A Pilot Study of the Effects of Contaminants on Shiner Surf Perch in

the San Francisco Bay-Estuary

Robert Spies, AMSRobert Spies, AMS

Kathrine Springman, UC Davis Kathrine Springman, UC Davis

July 2006July 2006

Problem

Populations of many fish in the SF Estuary Populations of many fish in the SF Estuary and its watershed are in a long-term decline.and its watershed are in a long-term decline.

There are many possible causes of the these There are many possible causes of the these declines including habitat loss, harvest, declines including habitat loss, harvest, introduced species, and contaminants.introduced species, and contaminants.

Contaminants probably are having a negative Contaminants probably are having a negative effect on some populations.effect on some populations.

Early gametogenesis:yolk

Late gametogenesis:

Internal fertilization

females males

implantation

gestation

birth

Recreational fishing

Habitat damage

Disease resistance

Growth

Mating

Foraging

sewage, street runoff, Ag runoff, atmospheric sources, etc.

PAHorganophos-phatepesticidespharma-ceuticals,DDTs, PCBs, chlorinatedpesticides,carbamatepesticides

Breeding population

Conceptual model for shiner surf perch declines

Objective Determine if shiner surfperch (Cymatogaster

aggregata; Embiotocidae) show effects of contamination on some aspect of their fitness, growth or reproduction.

Strategy2005-2006: 2005-2006:

1.1. Field studies relating exposure to fitness, Field studies relating exposure to fitness, growth, and reproduction.growth, and reproduction.

2.2. Establish surfperch culture for possibleEstablish surfperch culture for possible

laboratory exposure experiments.laboratory exposure experiments.

Today’s report

2006 goals2006 goals Collection and analyses Collection and analyses

to dateto date Culture startedCulture started Sex ratiosSex ratios Condition indicesCondition indices Number youngNumber young Weight youngWeight young Gonadosomatic indicesGonadosomatic indices

HistopathologyHistopathology E2 injection experimentE2 injection experiment Choriogenin measuresChoriogenin measures EROD activitiesEROD activities Genetic analysesGenetic analyses

Field studies-2006 Goals Collect 20+ fish each from 2 contaminated; 1 Collect 20+ fish each from 2 contaminated; 1

less-contaminated sites. less-contaminated sites. New control siteNew control site.. Measurements: length, weight, sex, no. Measurements: length, weight, sex, no.

young, size of young, gonad weight.young, size of young, gonad weight. Analyses: organic chemicals (carcass), Analyses: organic chemicals (carcass),

P4501A (adult liver,viscera of young).P4501A (adult liver,viscera of young). EROD (liver), choriogenin.EROD (liver), choriogenin. Mother-vs-offspring measures: Mother-vs-offspring measures:

morphometrics,P4501A, some morphometrics,P4501A, some histopathology.histopathology.

Progress to date I: Field studies in 2006

Collected 20+ adults by beach seine from Collected 20+ adults by beach seine from Oakland Middle Harbor (5/20/06).Oakland Middle Harbor (5/20/06).

Collected 26 fish by beach seine from State Collected 26 fish by beach seine from State Park Beach at Candlestick Park, So. San Park Beach at Candlestick Park, So. San Francisco (5/22/06).Francisco (5/22/06).

Collected 20 fish in by beach seine at Heart’s Collected 20 fish in by beach seine at Heart’s Desire Beach, SW Tomales Bay (6/1/05).Desire Beach, SW Tomales Bay (6/1/05).

All fish measured, weighed, dissected, sexed, All fish measured, weighed, dissected, sexed, young counted and weighed, tissues stored.young counted and weighed, tissues stored.

Progress to date III: Start of culture

Caught 56 shiners in Caught 56 shiners in Big River on 2/29/06.Big River on 2/29/06.

Transported to Bodega Transported to Bodega Bay Marine LaboratoryBay Marine Laboratory

Weighed ea fish.Weighed ea fish. 26 fish in ea of 2 tanks26 fish in ea of 2 tanks Fed 2% body wt./day.Fed 2% body wt./day. 98% survival.98% survival.

Progress to date III

Estradiol injection experiment performed 6/9/06.Estradiol injection experiment performed 6/9/06. Choriongenin assays completed at Bodega Marine Choriongenin assays completed at Bodega Marine

Laboratory on estradiol injected fish.Laboratory on estradiol injected fish. Histopathological analyses completed on 2005 fish at Histopathological analyses completed on 2005 fish at

Duke University.Duke University. EROD assays on 2006 fish completed.EROD assays on 2006 fish completed. Genetic analyses completed on 2005 fish at Tiburon Genetic analyses completed on 2005 fish at Tiburon

Center.Center.

General observations All of the fish caught in SF and Tomales Bay were sexually All of the fish caught in SF and Tomales Bay were sexually

mature. Smaller fish >8 g from Big River in 2005 were mature. Smaller fish >8 g from Big River in 2005 were generally not sexually mature.generally not sexually mature.

All female fish from SF Bay were pregnant except 5 (17%) All female fish from SF Bay were pregnant except 5 (17%) large females from Candlestick (2005-2006).large females from Candlestick (2005-2006).

Sex ratio (M:F) (2005-2006) more skewed in SF Bay towards Sex ratio (M:F) (2005-2006) more skewed in SF Bay towards females. Oakland Middle Harbor: 1:10.5;Candlestick Park females. Oakland Middle Harbor: 1:10.5;Candlestick Park 1:2.02; Tomales bay 1:1.08 Big River: 1:1.161:2.02; Tomales bay 1:1.08 Big River: 1:1.16

Heart’s Desire Beach, Tomales Bay appears to be a better Heart’s Desire Beach, Tomales Bay appears to be a better control than Big River. control than Big River.

Big River was colder than SF Bay (05 finding).Big River was colder than SF Bay (05 finding). Big River fish are exposed to p4501A inducers (2005 finding).Big River fish are exposed to p4501A inducers (2005 finding).

0

5

10

15

20

25

30

Nu

mb

er

male

fem

ale

male

fem

ale

male

fem

ale

male

fem

ale

Oak SFC BR TB

Sex within Location

0.5

1.0

1.5

2.0

2.5

3.0adju

ste

d c

ondit

ion f

acto

r (w

-wt.

young/l^

3)

male

fem

ale

male

fem

ale

male

fem

ale

male

fem

ale

Oak SFC BR TB

Sex within Location

ad

just

ed

con

dit

ion

fact

or

(w-w

t. y

ou

ng

/l^

3)

0.5

1

1.5

2

2.5

3

BR Oak SFC TB

Location

Each PairStudent's t 0.05Sex p=0.004

EROD p=0.0196

num

ber

of

young

0

10

BR Oak SFC TB

Location

Location p=0.007Weight of mother p=0.001EROD N.S.

wt.

/off

spri

ng

0

0.5

1

1.5

2

BR Oak SFC TB

Location

Location p=0.02Wet wt mother p<0.0001EROD p=0.001

y = 0.0123x - 0.0187

R2 = 0.1616

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

0 10 20 30 40 50 60

Wet weight mother

We

igh

t/o

ffsp

rin

g

-1

0

1

2

3

4

5

6

7

8

9

10

11

GS

I

Oak SFC BR TB Oak SFC BR TB

male female

Location within Sex

GSI vs. Condition index

y = 1.6311x - 0.3034

R2 = 0.2775

0

2

4

6

8

10

12

0 0.5 1 1.5 2 2.5 3 3.5

Adj. Condition Indx.

GS

I (w

ho

le w

t./g

on

ad

wt.

*(1

00

)

EROD Activity, Surfperch 2005

0

20

40

60

80

100

120

140

Oakland MiddleHarbor, male

(5)

Oakland MiddleHarbor, female

(14)

CandlestickPark, male (3)

CandlestickPark, female

(10)

Big River, male(8)

Big River,female (10)

Site, gender

ER

OD

Me

an

(p

mo

l/m

g/m

in)

2006 EROD activity by location & sex

-1

0

1

2

3

4

5

6

7

8

9

ER

OD

male

fem

ale

male

fem

ale

male

fem

ale

Oak SFC TB

Sex within Location

Location p=0.0009Sex p<0.0001

Examination and scoring of fish from 2005 field Examination and scoring of fish from 2005 field collections.collections.

1.1. Liver, heart, gills and gonad examined. Scored from 0 Liver, heart, gills and gonad examined. Scored from 0 (normal) to 5 (severe) lesions.(normal) to 5 (severe) lesions.

2.2. Few lesions found. Few lesions found. Branchitis:Branchitis:

5/20 fish at Candlestick with branchitis. One w. lamellar aneurisms5/20 fish at Candlestick with branchitis. One w. lamellar aneurisms0/20 fish at Big River; 0 of 2 at Oakland0/20 fish at Big River; 0 of 2 at Oakland

Ovary:Ovary:One intersex morphological female from Candlestick Park.One intersex morphological female from Candlestick Park.Two females with pale ovaries with decreased cell density.Two females with pale ovaries with decreased cell density.

Liver:Liver:One fish ea from Candlestick and Big River with fatty change.One fish ea from Candlestick and Big River with fatty change.

Heart:Heart:One fish from Oakland w mild macrophage aggregatesOne fish from Oakland w mild macrophage aggregates

Histopathology results

Preliminary results Livers of All field fish tested strongly positive Livers of All field fish tested strongly positive with 2 anti-bodies: anti-salmon (Biosense) and with 2 anti-bodies: anti-salmon (Biosense) and anti-herring (BML).anti-herring (BML). 48-hr E48-hr E22 injection experiment with 5 mg kg injection experiment with 5 mg kg-1-1 to induce ZRP; uninjected controls, solvent to induce ZRP; uninjected controls, solvent control, injected w Econtrol, injected w E22. Fish from Big River . Fish from Big River were used. Plasma of 2 injected fish and were used. Plasma of 2 injected fish and controls reacted very weakly with anti-herring controls reacted very weakly with anti-herring choriogenin.No reaction from females.choriogenin.No reaction from females.

Choriogenin marker

Preliminary choriogenin results (cont’d) Livers of All field fish tested strongly positive Livers of All field fish tested strongly positive

with 2 anti-bodies: anti-salmon (Biosense) and with 2 anti-bodies: anti-salmon (Biosense) and anti-herring (BML).anti-herring (BML).

48-hr E48-hr E22 injection experiment with 5 mg kg injection experiment with 5 mg kg-1-1 to to induce ZRP; uninjected controls, solvent induce ZRP; uninjected controls, solvent control, injected w Econtrol, injected w E22. Shiners from Big River . Shiners from Big River were used. Plasma of injected fish and controls were used. Plasma of injected fish and controls reacted with anti-herring, but not anti-salmon.reacted with anti-herring, but not anti-salmon.

Antibody reacts to choriogenin induced by EAntibody reacts to choriogenin induced by E22 in in 2/5 fish. Marker is not ready to use ion field.2/5 fish. Marker is not ready to use ion field.

TAACCGTTAGTCTTAAGAAATCAACTGATGGTAGGCTCTTACTACATTAAAATTTGGTTCTTGACGAGATGTTGAA

TCTTGGTATAACTTGACTTATGAATAGTGTGTTCGGTCTTAAGTTTCTTCCAATTTAATTGAATTTAAAAGTAAAT GTAATAATAATATGTTTTAAGTATATCTTAGGGTTGTGTACTAAACAGAAATGGTTAATATCGTTTAATGGTGATTA G ATACAT ATATATGC ATATATGC ATATATGC ATATATATA--CAT ATATATAAAATTGGCGGAGCCCGGCAAA CAT ATATATGC ATATATGC ATATATGC ATATATATA--TAT ATATATAAAA CAT ATATATGC ATATATGC ATATATGC ATATATATACATAT ATATATAAAA CAT ATATATGC ATATATGC ATATATGC ATATATATATACAT ATATATAAAA CAT ATATATGC ATATATGC ATATATGC ATATATATA----C ATATATAAAA CAT ATATATGC ATATATGC ATATATGC ATATATATA----T ATATATAAAA CAT ATATATGC ATATATGC ATATATGC ATATATACA----T ATATATAAAA

GAATAGTTTAGCTTAAGAATCCTAGCTTTGGGA

TACCACAGCAGCATTTCTGGCGAGCAACAAAGCAAGACAGAGGGTTTTGATGGCTTGTTAAGTGGTGACAC A TTGGCACATTGTGTGTAACGGTGAGGCGGGGAGATGCAACGGCAAAACGACCGGTTTTGACCCTCTTCTGG CTACCTTGGACTCTCTGTCTCTGGACGGAGAGGAAACATGCTCCAACAGTGAGCTCTTCAGCGCTTTGGAG TAATCTGGGCCTGAACGCTGAGGATCTGGAGCTGCTGCTTCTGGATGAGAGGATGATCCAGGTGGAGCTGGA

CCCCAACCACATCCCCACACTCAGTGACCTTCTTACCAACAACGAAATCCTTTCCTACATCCAGGACTCTC

TGGATAGCGGGGCAGAGGCAAAGGGACAACAGGACAGAGGTGGGTATGGACCTTCGAGCCATCCATCAAAC TCCAGACTCCTCCCAAAGTGTCTTCCAGCAGGCATGTTTGACGCCTGCTGTCCCTCCAACTCTTCCCAGCAG

CAGGCAGCCCATCGTCCAGCTGTCCSample D-loop Sequence

(polymorphisms shown below)

Sample AHR1 Sequence(polymorphisms shown below)

Single Nucleotide Polymorphisms (SNPs)

Length Variation

Mitochondrial genetic variation in the dloop region for population connectivity estimates in shiner surfperch

(Cohen, Schinske, and Spies)

Bodega Harbor

Big River

Distribution of AHR1 Alleles at 3 SitesDifferent colors in pie charts represent different alleles.

n = 12

n = 9

n = 18

Cohen, Schinske, Spies

Bodega Harbor

Big River

Distribution of D-Loop Alleles at 3 SitesSNP Alleles (Left) and Length Variation Alleles (Right)

n = 18

n = 10

n = 19

Cohen, Schinske, and Spies

Current surfperch genetic data

AHR1AHR1

Low variation in initial exon 10 transcriptional control region. Low variation in initial exon 10 transcriptional control region. Consider catalytic and other functional regions/loci, wrt Consider catalytic and other functional regions/loci, wrt functional assays.functional assays.

D-loopD-loop

No indication of population subdivision between Big River, Bodega, and San No indication of population subdivision between Big River, Bodega, and San Francisco based on dloop SNPs. Additional sampling would make repeat Francisco based on dloop SNPs. Additional sampling would make repeat region data informative.region data informative.

Observations and thoughts Sex ratios continue to suggest some possible Sex ratios continue to suggest some possible

estrogenic effects in SF Bay. When is sex of young estrogenic effects in SF Bay. When is sex of young fixed? Tools?fixed? Tools?

Males in SF Bay: fewer, lower condition and lower GSI Males in SF Bay: fewer, lower condition and lower GSI than controls. Is there poor survival of males in SF than controls. Is there poor survival of males in SF Bay? Endocrine disruption effect?Bay? Endocrine disruption effect?

Females from Oakland with greater number of young Females from Oakland with greater number of young (accounting for effect of mother’s weight).(accounting for effect of mother’s weight).

Condition index and weight of offspring are correlated Condition index and weight of offspring are correlated with EROD.with EROD.

Are EDCs accelerating sexual maturity and fecundity? Are EDCs accelerating sexual maturity and fecundity? Is there a cost to the population? Is there a cost to the population?

.15

.20

.25

.30

.35

.40

.45

.50

.55

.60M

ax o

ocy

te d

ia (

mm

)

control low DDT high DDT PCB

(15)(14) (13) (13)

Acceleration of oogenesis in kelp bass dosed with DDT and PCB

Priorities for additional work in 2006

1.Histology of gonads to look for abnormalities. Status of developing oocytes.

2. Chemical analyses of carcasses.

3. Testosterone and estradiol in plasma.

4. Otolith work to age fish. Age structure of populations.

5. Analysis of EROD data.

The Problem The relative binding capacity of synthetic chemicals to hormone

receptors may be 1/100 to 1/1000 of that of the natural ligand (e.g., estrogen) and the chemical concentration may exceed the effective

concentration by 100 to 10,000 X.

The Problem: there are numerous and complex endocrine interactions in reproduction

and growth of fishes

(sensory input-endocrine cascades--feedback loops)

Agonist for gonadotropin I acceleration of gametogenesis

mature female fish

pre-vitellogenic oocytes

vitellogenic oocytes

ovulated eggseggs

fertilization success

hatching success

yolk sac larvaefry

yolk sac adsorption

juvenile

growth

metamorphosis

embryological development

male fish

spawning success

sperm`

first feeding

Sex determination

Starry flounder in SF Bay

Reproductive success in

relationship to a biomarker

of contaminant

exposure

Shiner surf perch is a good choice

Accumulate high concentrations of contaminants (Davis et al., 2001). High site fidelity (Fritzsche and Collier 2001). Occur from Alaska to Mexico. Habitat is in shallows and channels where RMP chemical data taken

since 1993. Analyzed by the sport fish program, included in the Pacific States

Marine Recreational Fisheries Statistics Survey, & the IEP. Favorite of urban fishermen. Populations in decline. Live-bearers --annual reproductive output (4-36 young annually) can be determined

in early spring and summer.

The evidence so far: contaminants

1. Starry flounder reproductive dysfunction linked to P4501A and PCBs (Spies & Rice, 1988).

2. Sex reversal and estrogenic effects in longjaw mud suckers (Cherr et al., unpubl.).

3. Chinook salmon in upper watershed show high proportions of females that are phenotypically male (Williamson and May, 2002).

3. Striped bass larval growth and histology negatively affected (Ostrach, unpubl.).

4. Speckeld sanddab histology (Spies et al., 1993; Gunther et al. 1997).

5. Herring egg abnormal development (Vines et al., 2000).

0.0

0.1

0.2

0.3

0.4

0.5

0.6

Wt.

/off

spri

ng (

mean)

Oak SFC BR TB

Location