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)


Rela

tive t

ransc

riptio

n

0

200

400

600

800

B

C

2D Graph 6

X Data


Y D

ata

0

50

100

150

200

250

300

Norm to control

Treatment (nM)


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)


Relative transcription

0

5

10

15

20

Treatment (nM)



0

200

400

600

800

B

C

2D Graph 6

X Data


Y D

ata

0

50

100

150

200

250

300

Norm to control

Treatment (nM)



0

500

1000

1500

2000 D

BA

Control 0.004 0.04 0.4 0.04 0.4 4.0 4.0 40 400


Control 0.004 0.04 0.4 0.04 0.4 4.0 4.0 40 400


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)


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)


Re

lativ

e t

ra

nscrip

tio

n

0

5

10

15

20

Treatment (nM)


Re

lative

tran

scrip

tio

n

0

200

400

600

800

B

C

2D Graph 6

X Data


Y D

ata

0

50

100

150

200

250

300

Norm to control

Treatment (nM)


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


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)



0

5

10

15

20

Treatment (nM)



0

200

400

600

800

B

C

2D Graph 6

X Data


Y D

ata

0

50

100

150

200

250

300

Norm to control

Treatment (nM)



0

500

1000

1500

2000 D

BA

Control 0.004 0.04 0.4 0.04 0.4 4.0 4.0 40 400


Control 0.004 0.04 0.4 0.04 0.4 4.0 4.0 40 400


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)


Nonylphenol (NP)

controlAtlantic salmon vitellogenin mRNAAtlantic salmon vitellogenin mRNA

Dose (nM)Dose (nM)

Treatment (nM)


Rel

ativ

e tr

ansc

riptio

n

0

5

10

15

20

Treatment (nM)


Rel

ativ

e tra

nscr

iptio

n

0

200

400

600

800

B

C

Treatment (nM)


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


Control 0.004 0.04 0.4 0.04 0.4 4.0 4.0 40 400


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)


Nonylphenol (NP)

controlAtlantic salmon vitellogenin mRNAAtlantic salmon vitellogenin mRNA

Treatment (nM)


Rel

ativ

e tr

ansc

riptio

n

0

5

10

15

20

Treatment (nM)


Rel

ativ

e tra

nscr

iptio

n

0

200

400

600

800

B

C

Treatment (nM)


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


Control 0.004 0.04 0.4 0.04 0.4 4.0 4.0 40 400


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)


Rel

ativ

e tr

ansc

riptio

n

0

5

10

15

20

Treatment (nM)


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)


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


Control 0.004 0.04 0.4 0.04 0.4 4.0 4.0 40 400


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


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


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

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