Prepared in cooperation with the U.S. Environmental Protection Agency

Assessment of Microscopic Pathology in Fishes Collected at 

Sites Impacted by Wood Tar in Pennsylvania

Open-File Report 2020–1024

U.S. Department of the Interior

U.S. Geological Survey

Assessment of Microscopic Pathology 

in Fishes Collected at Sites Impacted by 

Wood Tar in Pennsylvania

By Heather L. Walsh, Vicki S. Blazer, Patricia M. Mazik, Adam J. Sperry, and 

Diana Pavlick

Prepared in cooperation with the U.S. Environmental Protection Agency

Open-File Report 2020–1024

U.S. Department of the Interior

U.S. Geological Survey
U.S. Department of the Interior

DAVID BERNHARDT, Secretary

U.S. Geological Survey

James F. Reilly II, Director

U.S. Geological Survey, Reston, Virginia: 2020

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Suggested citation:

Walsh, H.L., Blazer, V.S., Mazik, P.M., Sperry, A.J., and Pavlick, D., 2020, Assessment of microscopic pathology in 

fishes collected at sites impacted by wood tar in Pennsylvania: U.S. Geological Survey Open-File Report 2020–1024, 

14 p., https://doi.org/ 10.3133/ ofr20201024.

ISSN 2331-1258 (online)
iii

Acknowledgments

This project was funded by the U.S. Environmental Protection Agency (EPA), Region 3, and by the 

U.S. Geological Survey’s Environmental Health (Contaminant Biology) and Cooperative Fish and 

Wildlife Research Unit programs. The authors thank the EPA Wheeling office personnel for fish 

collections and associated field data.

v

Contents

Acknowledgments........................................................................................................................................iii

Abstract...........................................................................................................................................................1

Introduction.....................................................................................................................................................1

Methods...........................................................................................................................................................3

Field Methods........................................................................................................................................3

Histopathology.......................................................................................................................................3

Results .............................................................................................................................................................4

Meade Run.............................................................................................................................................4

Threemile Run........................................................................................................................................4

Kinzua Creek..........................................................................................................................................8

Summary........................................................................................................................................................14

References Cited..........................................................................................................................................14

Figures

1. Maps showing fish collection sites on Kinzua Creek (KC), Threemile Run (TR), 

and Meade Run (BG), Pennsylvania ..........................................................................................2

2. Microscopic observations in Redside Dace and Creek Chub collected at the 

Threemile Run upstream site (TR1) ............................................................................................6

3. Microscopic observations in Common Shiner collected at the Mt. Alton site 

(TR2) of Threemile Run .................................................................................................................8

4. Microscopic observations in Creek Chub collected at Kinzua Creek ...............................11

Tables

1. Site information for fish collections at potential wood tar impacted sites .........................1

2. Summary of fish species, sample number, and visible abnormalities observed at 

wood tar assessment sites .........................................................................................................3

3. Meade Run (BG1) site Mottled Sculpin observations ............................................................4

4. Observations of fish collected at the Threemile Run upstream site (TR1) ..........................5

5. Observations of Common Shiner collected at the Threemile Run Mt. Alton 

site (TR2).........................................................................................................................................7

6. Threemile Creek Mt. Alton site (TR2) Western Blacknose Dace observations .................9

7. Kinzua Creek upstream site (KC1) Western Blacknose Dace observations .....................10

8. Kinzua Creek upstream site (KC1) Creek Chub observations ..............................................12

9. Kinzua Creek Backus site (KC2) Creek Chub observations .................................................13
vi

Conversion Factors

U.S. customary units to International System of Units

Multiply By To obtain

Length

inch (in.) 2.54 centimeter (cm)

inch (in.) 25.4 millimeter (mm)

foot (ft) 0.3048 meter (m)

mile (mi) 1.609 kilometer (km)

yard (yd) 0.9144 meter (m)

Volume

ounce, fluid (fl. oz) 0.02957 liter (L)

pint (pt) 0.4732 liter (L)

quart (qt) 0.9464 liter (L)

gallon (gal) 3.785 liter (L)

Mass

ounce, avoirdupois (oz) 28.35 gram (g)

pound, avoirdupois (lb) 0.4536 kilogram (kg)

Temperature in degrees Celsius (°C) may be converted to degrees Fahrenheit (°F) as follows:

 °F=(1.8×°C)+32

Temperature in degrees Fahrenheit (°F) may be converted to degrees Celsius (°C) as follows:

 °C=(°F–32)/1.8

Abbreviations

BG1 Meade Run site

KC1 Kinzua Creek site

KC2 Backus Creek site

PAHs polycyclic aromatic hydrocarbons

TR1 Threemile Run site

TR2 Mt. Alton site
Assessment of Microscopic Pathology in Fishes Collected 

at Sites Impacted by Wood Tar in Pennsylvania

By Heather L. Walsh,1 Vicki S. Blazer,1 Patricia M. Mazik,1 Adam J. Sperry,1 and Diana Pavlick2

Abstract

In an effort to determine whether fish populations in an 

area affected by wood tar waste exhibited health effects, fish 

were collected and analyzed with histopathology. Multiple 

species, including Mottled Sculpin (Cottus bairdii), Creek 

Chub (Semotilus atromaculatus), White Sucker (Catostumus 

commersonii), Redside Dace (Clinostomus elongatus), 

Common Shiner (Luxilus cornutus), and Western Blacknose 

Dace (Rhinichthys obtusus) were sampled from a reference 

site, Meade Run, and potentially affected streams, Kinzua 

Creek and Threemile Run, in northwestern Pennsylvania. A 

full histopathological evaluation was conducted to identify 

microscopic abnormalities potentially associated with wood 

tar exposure. The evaluation identified primarily parasites 

associated with tissue changes. These included microsporidian parasites in the ovaries of Common Shiner and Western 

Blacknose Dace; myxozoan cysts in the muscle of Common 

Shiner, Creek Chub, and Western Blacknose Dace; trematode cysts in the muscle of Creek Chub, Redside Dace and 

Common Shiner; and coccidia in spleen or pancreas of Creek 

Chub and Common Shiner. Microscopic abnormalities potentially associated with chemical exposure included ceroid/

lipofuscin deposits in the meninges of the olfactory lobe of the 

brain in Common Shiner, Western Blacknose Dace, and Creek 

Chub, as well as bile duct proliferation and a biliary tumor in 

1U.S. Geological Survey.

2West Virginia University.

Creek Chub. Overall, the findings did not reveal significant 

microscopic pathology consistent with exposure to wood 

tar waste.

Introduction

Kinzua Creek is a 42.6-kilometer tributary of the Allegheny River in northwestern Pennsylvania. The former Day 

Chemical Company facility located in the area converted 

lumber to charcoal, methanol, and acetic acid. Wood tar is a 

tar-like waste generated by the chemical process and deposited into the ground. The material, which contains phenolic 

compounds and polycyclic aromatic hydrocarbons (PAHs), 

formed lagoons and flowed into the creek. Land use in the 

watershed is primarily forested with some residential area 

bordering the forests. Two sites (Mt. Alton and Backus) were 

identified by the U.S. Environmental Protection Agency (EPA) 

Region 3 Superfund Biological Technical Advisory Group for 

remediation of wood tar waste. The assessment plan was to 

sample fish for health-surveys from a site upstream from the 

Backus site on Kinzua Creek (KC1) and immediately below 

the impacted Backus site (KC2). Additionally, a site upstream 

from the Mt. Alton site on Threemile Run (TR1) and immediately below the Mt. Alton site (TR2) on Threemile Run were 

sampled. Meade Run (BG1), a tributary of Kinzua Creek 

not impacted by wood tar waste, was sampled as a reference 

(table 1; fig. 1).

Table 1. Site information for fish collections at potential wood tar impacted sites.

Site designation Stream Site Latitude 

(decimal degrees)

Longitude 

(decimal degrees)

BG 1 Meade Run Reference 41.73869 78.78649

TR 1 Threemile Run Upstream of Mt. Alton 41.79409 78.62258

TR 2 Threemile Run Mt. Alton 41.78517 78.60868

KC 1 Kinzua Creek Upstream of Backus 41.81312 78.58243

KC 2 Kinzua Creek Backus 41.79850 78.57615
2 Assessment of Microscopic Pathology in Fishes Collected at Sites Impacted by Wood Tar in Pennsylvania

!

!

!

!

!

!

!

!

!

!

!

!

Area shown

in detail

MARYLAND

New Jersey

Delaware

DELAWARE DISTRICT OF

COLUMBIA

Buffalo

Warren

Altoona

Bradford

Morgantown

Martinsburg

Chambersburg

Gaithersburg

State College

78°0'W 76°0'W 74°0'W

42°0'N

40°0'N

PENNSYLVANIA

MARYLAND

WEST VIRGINIA

VIRGINIA

NEW YORK

N

BG 1

TR 2

TR 1

KC 2

KC 1

Meade Run

Kinzua Creek

Threemile Run

78°45'W 78°40'W

41°50'N

41°45'N

41°40'N

Gifford

Cyclone

Lewis Run

Lafayette Twp

Mt. Jewett

Base from U.S. Geological Survey digital data

BG 1

EXPLANATION

Location of fish collection 

site and identifier

0 1 2 3 KILOMETERS

0 1 2 3 MILES

Figure 1. Fish collection sites on Kinzua Creek (KC), Threemile Run (TR), and Meade Run (BG), Pennsylvania.
Methods 3

Methods

Field Methods

Sites were selected by EPA Region 3 and the fish were 

sampled by the EPA Freshwater Biology team from Wheeling, West Virginia. The incidence of deformities, fin erosion, 

lesions, and tumors (DELTs), as described by Sanders and 

others (1999), were documented. The EPA team preserved the 

fish in a 10-percent buffered formalin to be used for histopathological assessment. For the histopathology subsamples, 

as many as 30 random fish of the two most numerous species 

at each site were examined and preserved. The following species were collected for histopathology: Creek Chub (Semotilus 

atromaculatus), White Sucker (Catostumus commersonii), 

Common Shiner (Luxilus cornutus), Western Blacknose Dace 

(Rhinichthys obtusus), Mottled Sculpin (Cottus bairdii), and 

Redside Dace (Clinostomus elongatus). Individual species and 

sample sizes varied among sites (table 2). Preserved samples 

were transported to the U.S. Geological Survey Leetown 

Science Center. Total body lengths were only provided by the 

field crew for the Meade Run fishes.

Histopathology

Preserved samples were processed in two ways. If fish 

were small (<10 centimeters), whole fish were placed into 

decalcification solution (Thermo Scientific Richard-Allan 

Scientific Decalcifying Solution, ThermoFisher Scientific, 

Waltham, Mass.) for 1–3 days. Once decalcified, a cut was 

made behind the opercles to remove the head. The head was 

cut longitudinally into two pieces and placed into 1–2 cassettes. The tail area was removed, and the body cut longitudinally and both pieces placed in 1–2 cassettes depending 

on size. For larger fish, individual organs (liver, posterior 

and anterior kidney, spleen, heart, gills, any specific lesions) 

were removed.

Tissue samples were placed in cassettes, routinely processed through graded alcohols, and embedded into paraffin. 

Blocks were sectioned at 5 micrometers (μm) and stained with 

hematoxylin and eosin (Luna, 1992). Slides were examined 

for any abnormalities at the microscopic level.

Table 2. Summary of fish species, sample number, and visible abnormalities observed at wood tar assessment sites.

[DELTs, deformities, fin erosions, lesions, or tumors]

Site Site designation Species Total number of 

individuals

Number of fish 

with DELTs

Meade Run BG 1 Mottled Sculpin 23 0

Threemile Creek upstream TR 1 Creek Chub 27 1

TR 1 White Sucker 1 1

TR 1 Redside Dace 4 0

Threemile Creek Mt. Alton TR 2 Common Shiner 35 0

TR 2 Western Blacknose Dace 30 0

TR 2 White Sucker 1 1

Kinzua Creek upstream KC 1 Western Blacknose Dace 30 0

KC 1 Creek Chub 30 0

Kinzua Creek Backus KC 2 Creek Chub 30 0
4 Assessment of Microscopic Pathology in Fishes Collected at Sites Impacted by Wood Tar in Pennsylvania

Results

The same species were not consistently collected at both 

upstream and downstream sites or at the reference site. Species 

collected include Mottled Sculpin (BG1), White Sucker (TR1, 

TR2), Western Blacknose Dace (TR2, KC1), Creek Chub 

(TR1, KC1, KC2), and Common Shiner (TR2). Detailed information and histological findings are detailed by site below.

Meade Run

At the reference site, Meade Run (BG1), only Mottled 

Sculpin were collected on October 1, 2018, and preserved 

for histopathology. These were not collected at either Kinzua 

Creek or Threemile Run sites, making a true comparison to a 

reference site impossible. Nine male, 13 female, and 1 unidentified Mottled Sculpin, ranging in length from 50 to 115 millimeters, were collected. None were observed to have external 

abnormalities or significant internal lesions (table 3).

Threemile Run

Fish were sampled at two sites on Threemile Run on 

October 3, 2018, an upstream site (TR1) and the Mt. Alton 

impacted site (TR2). One White Sucker, 4 Redside Dace, and 

27 Creek Chub were collected at TR1 (table 4).

At the upstream site (TR1), the White Sucker was noted 

as having a tumor by visual examination. Microscopically, no 

actual neoplasia was noted; however, there were some areas of 

epidermal hyperplasia. There were two male and two female 

Redside Dace collected; the ovaries of the females contained 

many atretic eggs (fig. 2A) with no obvious cause.

There were 13 female, 13 male, and 1 undetermined 

Creek Chub. Ovaries from two of the females had atretic eggs. 

All of the males were immature with undeveloped testes. One 

fish had coccidia parasites in the spleen (fig. 2B). Ceroid/

lipofuscin accumulations, indicative of oxidative damage, 

were present within the meninges of the olfactory lobe of the 

brain (fig. 2C) of 15 percent (4 out of 27) of the Creek Chub. 

The most common observation at this site was the presence of 

Table 3. Meade Run (BG1) site Mottled Sculpin observations.

[F, female; M, male]

Fish number Length (millimeters) Sex External abnormalities Microscopic observations

BG1-1 115 M None None

BG1-2 105 F None None

BG1-3 107 M None None

BG1-4 90 M None None

BG1-5 92 F None None

BG1-6 75 M None None

BG1-7 84 F None None

BG1-8 82 M None None

BG1-9 72 M None None

BG1-10 62 M None None

BG1-11 82 Unknown None None

BG1-12 73 F None None

BG1-13 75 F None None

BG1-14 80 F None None

BG1-15 61 M None None

BG1-16 64 F None None

BG1-17 55 F None None

BG1-18 52 M None None

BG1-19 66 F None None

BG1-20 61 F None None

BG1-21 52 F None None

BG1-22 50 F None None

BG1-23 54 F None None
Results 5

Table 4. Observations of fish collected at the Threemile Run upstream site (TR1).

[DELTs, deformities, fin erosions, lesions, or tumors; F, female; M, male; NG, no gonad]

Fish 

number Species Sex External DELTs Microscopic observations

Trematode cysts Other

TR1-CC1 White Sucker F Tumor No Epidermal hyperplasia

TR1-CS1 Redside Dace M None Yes None

TR1-CS2 Redside Dace F None Yes Atretic eggs, inflammation

TR1-CS3 Redside Dace M None Yes None

TR1-CS4 Redside Dace F None Yes Atretic eggs

TR1-SA1 Creek Chub M None No None

TR1-SA2 Creek Chub F Lesion Yes Erosion and inflammation

TR1-SA3 Creek Chub F None Yes Atretic eggs, inflammation

TR1-SA4 Creek Chub M None Yes None

TR1-SA5 Creek Chub F None No Brain ceroid

TR1-SA6 Creek Chub F None No None

TR1-SA7 Creek Chub F None No Brain ceroid

TR1-SA8 Creek Chub M None No Coccidia in spleen

TR1-SA9 Creek Chub F None No None

TR1-SA10 Creek Chub M None Yes None

TR1-SA11 Creek Chub F None No Brain ceroid

TR1-SA12 Creek Chub F None No None

TR1-SA13 Creek Chub NG None No None

TR1-SA14 Creek Chub F None Yes None

TR1-SA15 Creek Chub M None No None

TR1-SA16 Creek Chub M None No None

TR1-SA17 Creek Chub M None Yes None

TR1-SA18 Creek Chub M None No None

TR1-SA19 Creek Chub M None No None

TR1-SA20 Creek Chub M None No None

TR1-SA21 Creek Chub M None No None

TR1-SA22 Creek Chub M None No None

TR1-SA23 Creek Chub F None No None

TR1-SA24 Creek Chub M None Yes None

TR1-SA25 Creek Chub M None Yes None

TR1-SA26 Creek Chub F None Yes Brain ceroid, atretic eggs

TR1-SA27 Creek Chub F None No None
6 Assessment of Microscopic Pathology in Fishes Collected at Sites Impacted by Wood Tar in Pennsylvania

A

a

b

c

B

a

a

b

b

b

C

a

b

D

a

b

c

Figure 2. Microscopic observations in Redside Dace and Creek Chub collected at the Threemile Run 

upstream site (TR1). A. Ovarian tissue of Redside Dace with atretic eggs (a) with chronic inflammation 

within the oocyte (arrows) and in the surrounding tissue (b). Normal oocytes in the cortical alveolar stage 

(c) are present. Scale bar equals 50 micrometers (μm). B. Coccidial parasites (a) within the spleen (b) of 

a Creek Chub. Scale bar equals 50 μm. C. Yellowish-brown ceroid/lipofuscin pigment (a) in the meninges 

of the olfactory lobe (b) of the brain of a Creek Chub. Scale bar equals 50 μm. D. Large trematode (a) with 

a sucker (b). located under the musculature (c) around the gills. The trematode is within a cyst (arrows). 

Scale bar equals 50 μm.

infected (fig. 3B). The parasite location, appearance, and 

the inflammatory response induced (fig. 3C) were similar 

to Ovipleistophora ovariae (previously described as Pleistophora ovariae; Pekkarinen and others, 2002) reported from 

Golden Shiner, Notemigonus crysoleucas (Summerfelt, 1964). 

Ceroid/lipofuscin deposits in the olfactory lobe meninges were 

observed in 29 percent (10 out of 35) of the Common Shiners. 

Incidental findings included myxozoan cysts in the cartilage 

and gills of two fish, and coccidia in the spleen and pancreas 

of two fish (table 5).

Fourteen females, 13 males, and 3 unidentified Western Blacknose Dace were also collected at the Mt. Alton site 

(table 6). The most common finding (30 percent; 9 out of 30) 

was ceroid/lipofuscin deposits in the meninges of the olfactory lobe of the brain, similar to those observed in the Common Shiner (fig. 2C). Two of the ovaries also had atretic eggs 

with microsporidian parasites similar to the Common Shiner 

(fig. 3).

trematode cysts (fig. 2D). Encysted trematode metacercariae 

were present in all of the Redside Dace and 33 percent (9 out 

of 27) of the Creek Chub (table 4).

At the Mt. Alton site on Threemile Run (TR2), 1 White 

Sucker and 35 Common Shiner were collected on October 3, 

2018. The White Sucker, a female, had a visible lesion that 

was microscopically identified as epidermal erosion with 

inflammation. No visible abnormalities were observed on 

the Common Shiners. All but two of the shiners were female 

(table 5).

Large encysted trematode metacercariae were observed 

in 23 percent (8 out of 35) of the Common Shiners, primarily in the musculature and connective tissue around the gills. 

The ovaries of 45 percent (15 out of 33) Common Shiners 

contained ovarian lesions, including atretic eggs and granulomatous inflammation in association with the presence of a 

microsporidian parasite within the oocytes (table 5). In some 

fish the majority of oocytes were normal with a few infected 

(fig. 3A); whereas in others the majority of the oocytes were 
Results 7

Table 5. Observations of Common Shiner collected at the Threemile Run Mt. Alton site (TR2).

[F, female; M, male; NA, not applicable]

Fish number Sex External

abnormalities

Microscopic observations

Trematode cysts Ovarian lesions1 Other

TR2-LC1 F None Yes Yes Myxozoan in cartilage

TR2-LC2 F None No No None

TR2-LC3 F None No No Inflammation around eye

TR2-LC4 F None Yes Yes Brain ceroid

TR2-LC5 F None No No None

TR2-LC6 F None No No Brain ceroid

TR2-LC7 F None No Yes Brain ceroid

TR2-LC8 F None No Yes Brain ceroid

TR2-LC9 F None Yes Yes Brain ceroid

TR2-LC10 F None Yes Yes None

TR2-LC11 F None No No Brain ceroid, coccidia in pancreas

TR2-LC12 F None No No Myxozoan cyst in gill

TR2-LC13 F None No No None

TR2-LC14 F None Yes Yes None

TR2-LC15 F None No No None

TR2-LC16 F None Yes Yes None

TR2-LC17 F None Yes Yes None

TR2-LC18 F None Yes Yes None

TR2-LC19 M None No NA None

TR2-LC20 M None No NA None

TR2-LC21 F None No Yes Coccidia in spleen

TR2-LC22 F None No Yes Brain ceroid

TR2-LC23 F None No No Brain ceroid

TR2-LC24 F None No No None

TR2-LC25 F None No No None

TR2-LC26 F None No No None

TR2-LC27 F None No No None

TR2-LC28 F None No Yes None

TR2-LC29 F None No No None

TR2-LC30 F None No No None

TR2-LC31 F None No No None

TR2-LC32 F None No No None

TR2-LC33 F None No Yes Brain ceroid

TR2-LC34 F None No Yes None

TR2-LC35 F None No No Brain ceroid

1Ovarian lesions included atretic oocytes containing microsporidian parasites and associated inflammation.
8 Assessment of Microscopic Pathology in Fishes Collected at Sites Impacted by Wood Tar in Pennsylvania

A

a

a

b

b

B

b

a

c

C

a

b

Figure 3. Microscopic observations in Common Shiner collected at the Mt. Alton site (TR2) of 

Threemile Run. A. Atretic oocytes (a) among the normal oocytes (b) within the ovary. Scale bar equals 

10 micrometers (μm). B. Oocytes (a) filled with microsporidian parasites Large areas of the ovary 

are replaced by chronic inflammatory response (b) with small areas of normal oocytes (c). Scale bar 

equals 50 μm. C. Higher magnification of an infected oocyte (a) illustrating the small microsporidian 

spores (arrows) and surrounding inflammation (b). Scale bar equals 20 μm.

Kinzua Creek

Twenty female and 10 male Western Blacknose Dace 

were collected at the upstream site on Kinzua Creek (KC1). 

Ceroid/lipofuscin deposits within the olfactory lobe meninges 

were observed in 17 percent (5 out of 30) of the individuals. 

Incidental findings included trematode and myxozoan cysts. 

Focal granulomas, indicative of a chronic inflammatory reaction, were noted in 27 percent (8 out of 30) of the Western 

Blacknose Dace from this site (table 7). The cause of this 

inflammatory reaction was not determined.

Thirteen female, 13 male, and 4 undetermined Creek 

Chub were collected at the upstream Kinzua Creek site. Small 

focal areas of inflammation of unknown etiology in the muscle 

(fig. 4A) or skin, myxozoan cysts in the muscle (fig. 4B), a 

cholangiocarcinoma (bile duct neoplasm; fig. 4C), and trematode cysts were observed (table 8).

At the Kinzua Creek Backus site (KC2), 8 male and 

22 female Creek Chub were collected (table 9). Similar to 

Creek Chub collected at KC1, bile duct proliferation in the 

liver, myxozoan and trematode cysts, and inflammation in the 

muscle were observed. Additionally, brain ceroid was also 

observed in two individuals (7 percent) and pancreatic inflammation in two individuals. An unusual response was observed 

in the ovary of one fish, with microsporidian parasites within 

the oocytes where numerous rodlet cells were observed within 

the inflammation (fig. 4D).
Results 9

Table 6. Threemile Creek Mt. Alton site (TR2) Western Blacknose Dace observations.

[F, female; M, male; NG, no gonad]

Fish number Sex Microscopic observations

TR2-RA1 F Myxozoan cyst in gills

TR2-RA2 F Brain ceroid, gill trematode

TR2-RA3 F Brain ceroid

TR2-RA4 F Brain ceroid, atretic eggs with microsporidian

TR2-RA5 M None

TR2-RA6 F Brain ceroid, atretic eggs with microsporidian

TR2-RA7 NG Pancreatic inflammation

TR2-RA8 M None

TR2-RA9 F None

TR2-RA10 F None

TR2-RA11 F None

TR2-RA12 M Brain ceroid

TR2-RA13 M None

TR2-RA14 F Brain ceroid, epidermal hyperplasia with giant cells in skin

TR2-RA15 M Brain ceroid

TR2-RA16 M Brain ceroid

TR2-RA17 M Brain ceroid

TR2-RA18 F None

TR2-RA19 M None

TR2-RA20 M None

TR2-RA21 F Pancreatic inflammation

TR2-RA22 F None

TR2-RA23 NG None

TR2-RA24 NG None

TR2-RA25 M None

TR2-RA26 F None

TR2-RA27 F None

TR2-RA28 M None

TR2-RA29 M None

TR2-RA30 M None
10 Assessment of Microscopic Pathology in Fishes Collected at Sites Impacted by Wood Tar in Pennsylvania

Table 7. Kinzua Creek upstream site (KC1) Western Blacknose Dace observations.

[F, female; M, male]

Fish number Sex Microscopic observations

KC1-RA1 M None

KC1-RA2 F Brain ceroid

KC1-RA3 F Bile duct myxozoan

KC1-RA4 M None

KC1-RA5 M Brain ceroid

KC1-RA6 F Focal inflammation in skin

KC1-RA7 M Brain ceroid

KC1-RA8 M Focal inflammation in muscle

KC1-RA9 F Bile duct myxozoan

KC1-RA10 M Brain ceroid

KC1-RA11 F None

KC1-RA12 F None

KC1-RA13 F None

KC1-RA14 F Focal inflammation in muscle

KC1-RA15 F Brain ceroid, focal inflammation in fin

KC1-RA16 F None

KC1-RA17 F None

KC1-RA18 F None

KC1-RA19 M None

KC1-RA20 F Focal inflammation in muscle

KC1-RA21 F None

KC1-RA22 F None

KC1-RA23 M None

KC1-RA24 F None

KC1-RA25 F Focal inflammation in muscle

KC1-RA26 F None

KC1-RA27 M None

KC1-RA28 M Focal inflammation in muscle

KC1-RA29 F None

KC1-RA30 F Focal inflammation in skin
Results 11

A

a

b

b

a

a

b

b

C

a

a

B

a

D

Figure 4. Microscopic observations in Creek Chub collected at Kinzua Creek. A. Focal areas of 

acute inflammation (a) around the skeletal muscle bundles (b). Scale bar equals 50 micrometers 

(μm). B. Myxozoan spores (arrows) within a muscle bundle (a). Scale bar equals 20 μm. C. 

Cholangiocarcinoma (a) composed of disorganized, proliferating bile ducts separated from 

surrounding hepatic tissue (b) by a poorly defined border (arrows) was observed at the upstream 

site (KC1). Scale bar equals 50 μm. D. Microsporidian parasites infecting oocytes (a), surrounded by 

inflammation with the presence of rodlet cells (arrows) were observed at the Backus (KC2) site. Scale 

bar equals 30 μm.
12 Assessment of Microscopic Pathology in Fishes Collected at Sites Impacted by Wood Tar in Pennsylvania

Table 8. Kinzua Creek upstream site (KC1) Creek Chub observations.

[F, female; M, male; NG, no gonad]

Fish number Sex Microscopic observations

KC1-SA1 M None

KC1-SA2 F Inflammation in skin

KC1-SA3 F None

KC1-SA4 M None

KC1-SA5 M Trematode cysts in muscle

KC1-SA6 M None

KC1-SA7 F None

KC1-SA8 M None

KC1-SA9 F Muscle myxozoan

KC1-SA10 NG None

KC1-SA11 F Inflammation in muscle

KC1-SA12 F Trematode cysts in muscle

KC1-SA13 M Inflammation in muscle, myxozoan cysts

KC1-SA14 M Cholangiocarcinoma

KC1-SA15 M Muscle myxozoan

KC1-SA16 F None

KC1-SA17 M None

KC1-SA18 M None

KC1-SA19 NG None

KC1-SA20 F None

KC1-SA21 NG None

KC1-SA22 NG None

KC1-SA23 M None

KC1-SA24 F Inflammation in muscle

KC1-SA25 F Bile duct proliferation

KC1-SA26 M None

KC1-SA27 F None

KC1-SA28 F Inflammation in muscle

KC1-SA29 F Ovarian atresia with inflammation

KC1-SA30 M None
Results 13

Table 9. Kinzua Creek Backus site (KC2) Creek Chub observations.

[F, female; M, male]

Fish number Sex Microscopic observations

KC2-SA1 F None

KC2-SA2 F None

KC2-SA3 M Brain ceroid

KC2-SA4 F Pancreatic inflammation, bile duct proliferation

KC2-SA5 M Trematode cyst

KC2-SA6 F Pancreatic inflammation

KC2-SA7 M None

KC2-SA8 F None

KC2-SA9 F Myxozoan in muscle

KC2-SA10 F Inflammation in muscle

KC2-SA11 F None

KC2-SA12 F None

KC2-SA13 M Brain ceroid

KC2-SA14 M None

KC2-SA15 M None

KC2-SA16 F None

KC2-SA17 F Atretic eggs, inflammation and rodlet cells in ovary

KC2-SA18 F None

KC2-SA19 F None

KC2-SA20 F None

KC2-SA21 M None

KC2-SA22 F None

KC2-SA23 F None

KC2-SA24 F Myxozoan in cartilage

KC2-SA25 F None

KC2-SA26 F None

KC2-SA27 F Trematode cyst

KC2-SA28 F None

KC2-SA29 M None

KC2-SA30 F None
14 Assessment of Microscopic Pathology in Fishes Collected at Sites Impacted by Wood Tar in Pennsylvania

Summary

The majority of microscopic abnormalities observed 

in this study were related to parasitic infections. There were 

virtually no observable differences in types and severity of 

parasitic infections and host responses between fish of the 

same species sampled at the upstream sites and at the impacted 

sites. The other commonly observed abnormality, ceroid/lipofuscin deposits within the meninges of the olfactory lobe, was 

also observed in fish collected at both impacted and upstream 

sites. Although the incidence at the impacted sites was higher, 

the species of fish was not the same, which confounds the findings. The cause of this type of lesion, an accumulation of autofluorescent chromolipids, is unknown. Lipofuscins have traditionally been considered accumulations of polymerized lipid 

material associated with aging, whereas ceroids are considered 

a pathological accumulation of polymerized or oxidized lipid 

(Porta and others, 2002; Seehafer and Pearce, 2006). A variety 

of contaminants and other environmental stressors can cause 

oxidative stress in fish (Srivastava and Reddy, 2017).

Microsporidia, myxozoan, and trematode parasites were 

observed in numerous fish in this study. The significance 

of these findings on the health of these fish populations is 

unknown. Parasitic infections in fish have been used as an 

indication of environmental health (Marcogliese, 2005) and 

there are environmental variables such as temperature, contaminants, stream drift, and water quality that affect parasite 

assemblages in fish (Landsberg and others, 1998; BlascoCosta and others, 2013). In this study, females with microsporidia infections in their ovaries exhibited a mild to severe 

inflammatory response and had atretic oocytes. However, 

the effect of these infections on reproductive health remains 

unknown. Myxozoan and trematode cysts, on the other hand, 

were observed mainly in the muscle and initiated minimal 

host response. In order to fully understand the impact of these 

parasites and pathogens on the fish sampled from Kinzua 

Creek and its tributaries, long-term monitoring would need to 

be considered in order to understand what types and severity 

of infections are considered normal for these fish populations.

To conclude, there was minimal evidence that the fish in 

this study responded to contaminants in the wood tar waste 

at the affected sampling sites. However, histopathology 

alone may not be an effective tool for analyzing the effects of 

exposure. In fish, molecular dysfunction, growth inhibition, 

and behavioral and reproductive effects could occur without 

histopathological changes.

References Cited

Blasco-Costa, I., Koehler, A.V., Martin, A., and Poulin, R., 

2013, Upstream-downstream gradient in infection levels 

by fish parasites—A common river pattern?: Parasitology, 

v. 140, no. 2, p. 266–274, accessed October 23, 2019, at 

https://doi.org/10.1017/S0031182012001527.

Landsberg, J.H., Blakesley, B.A., Reese, R.O., Mcrae, G., 

and Forstchen, P.R., 1998, Parasites of fish as indicators of environmental stress: Environmental Monitoring 

and Assessment, v. 51, no. 1/2, p. 211–232, accessed 

November 10, 2019, at https://doi.org/10.1023/

A:1005991420265.

Luna, L.G., 1992, Histopathologic methods and color atlas 

of special stains and tissue artifacts: Gaithersburg, Md., 

American Histolabs, Inc., 767 p.

Marcogliese, D.J., 2005, Parasites of the superorganism—

Are they indicators of ecosystem health?: International 

Journal for Parasitology, v. 35, no. 7, p. 705–716, 

accessed November 10, 2019, at https://doi.org/10.1016/

j.ijpara.2005.01.015.

Pekkarinen, M., Lom, J., and Nilsen, F., 2002, Ovipleistophora

gen. n., a new genus for Pleistophora mirandellae-like 

microsporidia: Diseases of Aquatic Organisms, v. 48, 

p. 133–142, accessed November 14, 2019, at https://doi.org/

10.3354/dao048133.

Porta, E.A., Berra, A., Monserrat, A.J., and Benavides, S.H., 

2002, Differential lectin histochemical studies on lipofuscin 

(age-pigment) and on selected ceroid pigment: Archives 

of Gerontology and Geriatrics, v. 34, no. 3, p. 193–203, 

accessed November 11, 2019, at https://doi.org/10.1016/

S0167-4943(01)00224-2.

Sanders, R.E., Miltner, R.J., Yoder, C.O., and Rankin, E.T., 

1999, The use of external deformities, erosions, lesions, and 

tumors (DELT anomalies) in fish assemblages for characterizing aquatic resources—A case study of seven Ohio 

streams, in Simon, T.P., ed., Assessing the Sustainability 

and Biological Integrity of Water Resources Using Fish 

Communities: Boca Raton, Fla., CRC Press, p. 225–2248.

Seehafer, S.S., and Pearce, D.A., 2006, You say lipofuscin, 

we say ceroid—Defining autofluorescent storage material: Neurobiology of Aging, v. 27, no. 4, p. 576–588, 

accessed November 11, 2019, at https://doi.org/10.1016/

j.neurobiolaging.2005.12.006.

Srivastava, B., and Reddy, P.B., 2017, Lipid peroxidation 

and DNA damage as biomarkers of pollution induced 

oxidative stress (OS) in fish: Life Sciences International 

Journal, v. 4, p. 194–198, accessed November 14, 2019, at 

https://doi.org/10.13140/RG.2.2.13114.80323.

Summerfelt, R.C., 1964, A new microsporidian parasite from 

the golden shiner, Notemigonus crysoleucus: Transactions 

of the American Fisheries Society, v. 93, no. 1, p. 6–10, 

accessed November 11, 2019, at https://doi.org/10.1577/

1548-8659(1964)93[6:ANMPFT]2.0.CO;2.
For more information, contact

Director, Leetown Science Center

U.S. Geological Survey

11649 Leetown Road

Kearneysville, WV 25430

Publishing support provided by the West Trenton and

Pembroke Publishing Service Centers
Walsh and others—Assessment of Microscopic Pathology in Fishes Collected at Sites Impacted by Wood Tar in Pennsylvania—OFR 2020–1024

ISSN 2331-1258 (online)

https://doi.org/10.3133/ofr20201024