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Hylebos Waterway Fish Injury Studies

Individual Data and Quality Assurance Results

CASE NARRATIVE

Toxicopathic Conditions in Flatfish

and Reproductive Toxicology in Flatfish

Liver and Kidney Histopathology

Histopathologist’s qualifications

Histologic sections of liver, kidney and gonad from individual fish were

examined by three histopathologists (Mark Myers, Carla Stehr, Paul Olson) with

at least three years of experience in the histopathologic examination of English

sole and rock sole. The latter two examiners have completed a one-year training

program under the direction of the chief pathologist, Mark S. Myers. Each

histopathologist examined approximately 33% of the total number of cases.

Diagnostic criteria

Criteria for microscopic lesion diagnosis were adopted from comparative

pathology textbooks (Robbins, Cotran, and Kumar, 1984) and various

publications in fish histopathology (Myers et al., 1987; Moore and Myers, 1994;

Johnson et al., 1991; Hinton et al., 1992).

Histologic slide labeling

All histologic slides were labeled with a random number code so that the

geographic site of capture was not known to the examiner.

Internal quality control procedures for histologic slide examination

Histologic slides of liver, kidney and gonad examined originally by Carla

Stehr and Paul Olson, and affected with suspected or diagnosed toxicopathic

lesion types or where lesion diagnosis was not obvious, were re-examined by the

chief pathologist, Mark Myers. In this way, the vast majority of fish with

significant toxicopathic hepatic lesions were re-exammed by the chief

histopathologist. In addition, histologic slides of liver, kidney and ovary from 20

female English sole were randomly selected for re-examination from the

Reproductive Injury study. Twenty cases of English sole or rock sole from the

Toxicopathic Injury study in which toxicopathic liver lesions had not been

detected in the original histologic examination were also randomly selected for

re-examination. Out of the approximately 2500 tissue sections of liver, kidney or

gonad examined (from 808 individual English sole and rock sole actually

examined histologically), 382 tissue sections of liver, kidney or gonad (-15.3%)

from 128 individuals (15.8%) were re-examined by the chief pathologist. The

original and final, resolved diagnoses for these re-examined individual cases are

set forth in Table 1.
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External quality control procedures

Only one problematic case was encountered during this study, involving a

tumor in the testis in specimen #94-3593. The differential diagnosis for this case

was a testicular seminoma/embryonal carcinoma. This lesion was coded into the

database using the more conservative diagnosis of a benign seminoma. This case

has been submitted for evaluation and confirmation to Dr. John Fournie of the

U.S. Environmental Protective Agency in Gulf Breeze, FL.

Coding of diagnoses into database

Each final histopathologic diagnosis was entered into the database by the

examining histopathologist, using a lesion coding system developed at the

Northwest Fisheries Science Center. Individual toxicopathic lesions in liver and

kidney were also grouped into broader categories of toxicopathic lesions. Lesion

categories include the following:

Liver-

-neoplasms (including hepatocellular adenoma, cholangioma, hepatocellular

carcinoma, cholangiocellular carcinoma, and mixed hepatobiliary

carcinoma [epithelial neoplasms]; mesenchymal neoplasms; all neoplasms

[including epithelial or mesenchymal neoplasms]

-foci of cellular alteration (basophilic focus, eosinophilic focus, clear cell focus)

-non-neoplastic proliferative lesions (hepatocellular regeneration, oval cell

proliferation, cholangiofibrosis, increased parenchymal mitotic activity)

- specific degenerative /necrosis (including megalocytic hepatosis and

hepatocellular nuclear pleomorphism)

-hydropic vacuolation of biliary epithelial cells/hepatocytes (including focal

hydropic vacuolation)

-nonspecific necrotic lesions

-spongiosis hepatis

- any toxicopathic liver lesion (any of the above lesions, excluding spongiosis

hepatis and nonspecific necrotic lesions)

Kidney-

-neoplasms

-proliferative lesions

-necrotic lesions

-fibrotic/sclerotic lesions

- any toxicopathic kidney lesion (any of the above)

Gonadgonad type (testis or ovary) and developmental stage are indicated in this case

narrative; detailed data on ovarian lesions are used only for female English sole in

the Reproductive Injury study, and are reported in a separate case narrative.

Missing data

Occasionally the cassette used to hold tissues from individual fish will open up in

the fixative container, resulting in the loss of the tissue; also, in the necropsy

procedure, occasionally tissues are inadvertently not collected. Infrequent labeling
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errors may also occur during the handling of a large number of fish. If the tissue is

lost, or there is a question about the labeling, lesion information is not used, and

missing data for a particular tissue is indicated by a blank record. Specimens

collected for these studies, but where histopathology data is not available are:

• 943231 - no data for liver

• 943037, 943051, 943084, 943374, 943486, 943531, 943532, 943562, 943699,

943732, 943742, 943806, 943812, 943843 - no data for liver, kidney or gonad

• 943702 - no liver or kidney

• 943061, 943146, 943171, 943191, 943206, 943221, 943224, 943487, 943662,

943829, 943585, 943662,’943757 - no data for kidney

• 943206, 943771, 943224, 943233, 943651, 943761 - no data for kidney or

gonad

•

943162,943169,943212,943214,943216,943281,943293,943296,943334,943338,

943339, 943342, 943343, 943344, 943346, 943349, 943351, 943353, 943354,

943356, 943379, 943390, 943445, 943482, 943572, 943390, 943445, 943572,

943706, 943712, 943799, 943800 - no data for gonad

Otoliths are sometimes lost or damaged during collection; therefore age data

cannot be obtained. This missing data is indicated by a blank record. Fish where

we were unable to obtain age data were specimens 943350, 943374 and 943649.

Sequential gaps in the specimen numbers do not represent missing fish, those

specimen numbers not listed in Table 2 were assigned to projects other than

Hylebos Waterway fish injury studies.

Revisions 2/20/97.

The primary difference between this case narrative and the version previously

submitted on 5/30/96, is that the data presentation has been reorganized. All the

specimens are now fisted in sequential order, and three new columns have been

added to indicate which data is included in one or more of the three data

groupings presented in the Interpretive reports. In addition, since detailed data on

the histopathology of ovarian lesions in English sole is only reported in the

Reproductive Injury Interpretive Report, those were placed in a separate case

narrative. Quality Assurrance has been expanded. Additional slides of fish

previously diagnosed to have no liver lesions were randomly selected and reread

by the chief pathologist as a check for false negatives. Additional missing data

was identified and is included in the list above.
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Citations

Toxicopathic Conditions in Flatfish and

Reproductive Toxicology in Flatfish

Liver and Kidney Histopathology

CASE NARRATIVE

Hinton, D.E., P.C. Baumann, G.C. Gardner, W.E. Hawkins, J.D. Hendricks, R.A. Murchelano, and

M.S. Okihiro. 1992. Histopathologic biomarkers. In R.J. Juggett, R.A. Kimerle, P.M.

Mehrle, Jr., and H.L. Bergman (eds), Biomarkers: Biochemical, Physiological and

Histological Markers of Anthropogenic Stress, p. 155-210. Society of Environmental

Toxicology and Chemistry Special Publication Series, Lewis Publishers, Chelsea, MI.

Johnson, L.L., E. Casillas, M.S. Myers, L.D. Rhodes, and O.P. Olson. 1991. Patterns of oocyte

development and related changes in plasma 17-β estradiol, vitellogenin and plasma

chemistry in English sole (Parophrys vetulus). J. Exp. Mar. Biol. Ecol. 152:161-185.

Moore, M.J., and M.S. Myers. 1994. Pathobiology of chemical-associated neoplasia. in fish. In D.C.

Malins, G.K. Ostrander (eds), Aquatic Toxicology; Molecular, Biochemical and Cellular

Perspectives. p. 327-386. Lewis Publishers, CRC Press Boca Raton FL.

Myers, M.S., L.D. Rhodes, and B.B. McCain. - 1987. Pathologic anatomy and patterns of occurrence

of hepatic neoplasms, putative preneoplasitc lesions and other idiopathic hepatic conditions

in English sole (Parophrys vetulus) from Puget Sound, Washington. U.S.A. J. Natl. Cancer

Inst. 78(2):333-363.

Robbins, S.L., R. S. Cotran, and V. Kumar. 1984. Pathologic Basis of Disease, 3rd edition.

Saunders, Philadelphia, PA, 1467 p
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Documentation and Abbreviations for Table 2.

Liver and Kidney Histopathology Data

for Hylebos Fish Injury Studies

This case narrative includes liver and kidney histopathology data for flatfish

collected during the Toxicopathic and Reproductive Injury Studies of the

Hylebos Fish Injury Studies, Phase I. Ovary lesion data is used only in the

Reproductive Injury Study, and is included in a separate case narrative.

Studies:

The case narrative includes three columns to identify the study where the

data is reported in the interpretive reports.

Study: Toxicopath column indicates data reported in the Toxicopathic

Injury Interpretive report.

Study: Reprod. column indicates data reported in the Reproductive Injury

Interpretive Report.

Study: Combined column indicates data reported in an additional section of

the Toxicopathic Injury study, where data from both the Toxicopathic

and Reproductive Injury Studies are combined. Please note that

specimens used in the combined studies also include male English sole,

and rock sole that were collected during the Reproductive Injury Study,

but the data was not used in the Reproductive Injury report (which

focused on female English sole)

Y= yes, data included in the indicated study

N= No, data not included in the indicated study

Date collected - Date fish were caught, and tissue samples collected.

Reader - indicates last name of the person who read the histopathology slides

for that specimen number. A blank record occurs when no tissue sections were

available to be read for that specimen.

Lesion abbreviations

Liver lesions

EPNEO = epithelial neoplasms (tumors of epithelial origin e.g. HEPCARC,

CHOLCARC, MIXCARC, HEPAD, CHOLAD as described below)

MESNEO = mesenchymal neoplasms (tumors not of epithelial origin, e.g.

hemangiomas, hemangiopericytic sarcoma, fibromas)

ALLNEO = indicates that the fish has any one of several types of neoplasms

including EPNEO and MESNEO

HEP.CARC = hepatocellular carcinoma

CHOLCARC = cholangiocellular carcinoma
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MIXCARC = mixed hepatobiliary carcinoma

HEPAD = hepatocellular adenoma

CHOLAD cholangiocellular adenoma

HEPNEO indicates fish has any type of hepatocellular neoplasm including

HEPCARC and HEPAD

CHOLNEO = indicates fish has any type of cholangiocellular neoplasm including

CHOLCARC and CHOLAD

BF = basophilic focus

EF = eosinophilic focus

CCF = clear cell focus

ALLFCA = indicates that fish has any one of several types of focal

cellular alterations, including BF, EF, CCF

MH = megalocytic hepatosis

NP = hepatocellular nuclear pleomorphism.

SDN = fish has any one of specific degenerative/necrotic lesions, including

MH and NP.

LIVPROL = liver proliferation; nonneoplastic proliferative lesions

SPONGHEP = spongiosis hepatis

HYDVAC = hydropic vacuolation

FOCAL HYDVAC = focal hydropic vacuolation

NECROSIS= nonspecific necrotic lesions

TOXLIV = indicates that fish has one or more lesions considered to be

toxicopathic, including ALLNEO, ALLFCA, SDN, LIVPROL, and

HYDVAC

LIVER= 1, indicates liver was examined

Kidney lesions

KIDNEO = Kidney neoplasms

NEPBPROL = proliferative lesions in elements of kidney nephrons

NEPHNEC necrotic lesions in elements of kidney nephrons

NEPHSCLR sclerotic lesions in elements of kidney nephrons

TOXKID = indicates fish has one or more of kidney lesions considered to be

toxicopathic, including NEPBPROL, NEPHNEC, NEPHSCLR (none of

the fish had MDNE0).

KIDNEY = 1, indicates kidney was examined

Gonad stages

OVSTAGE = stage of maturation of the ovary

OVARY = 1, indicates ovary was examined

TESTAGE = stage of maturation of the testes

TESTIS = 1, indicates testis was examined.

Sex:

1 = male

2 = female
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Length: total length (head to tail) reported in millimeters.

Weight: actual weight reported in grams.

Age:

Actual years of age as indicated by growth rings on otoliths.

Blank records indicate no age data available; otoliths lost or damaged.

Explanation of codes

Presence or absence of lesions:

0= tissue examined, no lesion observed

1= tissue examined, lesion observed

blank = tissue not examined

Gonad Stages

Female

1= regressed; oogonia and primary oocytes

2= late regressed; secondary oocytes

3= previtellogenic; vacuolated secondary oocytes

4= vitellogenic

5= some hydrated oocytes; no post-ovulatory follicles (POFs)

6= spawning; hydrated oocytes with POFs

7= spawned out

Male

1= regressed; spermatogonia and primary spermatocytes

2= early recrudescence; secondary spermatocytes

3= late recrudescence; secondary spermatocytes, to spermatids,

4= early spermiogenesis or sperm production

5= late spermiogenesis; spawning

6= spawned out; few mature sperm remaining.
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