July 2003 October 2003 April 2004

Stim

ulat

ion

Inde

x

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

*

*A

July 2003 October 2003 April 20041.0

1.2

1.4

1.6

1.8

2.0

2.2

2.4

2.6

2.8

3.0

ASHCON

*B

Supplemental Figure 1. Seasonal lymphocyte proliferative response of anterior kidney leukocytes cultured from brown bullhead inhabiting the Ashtabula River and Conneaut Creek following an 18 hour in vitro exposure to concanavalin A (A) and phytohemagglutinin-P (B). Data presented as mean ± standard error; * indicates a significant difference (P ≤ 0.05).

Supplemental Figure 2 – Microscopic appearance of macrophage aggregates in the spleen and liver of largemouth bass and brown bullhead. (A) Hepatic macrophage aggregates of brown bullhead (arrow). (B) Splenic macrophage arrogate (a) of largemouth bass replete with hemosiderin.

Supplemental Figure 3 – Microscopic appearance of hepatic lesions in brown bullhead. (A) Normal brown bullhead liver illustrating hepatopancrease tissue (a) that contains occasional bile ducts (arrows) within the hepatic parenchyma (b). (B) Bile duct proliferation (arrows). (C) Fibrosis (arrows) surrounding normal bile ducts. ((D) Altered cell foci (a) within the normal hepatic parenchyma(b), (E) Cholangioma, a benign neoplasm of the bile ducts (a). (F) Cholangiosarcoma, a malignant neoplasm of the bile ducts (a).

October 2002 July 2003 October 2003 April 20040

500

1000

1500

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2500

Pla

sma

test

oste

rone

(pg/

ml)

**

0

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Pla

sma

17-e

stra

diol

(pg/

ml)

**

0.05

0.10

0.15

0.20

0.25

0.30

Gon

osom

atic

Inde

x

*

0

1000

2000

3000

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5000

6000

October 2002 July 2003 October 2003 April 2004

0

1000

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3000

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7000

*

*

0.0

0.5

1.0

1.5

2.0

2.5

3.0

ASH CON A B

C D

E F

Supplemental Figure 4 - Seasonal comparison of reproductive end-points in brown bullhead collected from the Ashtabula River and Conneaut Creek. Gonadosomatic index (A, B) plasma 17β-estradiol (C, D) and testosterone (E, F) in in males and females respectively; mean ± standard error; * indicates a significant difference (P ≤ 0.05).

0

100

200

300

400

500

600

Pla

sma

17b-

estra

dio

-es

tradi

ol (p

g/m

l)

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6000

Gon

osom

atic

Inde

x0.0

0.1

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0.4

0.5 **

0

1

2

3

4

5

6

7

ASH CON

*

October 2002 July 2003 October 2003 April 20040

200

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1200

Pla

sma

test

oste

rone

(pg/

ml)

October 2002 July 2003 October 2003 April 2004

0

200

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600

800

1000

1200

1400

A B

C D

E F

Supplemental Figure 5 - Seasonal comparison of reproductive end-points in largemouth bass collected from the Ashtabula River and Conneaut Creek. Gonadosomatic index (A, B) plasma 17β-estradiol (C, D) and testosterone (E, F) in in males and females respectively; mean ± standard error; * indicates a significant difference (P ≤ 0.05).

ppm

(mg/

kg)

0.00

0.02

0.04

0.06

0.08

0.10

0.12

*

*

*

* *

*

**

*

**

*

*

ppm

(mg/

kg)

0.00

0.02

0.04

0.06

0.08

0.10

0.12ASHCON

*

*

* **

*

*

*

*

*

**

*

*

A

B

HCB

1,2,

3,4-

TCB

PCA

-BHC

-Chl

orda

neO

xych

lord

ane

Hept

achl

or e

poxid

e

Aldr

inDi

eldr

inp,

p-DD

To,

p-DD

Tp,

p-DD

Eo,

p-DD

Ep,

p-DD

D

Supplemental Figure 6. Cumulative mean body burden of organochlorine and polyaromatic hydrocarbons in brown bullhead (A) and largemouth bass (B) collected from the Ashtabula River and Conneaut Creek during October 2003 and April 2004; mean ± standard error; * indicates a significant difference (P ≤ 0.05).