDF,
and PeCDF, respectively. The relative potencies (RePs) of TCDF and PeCDF
compared with TCDD were 3.4 and 13.3, respectively. The ReP of PeCDF based
on hatchability of Japanese quail eggs is similar to a ReP value of 13-30 based
on induction of CYP1A activity in cultured Japanese quail hepatocytes
determined in a complimentary study. To our knowledge, this is the first in vivo
study indicating that a TCDD-like compound is substantially more toxic to birds
than TCDD. It would be of interest to determine if dietary exposure of Japanese
quail to TCDD-like compounds results in similar ReP values as determined in the
enzyme induction and egg injection studies.
Incidence
1Department
4.6
7.22
o To our knowledge, this is the first in vivo study indicating that a TCDD-like compound is substantially
more toxic in an avian species than TCDD
9.42
To develop and confirm toxicity reference values (TRVs) for Japanese quail Figure 1: Mortality of quail eggs injected with 2,3,4,7,8-PeCDF, 2,3,7,8-TCDF, or 2,3,7,8- Figure 2: Age of death of embryos exposed to 2,3,7,8-TCDD
TCDD. Probit analysis conducted with doses beginning with the highest dose below the
(Coturnix japonica) exposed to TCDD, TCDF and PeCDF.
(top), 2,3,4,7,8-PeCDF (middle), or 2,3,7,8-TCDF (bottom).
point of inflection.
Funding for this project was provided through a non-restricted grant from The Dow Chemical Co., Midland, MI.
Poster Number: WP210
Multiple Lines of Evidence Risk Assessment of Great Horned Owls (Bubo virginianus) exposed to
PCDF/DDs in Midland, MI
National Food Safety and Toxicology Center
1
1
1
1
1
1
1
2
2
1,3
Coefield, S.J. , Zwiernik, M.J. , Fredricks, T.B. , Seston, R.M. , Nadeau, M.W. , Tazelaar, D.L. , Moore, J.N. , Shotwell, M.S. , Kay, D.P. , Giesy, J.P.
1. Michigan State University, East Lansing, MI, USA. 2. ENTRIX, Inc. East Lansing, MI, USA. 3. Veterinary Biomedical Sciences & Toxicology Centre, University of
Saskatchewan, Saskatoon, SK, Canada.
ABSTRACT
The great horned owl (Bubo virginianus), a non-migratory top predator, was chosen as a terrestrialbased indicator species for the ecological risk assessment of the Tittabawassee River and floodplain.
The Tittabawassee River floodplain downstream of Midland, MI has elevated levels of polychlorodibenzofurans and polychlorodibenzo-p-dioxins (PCDF/DDs) in the sediments and soils. A multiple
lines of evidence approach was utilized to determine the risk posed to great horned owls (GHOs) in
the floodplain by examining dietary exposure, tissue-based exposure, and population health. The
site-specific dietary exposure was determined by analyzing prey remains found in and around GHO
nests and reconstructing the dietary composition. Dietary items identified in the prey remains were
then collected and analyzed for contaminant concentrations. The average daily intake (ADI) of
PCDF/DD was predicted by use of a weighted average dietary concentration expressed as 2,3,7,8
tetrachlorodibenzo-p-dioxin equivalents (TEQs). The geometric mean GHO ADI was 4.5 x100ng
TEQWHOAvian/kg/day in the study area and 7.0x10-2 ng TEQWHOAvian/kg/day in the reference areas.
For the tissue-based exposure plasma samples were collected from adult and nestling GHOs. The
geometric mean concentrations of PCDF/DDs in nestling plasma were 2.7x100ng TEQWHOAvian/L in
the study area and 5.9x10-1ng TEQWHOAvian/L in the reference areas. The mean concentrations of
PCDF/Ds in adult plasma were 8.4x100ng TEQWHOAvian/L in the study area and 3.1x100ng
TEQWHOAvian/L in the reference areas. Hazard quotients (HQs) for the least observable adverse effect
concentration (LOAEC) for PCDF/DDs in plasma were <1 for all GHO plasma samples. The great
horned owl productivity and relative abundance was greater in the study area than in the reference
areas, indicating the elevated PCDF/DD concentrations in the diet and tissues did not result in population level effects.
INTRODUCTION
The Tittabawassee River floodplain soils and sediments in Midland, MI are contaminated with elevated levels of PCDF/DDs. As non-migratory tertiary predators, great
horned owls (Bubo virginianus; GHOs) have the potential to be exposed to high levels
of these contaminants in the terrestrial ecosystem. The Aquatic Toxicology Laboratory
at Michigan State University chose the GHO as an indicator species in a multiple lines
of evidence risk assessment of PCDF/DD contamination in the floodplain. The risk assessment examines dietary exposure, tissue-based exposure, and GHO population
health and abundance.
METHODS: DIETARY EXPOSURE
METHODS: PRODUCTIVITY AND ABUNDANCE
METHODS: TISSUE-BASED EXPOSURE
 Dietary items were collected from great horned owl nests in the study area and identified to
the lowest taxonomic level possible.
 GHO territories were located with call-response surveys.
 Prey items were collected from the study area in locations adjacent to identified GHO nests.
Small mammals were sampled at two locations upstream and six locations downstream of
Midland, MI. Rabbits were sampled at two locations upstream and three locations
downstream of Midland, MI.
 Blood was drawn from nestling GHOs ~6wks post-hatch by accessing the nests and
lowering nestlings to the ground.
 After homogenization prey items were analyzed for PCDF/DD concentrations.
 Chemical extraction followed EPA method 3540C and 3541.
 Congener-specific PCDF/DD analysis was conducted with GC/high resolution MS following
EPA method 8290.
 Results are corrected based on recoveries and non-detect congeners = ½ detection limit.
 GHO food intake rate is based on literature values.
 GHO average potential daily dose estimated based on avian-specific World Health
Organization (WHOAvian) TCDD equivalency factors.
 GHO ADDpot was compared to toxicity reference values from the literature (Table 1) to
determine hazard quotients for the risk assessment
T a b le 1 . D ie ta ry-b a s e d to x ic ity re fe re n c e
v a lu e s (T R V s ) fo r 2 ,3 ,7 ,8 te tra c h lo ro d ib e n zo - p -d io x in e q u iv a le n ts
(T E Q W H O -A v ia n ) fo r g re a t h o rn e d o w l (B u b o
v irg in ia n u s ) p o te n tia l a v e ra g e d a ily d o s e
(A D D p o t )
 Chemical extraction followed EPA method 3540C and 3541.
 Congener-specific PCDF/DD analysis was conducted with GC/high resolution MS following
EPA method 8290.
 Results are corrected based on recoveries and non-detect congeners = ½ detection limit.
 Great horned owl plasma concentrations are estimated based on avian-specific World
Health Organization (WHOAvian) TCDD equivalency factors.
 GHO plasma values were converted to egg equivalents for comparison to toxicity reference
values (Table 2)
105
fe e d in g M c L a n e 1 9 8 0
NOAEC
1800
fe e d in g
NOAEC
LO AEC
14
140
i.p .
N osek 1992/
in je c tio n S a m p le 1 9 9 6
NOAEC
LO AEC
100
1000
egg
N osek 1993
in je c tio n
Eastern Cottontail Rabbit
Chart 1. GHO dietary composition by biomass (n=465)
5%
2%
M u s k ra t
5% 0.04%
0.09%
NOAEC
LO AEC
77
1130
1200
0
*
Avian TEQs (ng/kg)
Avian TEQs (ng/kg)
Avian TEQs (ng/kg)
1000
M u s k ra t
E a s te rn C o tto n ta il
M e a d o w V o le
*
Reference (Upstream)
5
0.594
0.007
Nestling Egg Equivalent
(ng/kg)
O th e r h e rb iv o ro u s s m a ll
m a m m a ls
W a te rfo w l*
400
0
Nestling Plasm a (ng/L)
S h o rt-ta ile d S h re w
S ta r-n o s e d M o le *
200
*
1.20
0.009
14
2.67
0.115
B ird s
3
2
1
0
0.07
Chart 3. GHO ADDpot of PCDF/DDs
in reference and study areas
7
2
10
4
17
2
11
4
10
13
51
1.00 1.11
1.33 1.55
0.50 1.10
1.33 1.00
1.00
1.21
#Fledglings/Territorial Pair
0.33 0.40
0.50 1.11
0.67 1.31
0.40 1.00
N /A 0.91
0.62
1.04
M ayfield probability estim ate
of survival 77 days from egg N /A N /A
b
to fledge
N /A 1.00
1.00 0.62
0.22 0.60
1.00 0.50
0.72
0.65
M ayfield m ean num ber of
young/breeding pair
N /A 2.0
1.6
0.3
2.0
1.3
1.2
N /A N /A
1.2
14
0.7
0.6
0.5
0.4
0.3
0.2
12
10
8
6
4
0.1
2
0
0
M c Lane
1.1
0.8
a
N o survey data for 2008
b
not applicable for 2005 reference (only 1 observation in reference area), no breeding season observations for 2004
GHO productivity in the Tittabawassee River floodplain (Table 4) is similar to productivity considered
normal for the temperate forest. In a survey of 1,236
nesting attempts over 28 years, Holt observed a mean
productivity of 1.3 young/breeding attempt in the Cincinnati area.
Tissue-based exposure
Great horned owl plasma PCDF/DD concentrations downstream of Midland, MI, are higher
than those upstream (Chart 5). Adult and nestling plasma concentrations are significantly different (one-sided student t-test, p<0.05), and nestling PCDF/DD concentrations in the study
area are nearly identical to adult concentrations in the reference area, despite having different
congener profiles (Chart 8). This is likely due to the duration of exposure, growth dilution, and
metabolic differences between nestlings and adults. Great horned owls have been known to
survive for over 28 years in the wild, during which time even background concentrations of bioaccumulative compounds such as furans and dioxins can result in elevated tissue concentrations. In addition, nestling great horned owls are growing at a fast pace; the PCDF/DD plasma
concentrations are naturally diluted as the nestling’s mass increases.
The higher productivity and overall abundance in the
study area may be due to differences in microhabitat
and prey availability.
Few laboratory studies have been conducted that examine PCDF/DD concentrations in avian
plasma. Therefore, plasma concentration data were converted to egg concentration equivalents for the purpose of comparing the results to egg-based TRVs and determining hazard
quotients (Table 3).
Because there is no single definitive toxicity reference value for GHO exposure to PCDF/DDs,
TRVs for GHO tissues were derived from a combination of chronic laboratory studies (Table
2). The studies were chosen because they measured ecologically relevant endpoints such as
reproductive success and hatch success and concentrations were measured in comparable
tissues. The HQs for adult GHO plasma indicate PCDF/DD concentrations in the study area
are not likely to cause adverse effects (Chart 7).
Known GHO territory
Reference territory
with productivity data
Study territory with
productivity data
CONCLUSIONS
 GHOs in the study area are exposed to ~100X
higher PCDF/DD concentrations in their diet
than GHOs upstream, but not at levels expected to cause adverse effects.
 PCDF/DD concentrations in GHO plasma are
higher downstream than upstream but not at
levels expected to cause adverse effects.
 GHO relative abundance and productivity is
higher in the study area than the reference
area, and the popuation appears to be healthy.
 Further work will include incorporating incidental soil ingestion in GHO ADDpot and using a
Monte Carlo analysis to further examine GHO
dietary exposure to PCDF/DDs.
The primary congeners in the reference area are 1,2,3,7,8-PCDD, 2,3,4,7,8-PCDF, and
2,3,7,8-TCDD. In contrast, the primary congener in the study area is 2,3,4,7,8-PCDF. These
profiles are consistent with samples in different matrices in the floodplain, including the great
horned owls’ dietary items (Chart 8). The match in congener profile between diet and plasma
indicates the owls’ exposure can be attributed to their feeding habits.
4.43
0.124
100%
N os ek
Chart 4. Hazard quotients for GHO
potential average daily dose of
PCDF/Ds
R eference N estling
R eference A dult
S tudy N estling
S tudy A dult
0 .7
Chart 5. Geometric mean (95% UCL) Avian TEQs
in GHO plasma
2378-TC D F
2378-TC D D
23478-P eC D F
0.45
0.40
0 .6
GHO responses/river km
4
2
The GHO dietary exposure to PCDF/DDs is significantly higher in the floodplain downstream
of Midland, MI (4.55 ng/kg/day versus 0.070 ng/kg/day upstream). However, the concentrations of PCDF/DDs in the GHO diet is well below the NOAEC TRVs derived from laboratory
feeding studies (Table 1) and would not be expected to induce adverse effects in great horned
owls (Chart 4).
80%
Hazard Quotient (95% LCL/UCL)
4.55
4
Twenty-three GHOs territories (10 reference, 13
study) were identified in the reference and study areas
with a combination of surveys and direct observations
(Figure 1).
C ra yfis h
Avian TEQs (ng/L)
5
Study
1
Study (Downstream)
Hazard Quotient (95% LCL/UCL)
Funding was provided through an unrestricted grant from The
Dow Chemical Company to Michigan State University.
Avian TEQs (ng/kg)
ACKNOWLEDGMENTS
6
3
Figure 1. GHO territories in the Tittabawassee River
floodplain
16
R eference
1
1.00 1.00
*N. Short-tailed Shrew TEQs used to estimate Star-nosed Mole, Turkey (with skin on) TEQs used to estimate Waterfowl
7
0
#Fledglings/Breeding Pair
Table 3. G eom etric m ean and 95% UCL of 2,3,7,8-tetrachlorodibenzo- p -dioxin
equivalents (TEQ W H O Avian ) in great horned owl ( Bubo virginianus ) adult and nestling
plasm a and egg equivalents from the reference and study areas along the
Tittabawassee River in M idland, M ichigan, USA
Reference
Study
G eom etric
G eom etric
n
n
M ean 95% UCL
M ean 95% UCL
Tissue
Adult Plasm a (ng/L)
5
3.11
4.67
9
8.37
18.8
0.104
0.143
485
509
Adult Egg Equivalent
(ng/kg)
E a s te rn F o x S q u irre l
2
0
The mean GHO relative abundance for 2004-2007
was significantly greater in the study area downstream
of The Dow Chemical Co. (0.31 responses/river km)
than in the reference areas (0.11 responses/river km)
(Mann-Whitney U, p=0.043) (Chart 7).
M e a d o w V o le
C ra yfis h
Chart 2. Geometric mean (95% UCL) PCDF/DD concentrations in GHO
dietary components
4
2
0
Call-response surveys were conducted summers of
2004-2007. For the purposes of the study juvenile
and adult responses were grouped together, so the
relative abundance relates more directly to territory
abundance than actual number of GHOs present.
log10(µgPCDD/DFegg/g wet wt) = 1.647[log10(ng PCDD/DFplasma/ml)] – 2.578
O th e r h e rb iv o ro u s s m a ll
m a m m a ls
W a te rfo w l*
58%
600
M cLane 1980
1
N /A
Productivity and Abundance
The GHO potential daily dietary exposure to PCDF/DDs (Chart 3) was calculated using the
daily wildlife dose equation for dietary exposure (the GHO’s normalized ingestion rate is
72g/kg/day).
m
ADDpot = Σ(Ck x FRk x NIRk)
k=1
E a s te rn F o x S q u irre l
B ird s
6
N /A N /A
Fledglings
egg
H o ffm a n 1 9 9 8
in je c tio n
S ta r-n o s e d M o le *
2%
*
Failed N ests
S h o rt-ta ile d S h re w
6%
800
Table 4. G reat horned owl ( Bubo virginianus ) productivity in the Tittabawassee R iver floodplain reference and study
areas for 2004-2008
2004
2005
2006
2007
2008
All years
R ef Study R ef Study
R ef Study
R ef Study
R ef Study
R ef Study
a
O ccupied Territories
5
9
7 10
9 13
8 11
32
54
N /A 11
a
Territorial Pairs
3
5
4
9
6 13
5 11
21
49
N /A 11
Breeding Pairs
1
2
2
9
3 11
4 10
3 10
13
42
RESULTS AND DISCUSSION (CONT’D)
GHO pellets and prey remains were collected from 13 nests over the 2005 and 2006 breeding
seasons. Analysis of the dietary items (n=465) shows a preference for meadow voles, muskrat, rabbits, and birds in the GHO diet. Rabbits and muskrats contribute the majority of biomass consumed by the owls (chart 1) and as a result contribute the highest fraction of the
GHOs’ daily dietary exposure to PCDF/DDs. The two major congeners in the dietary items
are 2,3,4,7,8-PeCDF and 2,3,7,8-TCDF.
E a s te rn C o tto n ta il
22%
0.04%
8
 The Mayfield method for calculating nest success was used to account for nests that were
not observed during the breeding season.
T a b le 2 . T is s u e -b a s e d to x ic ity re fe re n c e
v a lu e s (T R V s ) fo r 2 ,3 ,7 ,8 -te tra c h lo ro d ib e n zo p -d io x in e q u iv a le n ts (T E Q W H O -A v ia n ) fo r g re a t
h o rn e d o w l (B u b o v irg in ia n u s ) e g g s
R e fe re n c e
10
 Productivity (#fledglings/territorial pair and #fledglings/breeding pair) was determined with a
combination of breeding season observations and responses from surveys.
 After collection, blood was stored in a heparanized Vacutainer. Within 2 hours after
collection blood was spun down in a centrifuge and plasma decanted for analysis.
NOAEC
12
 Relative GHO abundance was calculated as # responses/river km.
 Adult GHOs were caught in mist-nets by using a plastic decoy great horned owl and
broadcasting a territorial GHO call
D ie ta ry d o s e (n g /k g /d a y)
S tu d y
T yp e
 Call-response surveys were conducted from canoes during crepescular hours under
windless conditions. GHO hoots were broadcast every 500m along the river and any
responses were recorded.
 Artificial nesting platforms were installed in known GHO territories.
T is s u e
based
S tu d y
TRV
T yp e
R e fe re n c e
E g g T E Q c o n c e n tra tio n s (n g /k g w e t w t)
D ie ta ry
based
TRV
Dietary Exposure
RESULTS AND DISCUSSION
0 .5
0 .4
0 .3
0 .2
0 .1
0.35
R eferenc e
S tudy
0.30
40%
0.25
0.20
H o ffm a n
Chart 6. Hazard quotients for Avian
TEQ concentrations in adult GHO
plasma downstream of Midland, MI
12378-P eC D F
12378-P eC D D




20%
0.15
0.10

0%
0.00
N osek
234678-HxC D F


0.05
0
60%
REFERENCES
Chart 7. Relative abundance (responses/river km) of
GHOs in the reference and study areas 2004-2007
Reference
Reference
Nestlings
Reference
Reference
Adults
Reference
Reference
Diet Diet
Study
Target
Nestlings
Study
Target
Adults
Adults
Study
Target
DietDiet
Nestlings
Adults
Nestlings
Chart 8. Congener profiles of GHO nestling and adult plasma and dietary
items



Craighead JJ, Craighead FC. 1956. Hawks, Owls, and Wildlife Harrisburg, PA: Stackpole
Company
Hoffman DJ, Melancon MJ, Klein PN, Eisemann JD, Spann JW. 1998. Comparative developmental toxicity of planar polychlorinated biphenyl congeners in chickens, American kestrels,
and common terns Environmental Toxicology and Chemistry 17:747-57
Holt JB. 1996. A banding study of Cincinnati area great horned owls Journal of Raptor
Research 30:194-7
Mclane MAR, Hughes DL. 1980. Reproductive success of screech owls fed Aroclor 1248 Arch.
Environ. Contam Toxicol. 9:661-5
Nero RW. 1992. New great horned owl longevity record. The Blue Jay 50:91-2
Nosek JA, Craven SR, Sullivan JR, Hurley SS, Peterson RE. 1992. Toxicity and Reproductive
Effects of 2,3,7,8-Tetrachlorodibenzo-P-Dioxin in Ring-Necked Pheasant Hens Journal of
Toxicology and Environmental Health 35:187-98
Nosek JA, Sullivan JR, Craven SR, Gendronfitzpatrick A, Peterson RE. 1993. Embryotoxicity of
2,3,7,8-Tetrachlorodibenzo-P-Dioxin in the Ring-Necked Pheasant Environmental Toxicology
and Chemistry 12:1215-22
Sample BE, Opresko DM, Suter GW. 1996. Toxicologcial Benchmarks for Wildlife: 1996
Revision. Rep. ES/ER/TM-86/R3, USDOE
Strause KD, Zwiernik MJ, Im SH, Newsted JL, Kay DP et al. 2007. Plasma to egg conversion
factor for evaluating polychlorinated biphenyl and DDT exposures in great horned owls and
bald eagles Environ. Toxicol. Chem. 26:1399-409
[USEPA] US Environmental Protection Agency. 1993. Wildlife exposure factors handbook.
Rep. EPA 600/R-93/187b, USEPA, Washington, DC
Enzyme induction of several field collected avian species as part of a site-specific risk assessment on the Tittabawassee River, Midland, MI, USA
1
2
1
1
3
2
1
4
3
1
Timothy B. Fredricks , John L. Newsted , Rita M. Seston , Patrick W. Bradley , Steve B. Wiseman , Denise P. Kay , Steve J. Bursian , Sean W. Kennedy , John P. Giesy , Matthew J. Zwiernik
4. Environment Canada, NWRC, Ottawa, ON, Canada
Introduction
Objectives
 Nest box trail established on both study and reference areas in
the Tittabawassee River floodplain south of Midland, MI, USA
in field collected samples from the Tittabawassee floodplain
for several avian species
(Figure 1) in 2004 and has since been consistently monitored
 Study species include 3 passerine species
 Compare induction levels between these
MI
HOUSE WREN, HW;
EASTERN BLUEBIRD, EB)
N
literature values
200
150
1000
100
 Custer et al. [8] monitored TS exposed to PCBs in Green Bay, WI for
EROD/BROD (which were correlated) induction (ranged from
32.07-109.23 and 15.95-65.10 pmol/min/mg, respectively) but was not
correlated with TEQs or concentration data
50
0
0
 Custer et al. [9] monitored a TCDD exposed population of TS for EROD
Individual samples (2 individuals from reference areas and 4 individuals from study areas)
activity (maximum induction ~330 pmol/min/mg)
Figure 4. Belted kingfisher EROD and MROD maximum induction (pmol product/min/mg microsomal protein) in nestling
liver tissue collected from reference and study areas on the Tittabawassee River, Midland, MI, USA during 2007.
r
Riv
e
{
r
ive
Saginaw
0
4
8
16
Cass
Rive
r
Shi
a
wa
sse
eR
i ve
r
accumulating elevated
eR
Study
Areas
60
2
10
40
Kilometers
10
20
2,3,7,8-TCDF when compared to reference areas
 Research is part of a large collaborative congener specific project
5
0
2
EB
4
KF
5
HW
10
TS
4
EB
8
KF
Reference Areas
9
 EROD and MROD maximum induction was 5-fold greater in study
areas for belted kingfisher compared to reference areas (Figure 4)
 Tree swallow EROD induction was ~ 2-fold greater at study areas
while MROD induction was at or less than the detection limit (Figure 5)
HW
TS
 Eastern bluebird had the greatest EROD and MROD induction levels
0
10
for all study species (Figure 7)
reference areas (RAs) (Figure 1) from May through August 2007
Liver was removed in the field and snap frozen (subsequently stored at -80ºC)
Remaining nestling tissues were stored intact at -20ºC
Liver tissue was split with ~ 0.1 g for enzyme activity, ~0.025 g for CYP1A mRNA
quantification, and the remaining tissue for residues analyses
Residue Analyses
1200
Figure 2. Percent congener profiles and nestling TEQ Avian ranges with medians for associated nestling
samples collected in the Tittabawassee River floodplain, Midland, MI, USA in 2007.
Results
 Congener profiles at downstream study areas are composed of > 80%
furan compounds (Figure 2)
 WhoAvian TEQs are at least an order of magnitude greater at study
40
Legend
Reference Areas
Study Areas
30
800
20
400
 Enzyme induction profiles were greater at study areas and were
0
species specific (Figure 3)
0
Results were corrected based on recoveries and non-detected congeners equal ½
detection limit
TEQ concentrations are based on avian-specific World Health Organization
(WHOAvian) TCDD equivalency factors [1]
 Reconstituted whole body nestling concentrations were determined by
combining values for liver and nestling homogenates by individual
Enzyme Activity
 Liver microsome preparation and EROD/MROD analyses [2,3,4]
Figure 5. Tree swallow EROD and MROD maximum induction (pmol product/min/mg microsomal protein) in nestling
liver tissue collected from reference and study areas on the Tittabawassee River, Midland, MI, USA during 2007.
Legend
Reference Area
Target Area
1000
Belted Kingfisher
House Wren
Eastern Bluebird
Tree Swallow
5000
4000
Legend
Reference Areas
Study Areas
3000
2000
750
400
500
200
250
1000
 EROD assay wells averaged 28.5 ug/ml microsomal proteins (9.09-46.4 ug/ml)
 MROD assay wells averaged 53.6 ug/ml microsomal proteins (18.4-83.9 ug/ml)
 Results expressed as maximum pmol product/mg microsomal protein/min
Tree swallow (Tachycineta bicolor)
Eastern bluebird (Sialia sialis)
House wren (Troglodytes aedon)
Belted kingfisher (Ceryle alcyon)
0
2
4
6
8
10
12
14
16
18
20
22
1500
0
24
26
28
Conclusions
 Based on these results furan induction of CYP1A activity is greater
for these species than previously reported for both TCDD and PCB
field based exposures
 Incorporation of data into overall comparison of lab to field studies
Literature Cited
 ~ 0.1000 g liver homogenized (range 0.0934-0.1288 g)
 Induction measured in 96-well plates with a total well volume of 201 ul
500
should prove extremely interesting…stay tuned!
MROD (pmol/min/mg)
Chemical analyses followed EPA method 8290
3000
compare against percent maximal induction and total TEQs [10]
Individual samples (5 individuals from reference areas and 10 individuals from study areas)
6000
1000
 TEQs and EROD/MROD positively correlated for all species studied
 Need to finalize CYP1A mRNA quantification for these samples to
Nestlings were homogenized without feathers, lower legs, bill, and liver tissue
Chemical extraction followed EPA method 3540C & 3541
4500
Figure 7. Eastern bluebird EROD and MROD maximum induction (pmol product/min/mg microsomal protein) in nestling
liver tissue collected from reference and study areas on the Tittabawassee River, Midland, MI, USA during 2007.
10
rather than reference areas (Figure 2)
Nestling liver tissue was homogenized and analyzed individually
6000
Individual samples (5 individuals from reference areas and 10 individuals from study areas)
EROD (pmol/min/mg)
Near fledgling age nestlings were collected at four study areas (SAs) and 2
1500
0
Study Areas
research, AhR ligand binding domain sequencing, mRNA response
Methods
Legend
Reference Areas
Study Areas
and was only slightly greater in study areas (Figure 6)
involving a multi-species egg injection study, primary hepatocyte
quantification, and the field component partially presented here
7500
 House wren EROD and MROD induction varied among individuals
1
Figure 1. Map of research areas including reference and study areas in the Tittabawassee River floodplain,
Midland, MI, USA in 2007.
Results cont.
MROD (pmol/min/mg)
{
naw
r
i ve
Bay City
EROD (pmol/min/mg)
Midland
sse
wa
eR
Pin
3
10
80
Sag
i
er
a R iv
ippew
collected at study areas are

1500
primarily background EROD levels that ranged from 10.7-34.9
pmol/min/mg, ~4-22 pmol/min/mg, and 20.84-38.03 pmol/min/mg,
respectively, for field exposure TS to a variety of chemicals
MROD (pmol/min/mg)
{
Reference
Areas
Ch





250
Lake Huron
 Nestling bird tissues



 Bishop et al. [5] , Bishop et al. [6], and Gentes et al. [7] reported
300
500
100
BELTED KINGFISHER (BK)

Legend
2000
Midland Area
and the
2,3,4,7,8-PeCDF and
Other
2,3,7,8-TCDD
1,2,3,7,8-PeCDD
2,3,7,8-TCDF
2,3,4,7,8-PeCDF
Min/Median/Max
primarily furan exposed birds and existing
(TREE SWALLOW, TS;
concentrations of
Legend
ba
tta
Ti
Ethoxyresorufin-O-dealkylase (EROD) and methoxyresorufin-Odealkylase (MROD) activity was assayed in several avian species
nesting in the Tittabawassee and Chippewa River floodplains
near Midland, Michigan to examine the exposure and potential
effects of polychlorinated dibenzofurans (PCDFs) and
dibenzo-p-dioxins (PCDDs). Concentrations of PCDF/PCDDs in
biota have been found to be 10- to 20-fold greater downstream
(study areas) of Midland when compared to upstream (reference
areas) but the toxicological significance of these differences
relative to avian population health downstream of Midland is still
being investigated. Tree swallow (TS), eastern bluebird (EB),
house wren (HW), and belted kingfisher (BK) were chosen as
species of interest. In this study, maximum EROD and MROD
activity was assayed utilizing a kinetic assay in liver tissue
collected from nestlings prior to fledging. Seventeen 2,3,7,8
substituted PCDF and PCDD congeners were measured in whole
body nestling homogenates and converted to TEQs using
WHOAvian TEF values. EROD and MROD activities (pmol
product/mg microsomal protein/min) were greater at
downstream study areas and varied by species (EROD:
EB>KF>TS>HW and MROD: EB>KF=HW>TS). Avian TEQs at study
areas were similar for EB, KF, and HW while TS had slightly greater
nestling whole body accumulation. The study areas congener
profiles for whole body nestling homogenates were dominated
by 2,3,7,8-TCDF and 2,3,4,7,8-PeCDF (80-90%), but varied by
species with the EB profile dominated by PeCDF, TS profile
dominated by TCDF, and the KF and HW profile were a mixture of
PeCDF and TCDF. Enzyme induction levels reported here,
primarily from furan exposure, are greater than or equal to
previously reported induction levels for TS exposed to similar
TCDD dominated TEQ levels. Despite greater enzyme activity and
WHOAvian TEQs at study areas compared to reference areas
overall productivity measurements are comparable between
sites.
 Quantify both enzyme activity and residue concentrations
Discussion
350
Reference Areas
Study Areas
EROD (pmol/min/mg)
Abstract
2500
MROD (pmol/min/mg)
3. University of Saskatchewan, Saskatchewan, SK, Canada
EROD (pmol/min/mg)
2. Entrix, Inc., Okemos, MI, USA
Nestling TEQAvian (pg/g)
1. Michigan State University, East Lansing, MI, USA
pmol product/min/mg microsomal protein
Environment
C anada
Percent TEQAvian
E nvironm ent
C anada
30
Reaction Time (min)
Figure 3. Representative time-course curves for study species at both reference and study areas collected along the Tittabawassee
River, Midland, MI, USA in 2007 for EROD induction (pmol product formed/min/mg microsomal protein)
0
0
Individual samples (5 individuals from reference areas and 10 individuals from study areas)
Figure 6. House wren EROD and MROD maximum induction (pmol product/min/mg microsomal protein) in nestling
liver tissue collected from reference and study areas on the Tittabawassee River, Midland, MI, USA during 2007.
1. Van den Berg et al. 1998. Environ. Hlth. Persp. 106:775-792.
5. Bishop et al. 1998. J. Toxicol. and Environ. Hlth. Part A 55:531-559.
2. Kennedy, S.W. and Jones, S.P. 1994. Anal. Biochem. 222:217-223.
6. Bishop et al. 1999. Environ. Toxicol. Chem. 18:263-271.
3. Shipp et al. 1998. J. Toxicol. Environ. Health. 54(5):377-401.
7. Gentes et al. 2007. J. Toxicol. Environ. Hlth. Part A. 70:1182-1190.
4. Trudeau, S.F. and Maisonneuve, F.J. 2001. A method to determine
8. Custer et al. 1998. Environ. Toxicol. Chem. 17:1786-1798.
cytochrome P450A activity in wildlife microsomes. Technical
9. Custer et al. 2005. Environ. Toxicol. Chem. 24:93-109.
Report Series No 339E. Canadian Wildlife Service.
10. Head and Kennedy. 2007. Analyt. Biochem. 360:294-302.
Acknowledgements
We would like to thank all field/laboratory personnel that helped with this project, especially the
following: Arianne Neigh, Karl Strause, Mike Fales, Stephanie Plautz, Cassie Steiler, Melissa Palmer,
Megan Barker, Dave Hamman, Sarah Coefield, Emily Koppel, Lori Williams, Jeremy Moore, Mick Kramer,
Nozomi Ikeda, Casey Bartrem, Autumn Foutch, Nathan Hubbard, Tara Franey, Melanie Collins, Will
Folland, Bretton Joldersma, William Sterling, Mike Nadeau, Bethany Opperman, Mike Szor, Sanjeev
Mahabir, Susan Dalgarn, and Kelly Winchell. Additionally, this study would not have been possible
without the dedicated team of the employees at Entrix, Inc., Okemos, MI, especially Melissa Shotwell
and Kate Luce, and wonderful support staff at Michigan State University. Funding was provided
through an unrestricted grant from The Dow Chemical Company to Michigan State University.
For additional information please visit our website at http://riverwildlife.msu.edu
National Food Safety and Toxicology Center
Exposure of American robins (Turdus migratorius) to PCDF and PCDD
on the Tittabawassee River floodplain, MI, USA.
Dustin L Tazelaar1‡*, Rita M Seston1†*, Timothy B Fredricks1†*, Sarah J Coefield1†*, Michael W Nadeau1*,
Melissa S Shotwell2, Denise P Kay2, Steven J Bursian1‡, Matthew J Zwiernik1‡*, John P Giesy3, 1†*
¹Michigan State University, †Department of Zoology / ‡ Department of Animal Science / *National Food Safety and Toxicology Center, East Lansing, MI, USA; ²Entrix,
Inc., East Lansing, MI, USA; ³Department of Biomedical Veterinary Sciences and Toxicology Centre University of Saskatchewan, Saskatoon, Saskatchewan, Canada
ABSTRACT
Polychlorinated dibenzofuran (PCDF) and polychlorinated dibenzo-p-dioxin (PCDD)
concentrations in the tissues of receptor species are important assessment endpoints in evaluations
of ecological risk. During the spring and summer of 2005, 2006 and 2007, 67 American robin
eggs, 30 nestlings and 12 adults were collected from the Tittabawassee River floodplain from
upstream reference sites and study sites downstream of the city of Midland, MI, USA. Previous
studies have indicated that study sites had concentrations of PCDF and PCDD that were greater
than in nearby reference areas. Concentrations of the 17 2,3,7,8 substituted PCDFs and PCDDs
were quantified in American robin tissues and normalized to 2,3,7,8 dibenzo-p-dioxin using WHO
avian 1998 TEFs. Preliminary American robin egg TEQs ranged from 2.4 ng/kg ww to 1.5 x 101
ng/kg ww in reference areas and 2.5 x 101 ng/kg ww to 1.7 x 103 ng/kg wet weight in study areas,
while preliminary nestling tissue TEQs ranged from 1.0 ng/kg wet weight to 2.1 x 101 ng/kg ww in
reference areas and 4.7 x 101 ng/kg ww to 5.6 x 102 ng/kg ww in study areas.
RESULTS
Figure 1. Map of sampling locations in the
Tittabawassee River floodplain in Michigan, USA.
METHODS AND MATERIALS
• American robin tissues were collected in 2005, 2006, 2007 and 2008 from nests located within the
floodplains of target and reference areas of the Tittabawassee River floodplain
• Fresh egg samples were collected randomly prior to or during incubation
• Addled egg samples were collected opportunistically following hatch date or nest failure
• Egg sample TEQ concentrations based on calculated fresh mass minus the mass of shell (Hoyt,
diet items of the Tittabawassee River and composition.
Dietary
Item
Plant
Coleoptera
Lepidoptera
Misc.
Earthworm
INTRODUCTION
The American robin (Turdus migratorius) is a useful receptor for the ecological risk assessment of
study areas contaminated with bioaccumulative contaminants of concern (COCs) (Henning et al.,
2003). American robins have an intimate relationship with soil as a nest building material and soil
ingesting invertebrates as dietary components, including earthworms. Robins are common and
nesting distribution is widespread, making data collection and sampling realistic. The American
robin is an ideal representative passerine study species exposed to the soil-to-invertebrate food web
in the area of concern.
Table 1. Concentrations of TEQ-avian (ng/kg ww) measured in
Ref.
median
0.6
3.3
1.0
1.2
1.4
Ref.
max
1.8
16
1.5
4.5
2.4
Study Study
median max
2.7
13
410
1900
42
98
23
380
220
530
Figure 2. Congener contribution of avian TEQs for American robin
tissues in the Tittabawassee River floodplain.
CONCLUSIONS
• Median soil TEQ concentrations are more than 600 times greater in
study locations than in reference locations.
Table 2. Concentrations of TEQ-avian (ng/kg dw)
measured in surface soil of the Tittabawassee River
floodplain.
Reference
11
6.7
3.95
24.8
N
Median
Min
Max
• Median invertebrate dietary item TEQ concentrations are more than 1
order of magnitude greater in study areas than in reference areas.
• 2,3,4,7,8-PeCDF, 2,3,7,8-TCDF, 1,2,3,7,8-PeCDD and 2,3,7,8-TCDD
Study
27
4478
425
18800
account for more than 90% of the TEQs in American robin tissues in
both reference and study areas.
• 2,3,4,7,8-PeCDF predominates the congener profiles of both eggs and
nestlings collected from the study area.
• 1,2,3,7,8-PeCDD and 2,3,7,8-TCDD account for the greatest
percentage of reference egg congener profiles while reference
nestling profiles are predominately 2,3,7,8-TCDF.
1978)
•
•
•
•
TEQ concentrations in dietary items are greater in study areas than in
reference areas. Median TEQ concentrations in reference areas are as
great as 3.3 ng/kg ww while study area median TEQ concentrations are
as great as 4.1 x 102 ng/kg ww in the same taxonomic order, Coleoptera,
or beetles (Table 1). TEQ concentrations in soil are greater in study
areas than in reference areas. Concentrations measured in reference area
soils are as little as 4.0 ng/kg dw while study location soils exhibit
concentrations as great as 1.9 x 104 ng/kg ww (Table 2). Conger
profiles vary between reference and study areas with 2,3,4,7,8-PeCDF
contributing approximately 80% to the total TEQs in eggs and 70% in
nestlings in study areas, whereas 2,3,4,7,8-PeCDF contributes less than
20% to the total TEQs in eggs and less than 10% in nestlings in
reference areas (Figure 2). Median TEQ concentrations in American
robin tissues are greater in study areas than in reference areas. Median
TEQs are as little as 3.6 ng/kg ww in reference nestlings and as great as
1.8 x 102 ng/kg ww in study area eggs (Table 3).
• Median American robin egg TEQ concentrations are more than 25
times greater in study locations than in reference locations.
Nestling samples were collected approximately 12 d following hatch date
Nestling samples homogenized following removal of feathers, bill and legs below the tibiotarsus
Table 3. Concentrations of TEQ-avian (ng/kg ww) in American
Soil samples and dietary samples collected from the Tittabawassee River floodplain 2003-2006
robin egg and nestlings collected in the Tittabawassee River
floodplain.
Concentrations of TEQ in soil are expressed as ng/kg on a dry weight basis and ng/kg wet weight
for tissues
• Chemical extraction EPA methods 3540C and 3541
• Analyses of the 17 2,3,7,8 substituted PCDF/D congener concentrations in samples are conducted
Egg
Nestling
7.2
3.6
95% UCL
9.2
9.7
40 times greater in study locations than in reference locations.
• American robin tissue TEQ concentrations comparable to those in
Study Area
Reference Area
Median
• Median American robin nestling TEQ concentrations are more than
(n)
(16)
(8)
Median 95% UCL
183
157
391
253
(n)
(37)
(14)
at AgriQuality Limited (Lower Hutt, New Zealand) using EPA method 8290
• All TEQ values based on avian World Health Organization toxicity equivalency factors (Van den
Berg et al., 1998)
• The TEQ concentrations are calculated by assigning a proxy value of ½ the detection limit (DL)
for congeners below the DL
Study area median nestling TEQs
more than 40 times greater than
reference area
ACKNOWLEDGMENTS
We would like to thank all field/laboratory personnel that helped with this project, especially the following:
Jeremy Moore, Michael Nadeau, Anna Boegehold, Michelle Hodges, Clay Manntz, Chelse Grohman,
Nathan Hubbard, Melanie Collins, William Folland, Megan Barker, Michael Szor, Arianne Neigh, Karl
Strause, Bretton and Carrie Joldersma, Cyrus Park, Mike Fales, Meghan Mikesell, Ben Nessia, Jiachun Ge,
Lam Wong, Mick Kramer, Patrick Bradley, Nozomi Ikeda, Emily Koppel, Melissa Palmer, Cassie Stieler,
Bethany Opperman, William Sterling, Lacy Sharrard, Sandy Mazzoni, and Kelly Winchell. Additionally, this
study would not have been possible without the dedicated team of the employees at Entrix, Inc., East
Lansing, MI and wonderful support staff at Michigan State University. Funding was provided through an
unrestricted grant from The Dow Chemical Company to Michigan State University.
tree swallow (Tachycineta bicolor) tissues where hatching success
was negatively associated with concentrations of 2,3,7,8-TCDD in
eggs in of the Woonasquatucket River, Rhode Island, USA.
REFERENCES
1. Henning et al. (2003) Environ. Toxic. and Chem. 11: 2783-2788.
2. Hoyt (1979) The Auk. 96: 73-77.
3. Van den Berg et al. (1998) Environ. Health Perspect. 106: 775–79.
4. Custer et al. (2005) Environ. Toxic. and Chem. 1: 93-109.
2,3,4,7,8-PeCDF accounts for
greater than 70% of the total
TEQs in study area American
robin tissues
Impact of TCDD, PeCDF and TCDF Exposure on Hepatic Cyp1A4 Transcript Abundance in
Japanese Quail and Ring-Necked Pheasant in Ovo
Yinfei Yang1*, Steve Wiseman1, Steve Bursian2, Matt Zweirnik2, Patrick Bradley 2, Timothy B. Fredricks 2, Andrew Cohen-Brownhouse 2, Sean Kennedy3, Jessica Herve3,
John Newsted4, Yi Wan1, Paul D. Jones1 and John P. Giesy1
1. Toxicology Center, University of Saskatchewan, Saskatoon, Saskatchewan, Canada
2. Zoology Department, Center for Integrative Toxicology, Michigan State University, East Lansing, MI, USA.
3. Center for Advanced Research in Environmental Genomics, Department of Biology, University of Ottawa, Ottawa, Ontario, Canada
4. ENTRIX Inc. Okemos, Michigan, USA
Background
Hypothesis
-Dioxin-like compounds (DLCs) stimulate transcription of Cyp1A genes, including Cyp1A4, via the
AhR signalling pathway.
-Potencies of various DLCs may be orders of magnitude different.
-It is generally accepted that TCDD is the most potent DLC.
-Species differences in sensitivity to DLCs have been observed. In birds these differences are
observable amongst different species within the order Galliforms. For example,
- Chicken (Gallus gallus) – Sensitive (Type 1)
- Ring-Necked Pheasant (Phasianus colchicus) – Moderately Sensitive (Type 2)
- Japanese Quail (Coturnix japonica) – Insensitive (Type 3)
-The molecular basis of differential sensitivity appears to be rooted at the level of the amino acid
sequence of the AhR ligand binding domain (LBD).
Methods
As AhR controls Cyp1A4 transcription, and AhR plays a role in determining sensitivity to DLC, we
tested the hypothesis that the Cyp1A4 response to DLCs differs in differentially sensitive species.
Tissue Extraction from Chicks
Egg Injection
Incubate
Q-PCR
Specific Objectives
1.Intra-species comparison: Do TCDD, PeCDF, and TCDF differ in Cyp1A4 inducing potential within
Japanese Quail and Ring-Necked Pheasant ?
2. Interspecies comparison: Are the effects of TCDD, PeCDF and PCDF on Cyp1A4 transcript
abundance different between Japanese Quail and Ring-Necked Pheasant ?
Administered Doses (ng/g Egg)
Quail: 0, 0.13, 0.19, 0.49, 0.89, 1.47, 2.62, 7.22
Pheasant: 0, 0.04, 0.05, 0.09, 0.20, 0.34, 1.46, 4.33
B
*
300
250
200
150
*
100
50
C
700
600
*
500
400
300
200
100
0
0
0.24
0.40
0.92
1.82
3.56
0
0.14
0.31
0.62
0.89
1.81
3.80
200
150
*
100
*
50
0
0
7.58
0.13
[PeCDF] ng/g Egg
[TCDD] ng/g Egg
0.19
0.49
0.89
1.47
2.62
7.22
[TCDF] ng/g Egg
*
800
700
600
500
*
400
300
200
100
B
1400
Cyp1A4 Transcript
Abundance (% Control)
Cyp1A4 Transcript
Abundance (% Contol)
A
*
1200
1000
800
600
*
*
*
400
*
*
200
0
0
0.00
0.02
0.03
0.07
0.10
0.26
1.02
2.15
0.00
0.05
[TCDD] ng/g Egg
0.08
0.13
0.20
0.36
1.39
2.30
[PeCDF] ng/g Egg
C
500
450
400
350
300
250
200
150
100
50
0
A
*
*
*
0.00
0.04
0.05
0.09
0.20
0.34
1.46
4.33
[TCDF] ng/g Egg
Figure 1: Effect of A) TCDD, B) PeCDF, and C) TCDF on Cyp1A4 transcript abundance in Japanese Quail and Ringed-Necked Pheasant.
Significant changes (denoted by *) in transcript abundance (P < 0.1, Mann Whitney U Test) relative to the control were observed
within each DLC treatment group. Data is shown as percent control, however statistical analysis was performed on mean normalized
expression values where b-actin was used as the reference gene. Concentrations of DLCs are reported as administered doses
calculated from nominal concentrations of prepared injection solutions.
Results 3: LOEC and Potency Values for Cyp1A4 Induction
Estimated LOEC Value
[ng/g Egg]
1.60
1.40
1.20
B
Quail
Pheasant
1.00
0.80
1.35
0.90
0.75
0.60
0.40
0.20
0.20
0.15
0.01
0.00
TCDD
PeCDF
16.00
15
14.00
Relative Potency
A
300
200
100
0
0
-100
1
2
3
4
[TCDD] ng/g Egg
600
400
200
0
0
-200
1
2
3
4
[PeCDF] ng/g Egg
300
200
100
0
0.0
0.5
1.0
1.5
2.0
2.5
3.0
[TCDF] ng/g Egg
-100
-200
Ring-Necked Pheasant
Cyp1A4 Transcript
Abundance (% Control)
Ring-Necked Pheasant
400
C
800
Quail
600
400
200
0
0.0
-200
0.5
1.0
1.5
2.0
[TCDD] ng/g Egg
8.00
Results and Conclusions
Intra-species Comparisons
-Rank order of Cyp1A4 LOEC Values:
Japanese Quail: TCDD > PeCDF > TCDF ; Ring-Necked Pheasant: PeCDF > TCDD > TCDF
-Findings from Ring-Necked Pheasant, but not Japanese Quail, are in accordance with Hervé et
al., (Poster #MP33 ) who show that PeCDF is a more potent inducer of EROD than TCDD in vitro.
6.00
4.00
1.00 1.00
0.83
TCDD
PeCDF
0.56 0.75
PCDF
Figure 3: (A) In Ovo LOEC values for TCDD, PeCDF and TCDF based on Cyp1A4 induction as
determined by linear regression analysis (Figure 2). (B) Relative potencies of TCDD, PeCDF and
TCDF in Japanese Quail and Ring-Necked Pheasant.
2.5
C
1400
1200
1000
800
600
400
200
0
-200
0.0
0.5
1.0
1.5
2.0
2.5
600
500
400
300
200
100
0
-100
0
[PeCDF] ng/g Egg
1
2
3
4
5
[TCDF] ng/g Egg
Figure 2: Linear Regression analysis of changes in Cyp1A4 transcript abundance in response to A) TCDD, B) PeCDF, C) TCDF in
Japanese Quail and Ringed-Necked Pheasant. 95% Confidence intervals were plotted to the regression line. The point on the
X-axis where the lower confidence interval transects the axis was estimated as the Lowest Observed Effective Concentration
(LOEC) of chemical that induced Cyp1A4 transcript. LOEC values are represented in figure 3A. Concentrations of DLCs are
administered doses calculated from nominal concentrations of prepared injection solutions.
-Ring-Necked Pheasant: PeCDF is 15x more potent as an inducer of Cyp1A4 than TCDD.
10.00
0.00
PCDF
800
Pheasant
12.00
2.00
B
1000
Cyp1A4 Transcript Abundance
(Change From Control)
0.16
250
*
B
500
Cyp1A4 Transcript Abundance
(Change From Control)
0.07
*
A
Cyp1A4 Transcript Abundance
(Change From Control)
0
300
Cyp1A4 Transcript Abundance
(Change From Control)
350
Cyp1A4 Transcript
Abundance (% Control)
A
Cyp1A4 Transcript Abundance
(Change From Control)
Japanese Quail
Cyp1A4 Transcript Abundance
(Change From Control)
Japanese Quail
Cyp1A4 Transcript
Abundance (% Control)
Results 2: Regression Analysis of DLC Impact on Cyp1A4 Transcript Abundance
Cyp1A4 Transcript
Abundance (% Control)
Results 1: Impact of DLCs on Cyp1A4 Transcript Abundance
Interspecies Comparisons
-Assuming Cyp1A4 transcript abundance can be used as an measure of sensitivity to DLCs, our
results suggest that Ring-Necked Pheasant is more sensitive to each chemical than Japanese Quail.
- Based on relative potencies PeCDF is approximately 18x more potent as an inducer of Cyp1A4 in
ring-Necked Pheasant than in Japanese Quail.
Ongoing Research and Future Directions
A complementary study is currently being performed in Chicken. Cyp1A4 transcript abundance will
be assayed in samples from this study. Analysis of Cyp1A5 transcript abundance and EROD activity
is being conducted for each species. Tissue levels of TCDD, PeCDF and TCDF will be quantified in all
species. This data will be utilized when preparing data for manuscript preparation.
The utility of the mRNA approach as an indicator of species sensitivity and DLC impact will be
assessed in wild birds from contaminated sites (See Fredricks, poster # WP222).
Related Posters
Readers interested in this research are encouraged to view posters WP210 (CohenBarnhouse), MP33 (Herve et al), WP222 (Fredricks et al), and MP36 (Farmahin et al).
Acknowledgements
This work is funded by an unrestricted grant from The DOW Chemical Company.
POSTER # WP229