Tara Duffy and Stephen McCormick Conte Anadromous Fish Research Lab, Turners Falls, MA
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Transcript of Tara Duffy and Stephen McCormick Conte Anadromous Fish Research Lab, Turners Falls, MA
Tara Duffy and Stephen McCormickTara Duffy and Stephen McCormick
Conte Anadromous Fish Research Lab, Turners Falls, MAConte Anadromous Fish Research Lab, Turners Falls, MA
Differential life-stage response to Differential life-stage response to common endocrine disruptors in two common endocrine disruptors in two
endangered species, Atlantic salmon and endangered species, Atlantic salmon and shortnose sturgeonshortnose sturgeon
Tara Duffy and Stephen McCormickTara Duffy and Stephen McCormick
Conte Anadromous Fish Research Lab, Turners Falls, MAConte Anadromous Fish Research Lab, Turners Falls, MA
Differential life-stage response to Differential life-stage response to common endocrine disruptors in two common endocrine disruptors in two
endangered species, Atlantic salmon and endangered species, Atlantic salmon and shortnose sturgeonshortnose sturgeon
Funding
Salmon and sturgeon conservationSalmon and sturgeon conservation
Anadromous Atlantic salmon and Anadromous Atlantic salmon and shortnose sturgeon are listed as shortnose sturgeon are listed as endangered under the ESAendangered under the ESA
Conservation efforts traditionally Conservation efforts traditionally do not consider potential do not consider potential impacts of EDCsimpacts of EDCs
Habitat use and life strategies Habitat use and life strategies suggest these species may be suggest these species may be particularly sensitive to degraded particularly sensitive to degraded water qualitywater quality
EPA WET testing and surrogate species
WET: Whole Effluent Testing uses a single life-stageWET: Whole Effluent Testing uses a single life-stage4-day exposure, mortality is primary target4-day exposure, mortality is primary targetLong-term, Long-term, sublethalsublethal impacts are not routinely monitored impacts are not routinely monitored
Surrogate speciesSurrogate speciesDaphniaDaphnia, brook trout, fathead minnow, silversides, brook trout, fathead minnow, silversides
Sturgeon and salmon are verySturgeon and salmon are verysensitive to common endocrine sensitive to common endocrine disruptors and effluentdisruptors and effluent
Dwyer Dwyer et alet al. 2005, . 2005, Arch. Environ. Arch. Environ. Toxicology and ContaminationToxicology and Contamination
JuvenilesJuvenilesEmbryoEmbryo
Life-stage and life-history will influence Life-stage and life-history will influence contaminant responsecontaminant response
Gonad differentiation or other factor
LarvaeLarvae
Numerous life-stage sensitivity examplesNumerous life-stage sensitivity examples
At what life stage will this chemical do the At what life stage will this chemical do the most damage? most damage?
ObjectivesObjectives
Three year study:Three year study:
1.1. Assess which life-stage may be most responsive Assess which life-stage may be most responsive to common feminizing EDCsto common feminizing EDCs
2.2. Assess vitellogenin (Vtg) as an appropriate Assess vitellogenin (Vtg) as an appropriate biomarker for measuring contaminant sensitivitybiomarker for measuring contaminant sensitivity
3. Carry out chronic exposures (21-day) and assess 3. Carry out chronic exposures (21-day) and assess impacts to fitness (growth, gonad histology, impacts to fitness (growth, gonad histology, development, salinity tolerance)development, salinity tolerance)
MethodologyMethodology4-day exposures on early life-stages4-day exposures on early life-stages
1. Embryos1. Embryos2. Yolk-sac larvae2. Yolk-sac larvae3. Feeding fry3. Feeding fry4. Juveniles/smolts4. Juveniles/smolts
Compounds usedCompounds used
1. α-ethinylestradiol (EE2)1. α-ethinylestradiol (EE2)2. 17 2. 17 ββ-estradiol (E2)-estradiol (E2)3. 4-nonylphenol (NP)3. 4-nonylphenol (NP)
1515ooC target temperature C target temperature for all life-stages for all life-stages
(12(12ooC for salmon embryos)C for salmon embryos)
Concentrations used represent:Concentrations used represent:1.1.Positive control dosePositive control dose2.2.Concentration common to wastewater Concentration common to wastewater effluenteffluent3.3.Low dose similar to that found in an Low dose similar to that found in an urbanized river/estuaryurbanized river/estuary4.4.Solvent control (<0.001%)Solvent control (<0.001%)
Flow through system, 2 to 7 volumes dFlow through system, 2 to 7 volumes d-1-1
Vtg gene expression (qPCR) and development of a sensitive EIA Vtg gene expression (qPCR) and development of a sensitive EIA for Vtg protein concentration (from Peck for Vtg protein concentration (from Peck et alet al. 2011). 2011)
Whole body mRNA for early life-stages, liver in juvenilesWhole body mRNA for early life-stages, liver in juveniles
Mortality dataMortality data
MethodologyMethodology
Vitellogenin as an indicator of Vitellogenin as an indicator of life-stage sensitivitylife-stage sensitivity
Common biomarker of estrogenic contaminant exposureCommon biomarker of estrogenic contaminant exposure
Indicative of potential long-term reproductive impactsIndicative of potential long-term reproductive impacts
Could be used in the field in early life-stage fishCould be used in the field in early life-stage fish
Treatment (nM)
control aE 0.004 aE 0.04 aE 0.4 E2 0.04 E2 0.4 E2 4.0 NP 4.0 NP 40 NP 400
Re
lative
tra
nsc
rip
tion
0
5
10
15
20
Treatment (nM)
control aE 0.004 aE 0.04 aE 0.4 E2 0.04 E2 0.4 E2 4.0 NP 4.0 NP 40 NP 400
Rela
tive t
ransc
riptio
n
0
200
400
600
800
B
C
2D Graph 6
X Data
control aE 0.004 aE 0.04 aE 0.4 E2 0.04 E2 0.4 E2 4.0 NP 4.0 NP 40 NP 400
Y D
ata
0
50
100
150
200
250
300
Norm to control
Treatment (nM)
control aE 0.004 aE 0.04 aE 0.4 E2 0.04 E2 0.4 E2 4.0 NP 4.0 NP 40 NP 400
Re
lativ
e t
ran
scrip
tion
0
500
1000
1500
2000 D
BA
Control 0.004 0.04 0.4 0.04 0.4 4.0 4.0 40 400
α-ethinylestradiol 17 β-estradiolNonylphenol Treatment (nM)
Control 0.004 0.04 0.4 0.04 0.4 4.0 4.0 40 400
α-ethinylestradiol 17 β-estradiolNonylphenol Treatment (nM)Rel
ative e
xpress
ion
(norm
alized
to EF
-1α)
Dose (nM) Dose (nM)
Treatment (nM)
control aE 0.004 aE 0.04 aE 0.4 E2 0.04 E2 0.4 E2 4.0 NP 4.0 NP 40 NP 400
Relative transcription
0
5
10
15
20
Treatment (nM)
control aE 0.004 aE 0.04 aE 0.4 E2 0.04 E2 0.4 E2 4.0 NP 4.0 NP 40 NP 400
Relative transcription
0
200
400
600
800
B
C
2D Graph 6
X Data
control aE 0.004 aE 0.04 aE 0.4 E2 0.04 E2 0.4 E2 4.0 NP 4.0 NP 40 NP 400
Y D
ata
0
50
100
150
200
250
300
Norm to control
Treatment (nM)
control aE 0.004 aE 0.04 aE 0.4 E2 0.04 E2 0.4 E2 4.0 NP 4.0 NP 40 NP 400
Relative transcription
0
500
1000
1500
2000 D
BA
Control 0.004 0.04 0.4 0.04 0.4 4.0 4.0 40 400
α-ethinylestradiol 17 β-estradiolNonylphenol Treatment (nM)
Control 0.004 0.04 0.4 0.04 0.4 4.0 4.0 40 400
α-ethinylestradiol 17 β-estradiolNonylphenol Treatment (nM)
Atlantic salmon vitellogenin mRNAAtlantic salmon vitellogenin mRNAα-ethinylestradiol (EE2)
17 β-estradiol (E2)
Nonylphenol (NP)
control
EE2 and E2 show significant treatment response, but this trend is EE2 and E2 show significant treatment response, but this trend is not dose dependent (2-way ANOVA). NP is not significantly not dose dependent (2-way ANOVA). NP is not significantly
different from control.different from control.
Rela
tive
tran
scrip
tion
Rela
tive
tran
scrip
tion
(nor
mal
ized
to E
F-1
(nor
mal
ized
to E
F-1 αα
)) embryosembryos
Atlantic salmon vitellogenin mRNAα-ethinylestradiol (EE2)
17 β-estradiol (E2)
Nonylphenol (NP)
control
Significant treatment effects, but dose-dependent Significant treatment effects, but dose-dependent response is less clear.response is less clear.
Rela
tive
tran
scrip
tion
(nor
mal
ized
to E
F-1α
) embryosembryos*
*Treatment (nM)
control aE 0.004 aE 0.04 aE 0.4 E2 0.04 E2 0.4 E2 4.0 NP 4.0 NP 40 NP 400
Re
lativ
e t
ra
nscrip
tio
n
0
5
10
15
20
Treatment (nM)
control aE 0.004 aE 0.04 aE 0.4 E2 0.04 E2 0.4 E2 4.0 NP 4.0 NP 40 NP 400
Re
lative
tran
scrip
tio
n
0
200
400
600
800
B
C
2D Graph 6
X Data
control aE 0.004 aE 0.04 aE 0.4 E2 0.04 E2 0.4 E2 4.0 NP 4.0 NP 40 NP 400
Y D
ata
0
50
100
150
200
250
300
Norm to control
Treatment (nM)
control aE 0.004 aE 0.04 aE 0.4 E2 0.04 E2 0.4 E2 4.0 NP 4.0 NP 40 NP 400
Rela
tive t
ranscrip
tion
0
500
1000
1500
2000 D
BA
Control 0.004 0.04 0.4 0.04 0.4 4.0 4.0 40 400
α-ethinylestradiol 17 β-estradiolNonylphenol Treatment (nM)
Control 0.004 0.04 0.4 0.04 0.4 4.0 4.0 40 400
α-ethinylestradiol 17 β-estradiolNonylphenol Treatment (nM)Rela
tive
expr
essi
on
(nor
mal
ized
to E
F-1α
)
Dose (nM) Dose (nM)
Treatment (nM)
control aE 0.004 aE 0.04 aE 0.4 E2 0.04 E2 0.4 E2 4.0 NP 4.0 NP 40 NP 400
Relative transcription
0
5
10
15
20
Treatment (nM)
control aE 0.004 aE 0.04 aE 0.4 E2 0.04 E2 0.4 E2 4.0 NP 4.0 NP 40 NP 400
Relative transcription
0
200
400
600
800
B
C
2D Graph 6
X Data
control aE 0.004 aE 0.04 aE 0.4 E2 0.04 E2 0.4 E2 4.0 NP 4.0 NP 40 NP 400
Y D
ata
0
50
100
150
200
250
300
Norm to control
Treatment (nM)
control aE 0.004 aE 0.04 aE 0.4 E2 0.04 E2 0.4 E2 4.0 NP 4.0 NP 40 NP 400
Relative transcription
0
500
1000
1500
2000 D
BA
Control 0.004 0.04 0.4 0.04 0.4 4.0 4.0 40 400
α-ethinylestradiol 17 β-estradiolNonylphenol Treatment (nM)
Control 0.004 0.04 0.4 0.04 0.4 4.0 4.0 40 400
α-ethinylestradiol 17 β-estradiolNonylphenol Treatment (nM)
Yolk-sac Yolk-sac larvaelarvae*
*
**
*
All compounds show significant treatment effect, but suggestive All compounds show significant treatment effect, but suggestive dose-dependent response is non-significant. Vtg response is 200 dose-dependent response is non-significant. Vtg response is 200
to 500 times Vtg control transcription.to 500 times Vtg control transcription.
α-ethinylestradiol (EE2)
17 β-estradiol (E2)
Nonylphenol (NP)
controlAtlantic salmon vitellogenin mRNAAtlantic salmon vitellogenin mRNA
Dose (nM)Dose (nM)
Treatment (nM)
control aE 0.004 aE 0.04 aE 0.4 E2 0.04 E2 0.4 E2 4.0 NP 4.0 NP 40 NP 400
Rel
ativ
e tr
ansc
riptio
n
0
5
10
15
20
Treatment (nM)
control aE 0.004 aE 0.04 aE 0.4 E2 0.04 E2 0.4 E2 4.0 NP 4.0 NP 40 NP 400
Rel
ativ
e tra
nscr
iptio
n
0
200
400
600
800
B
C
Treatment (nM)
control aE 0.004 aE 0.04 aE 0.4 E2 0.04 E2 0.4 E2 4.0 NP 4.0 NP 40 NP 400
Rel
ativ
e tra
nscr
iptio
n
0
500
1000
1500
2000 D
BA
Control 0.004 0.04 0.4 0.04 0.4 4.0 4.0 40 400
α-ethinylestradiol 17 β-estradiolNonylphenol Treatment (nM)
Control 0.004 0.04 0.4 0.04 0.4 4.0 4.0 40 400
α-ethinylestradiol 17 β-estradiolNonylphenol Treatment (nM)
Rela
tive
tran
scrip
tion
Rela
tive
tran
scrip
tion
(nor
mal
ized
to E
F-1
(nor
mal
ized
to E
F-1 αα
)) Feeding Feeding fryfry
*
*
*
*
*
EE2 and E2 elicit significant, dose-dependent response. Vtg mRNA is EE2 and E2 elicit significant, dose-dependent response. Vtg mRNA is highest in this life-stage at 500-1500 times control transcription. highest in this life-stage at 500-1500 times control transcription.
α-ethinylestradiol (EE2)
17 β-estradiol (E2)
Nonylphenol (NP)
controlAtlantic salmon vitellogenin mRNAAtlantic salmon vitellogenin mRNA
Treatment (nM)
control aE 0.004 aE 0.04 aE 0.4 E2 0.04 E2 0.4 E2 4.0 NP 4.0 NP 40 NP 400
Rel
ativ
e tr
ansc
riptio
n
0
5
10
15
20
Treatment (nM)
control aE 0.004 aE 0.04 aE 0.4 E2 0.04 E2 0.4 E2 4.0 NP 4.0 NP 40 NP 400
Rel
ativ
e tra
nscr
iptio
n
0
200
400
600
800
B
C
Treatment (nM)
control aE 0.004 aE 0.04 aE 0.4 E2 0.04 E2 0.4 E2 4.0 NP 4.0 NP 40 NP 400
Rel
ativ
e tra
nscr
iptio
n
0
500
1000
1500
2000 D
BA
Control 0.004 0.04 0.4 0.04 0.4 4.0 4.0 40 400
α-ethinylestradiol 17 β-estradiolNonylphenol Treatment (nM)
Control 0.004 0.04 0.4 0.04 0.4 4.0 4.0 40 400
α-ethinylestradiol 17 β-estradiolNonylphenol Treatment (nM)
Rela
tive
tran
scrip
tion
Rela
tive
tran
scrip
tion
(nor
mal
ized
to E
F-1
(nor
mal
ized
to E
F-1 αα
))
Dose (nM)Dose (nM)
SmoltsSmolts
*
* *
*
50
SummarySummary
E2 and EE2 elicit robust E2 and EE2 elicit robust response in Vtg mRNA.response in Vtg mRNA.
Feeding fry show highest Feeding fry show highest Vtg upregulation in Vtg upregulation in response to NP.response to NP.
Overall expression is Overall expression is highest in salmon smolts highest in salmon smolts (liver vs. whole animal (liver vs. whole animal differences?).differences?).
Dose (nM)
Treatment (nM)
control aE 0.004 aE 0.04 aE 0.4 E2 0.04 E2 0.4 E2 4.0 NP 4.0 NP 40 NP 400
Rel
ativ
e tr
ansc
riptio
n
0
5
10
15
20
Treatment (nM)
control aE 0.004 aE 0.04 aE 0.4 E2 0.04 E2 0.4 E2 4.0 NP 4.0 NP 40 NP 400
Rel
ativ
e tra
nscr
iptio
n
0
200
400
600
800
B
C
2D Graph 6
X Data
control aE 0.004 aE 0.04 aE 0.4 E2 0.04 E2 0.4 E2 4.0 NP 4.0 NP 40 NP 400Y
Da
ta0
50
100
150
200
250
300
Norm to control
Treatment (nM)
control aE 0.004 aE 0.04 aE 0.4 E2 0.04 E2 0.4 E2 4.0 NP 4.0 NP 40 NP 400
Rel
ativ
e tra
nscr
iptio
n
0
500
1000
1500
2000 D
BA
Control 0.004 0.04 0.4 0.04 0.4 4.0 4.0 40 400
α-ethinylestradiol 17 β-estradiolNonylphenol Treatment (nM)
Control 0.004 0.04 0.4 0.04 0.4 4.0 4.0 40 400
α-ethinylestradiol 17 β-estradiolNonylphenol Treatment (nM)
Rela
tive
tran
scrip
tion
(nor
mal
ized
to E
F-1α
) Feeding fry
*
*
*
*
*
Dose (nM)Dose (nM)
Smolt plasma vitellogeninSmolt plasma vitellogenin
α-ethinylestradiol
17 β-estradiol
Nonylphenol
control
Significant overall treatment effect and dose-Significant overall treatment effect and dose-dependent responsedependent response
Response to NP is muted compared to estrogensResponse to NP is muted compared to estrogens
EIA shows a clearer response than qPCR dataEIA shows a clearer response than qPCR data
Treatment
control aE 0.004 aE 0.04 aE 0.4 E2 0.04 E2 0.4 E2 4.0 NP 4.0 NP 40 NP 400
Pla
sma
Vtg
(u
g/m
l)
0
15000
20000
a
c
b,e
d
b e
c
d
f
g
Control 0.004 0.04 0.4 0.04 0.4 4.0 4.0 40 400
α-ethinylestradiol 17 β-estradiol Nonylphenol
Treatment (nM)
Plas
ma
Vtg
(μg/
ml)
Plas
ma
Vtg
(μg/
ml)
400
*
*
*
*
*
*
* *
*
Sturgeon Vtg mRNASturgeon Vtg mRNA2D Graph 1
X Data
0 1 2 3 4 5 6 7 8 9 10 11
Y D
ata
0
2
4
6
8
10
12
14
16
18
20
avg: 1.0000 avg: 1.9278 avg: 1.4410 avg: 2.3418 avg: 2.7432 avg: 3.5669 avg: 9.6482 avg: 4.8970 avg: 1.6046 avg: 4.0810
Treatment
control aE 0.004 aE 0.04 aE 0.4 E2 0.04 E2 0.4 E2 4.0 NP 4.0 NP 40 NP 400
Pla
sma
Vtg
(ug
/ml)
0
15000
20000
a
c
b,e
d
b e
c
d
f
g
Control 0.004 0.04 0.4 0.04 0.4 4.0 4.0 40 400
α-ethinylestradiol 17 β-estradiol Nonylphenol
Treatment (nM)
Rela
tive
Vtg
tran
scrip
tion
Rela
tive
Vtg
tran
scrip
tion
Dose (nM)Dose (nM)
Preliminary dataPreliminary data
Vtg response less Vtg response less robust than in salmonrobust than in salmon
Full analysiFull analysis will demonstrate if Vtg is an appropriate biomarker in early life-stage sturgeon
Feeding fry
ConclusionsConclusions
E2 and EE2 elicited nearly identical responses, and E2 and EE2 elicited nearly identical responses, and exposure to these compounds elicited strong Vtg response. exposure to these compounds elicited strong Vtg response.
Plasma Vtg may be the most appropriate biomarker for Plasma Vtg may be the most appropriate biomarker for exogenous estrogen exposure.exogenous estrogen exposure.
Vtg protein demonstrated a clear pattern of dose-Vtg protein demonstrated a clear pattern of dose-dependent response, but not applicable to early life stages.dependent response, but not applicable to early life stages.
Sensitivity vs. responsiveness of each life stage? Sensitivity vs. responsiveness of each life stage?
Development of appropriate biomarker for sturgeon.Development of appropriate biomarker for sturgeon.
Future directionsFuture directions
What are the implications of long-term exposure?What are the implications of long-term exposure?
Which species is most sensitive?Which species is most sensitive?
Comparison to surrogate speciesComparison to surrogate species
Funding and acknowledgementsFunding and acknowledgements
McCormick labMcCormick lab Amy RegishAmy Regish Arne ChristensenArne Christensen Michael O’DeaMichael O’Dea Joe ChadwickJoe Chadwick
Barbara ArterBarbara Arter
Bill Kier-Kier AssociatesBill Kier-Kier Associates