MMT ISR Pathology Appendix DRAFT 1 March 29, 2013

Appendix F. Pathology Guidance

Section 1 MMT Histotechnology 

1.1 Euthanasia and necropsy

1.2 Histotechnology

1.2.1 Transverse-Oblique Sectioning (Subadults)

1.2.2 Sagittal Sectioning (Adult)

Section 2 MMT Pathology

2. General approach to reading studies

2.2 Data Recording Insert an example histopathology spreadsheet

2.3 Statistical analysis

RSCABS

2.3 Histopathology Report Format.

2.4 Gonadal staging criteria

2.4.1 Criteria for staging testes in Medaka.

2.4.2 Criteria for staging ovaries in Medaka.

2.5 Diagnoses of Pathologies Induced by Endocrine Disruptors

2.5.1 Testis Pathology

Increased interstitial cells

Increased germ cells

Testicular generation

Etc.

2.5.2 Ovary Pathology

Oocyte atresia

Decreased yolk formation

Granulosa cell hypertrophy/hyperplasia

Etc.

2.5.3 Intersex

2.5.4 Kidney Pathology

2.5.5 Liver Pathology

Section 3 OATPR (Online Aquatic Toxicology Pathology Reference)

Section 4 References
MMT ISR Pathology Appendix DRAFT 2 March 29, 2013

Section 1 MMT Histotechnology 

Medaka were sampled at two lifestages for MMT histology analysis. While the fish’s age at 

sampling varied slightly across the various MMTs, these differences did not have an impact on the 

histotechnique used. Initially, both adult and subadult fish were histologically processed using a 

transverse-oblique (frontal) sectioning protocol for both life-stages. The transverse-oblique sectioning 

plane provides excellent sections of the gonad in particular. However, to facilitate pathology 

assessments of multiple organs including the gonads, later versions of the MMT protocol employed 

sagittal sectioning of adult samples. Details of the histotechniques employed are outlined below.

Section 1.1 Euthanasia and necropsy

Objectives

(1) Provide for the humane sacrifice of fish.

(2) Obtain necessary body weights and measurements. 

(3) Excise liver tissue. (Sub-adult sampling)

(4) Provide for adequate fixation of the tissues within the carcass.

Euthanasia solution (MS-222; tricaine methanesulfonate)

Tricaine methanesulfonate 100 mg

Sodium bicarbonate 200 mg

Tank or reservoir water 1 L

The buffered MS-222 should be buffered to the pH of the test dilution water to avoid distress.

Fixation of tissues for histopathology

Techniques were selected that would most optimally:

(1) Preserve the cellular structure of target tissues;

(2) Maximize the amount of target tissues available for analysis;

(3) Sample the target tissues available for analysis;

(4) Allow the pathologist to examine at least 3 step sections of target tissues.

Gonads should be pre-fixed in situ and then be processed in the whole body. However, in order to 

allow optimal penetration of the fixative, tail and/or head portions should be removed by means of 

sharp razor blades or scissors.

Bouin’s fixative or Modified Davidson ́s fixative are the recommended fixatives. Compared to

Bouin’s fixative, the advantages of Modified Davidson’s fixative are as follows:

(1) the morphologic appearance of gonad sections is generally considered to be comparable to 

sections fixed in Bouin’s fixative and superior to sections fixed in formalin;

(2) compared to Bouin’s fixative, which contains picric acid, Davidson’s fixative is 

generally considered to be less noxious, less hazardous, and more easily disposed of;
MMT ISR Pathology Appendix DRAFT 3 March 29, 2013

(3) specimens fixed in Bouin’s fixative require multiple rinses in alcohol prior to transfer to 

formalin.

Formulation of Modified Davidson’s Fixative (1 L):

Formaldehyde (37 - 40 %) 220 ml

Glacial acetic acid 115 ml

95% Ethyl alcohol 330 ml

Distilled water 335 ml

Formulation of Bouin’s Fixative (1 L):

Saturated Picric acid solution 750 ml

Formaldehyde (37 - 40 %) 250 ml

Glacial acetic acid 50 ml

Please be aware that different recipes and products that are designated as “Davidson’s fixative”

may actually be modifications of the original formula; if an alternative Davidson’s fixative formulation 

is used, this should be noted by the laboratory. If necessary, a decalcification solution (EDTA, acetic

acid) may be used. Factors that may affect the need for decalcification include the size of the

individual fish, the length of time that the carcass was immersed in fixative, and the extent to which the

abdominal cavity came into contact with the fixative.

Necropsy Procedure

(1) Fish should be sacrificed within one to two minutes prior to necropsy. Therefore, unless

multiple prosectors are available, multiple fish should not be sacrificed simultaneously.

(2) Using a small dip net, a single fish is removed from the experimental chamber and

transported to the necropsy area in the transport container. For each test chamber, the sex of

each fish is determined by external body characteristics (shape of dorsal and anal fins).

(3) The fish is placed into buffered MS-222 (tricaine methanesulfonate) solution. The fish is 

removed from the solution when there is cessation of respiration and the fish is unresponsive

to external stimuli.

(4) The fish is wet weighed and measured according to protocol.

(5) The fish is placed on a corkboard on the stage of a dissecting microscope.

a) For subadult animals, two transverse cuts are performed to remove the head and tail. The 

head is removed by a transverse cut at the posterior margin of the operculum. The liver is 

collected by applying slight pressure on the abdomen and separating the liver from the 

body with forceps. Some liver tissue may also be contained in the head depending on the 

location of the cut. The liver is then placed in a microcentrifuge tube in *RNA stabilizing 

solution. The tail is removed by a transverse cut posterior to vent and saved for preparing 

the anal fins for counting papillae. The anal fin is flared out on a dish and a few drops of 

Davidson’s fixative are applied to the fin. After approximately 30 seconds to a minute, 
MMT ISR Pathology Appendix DRAFT 4 March 29, 2013

the tail is placed in a small microcentrifuge tube and filled with Davidson’s fixative.

b) Adult animals are prepared by having tails removed as in the subadults above, and then 

the abdomen is opened by a small incision along the ventral midline starting at the vent 

and ending midway along the abdomen. Using a syringe, approximately 0.5 mL of 

fixative is then gently perfused into the viscera and buccal cavity.

(6) The carcass and abdominal viscera are placed in an individual labeled histology cassette and 

placed in a bottle of fixative. The volume of fixative in the container should be at least 10

times the approximated volume of the tissues. The fixative container is gently agitated for 

five seconds to dislodge air bubbles from the cassette.

(7) All tissues remain in Davidson’s fixative overnight, followed by transfer to 10 % 

neutral buffered formalin the next day. Bouin’s fixed tissues remain in fixative for 72 hours, 

before being washed twice overnight with a 70% ethanol solution saturated with lithium 

carbonate and then transferred to 10% neutral buffered formalin. Fixative containers are

gently agitated for 5 seconds to ensure adequate penetration of solutions into cassettes.

Section 1.2.1 Preparation of slides from subadult specimens

Typically, subadult fish shall be sectioned such that the gonads are sectioned on their long axis 

along a plane roughly parallel to a frontal plane of the fish. The pathology evaluation shall be limited to 

diagnosing effects on kidney, gonadal development, and grading the gonadal phenotype. The trunkportion of each specimen shall be trimmed and embedded to produce sections that are roughly parallel 

to the fish’s frontal plane. The specific trimming protocol is illustrated below. Cuts 1, 2, and 3 are 

performed at necropsy. Cut 4 is done post-fixation prior to processing and embedment.

a. Trimming: The fish shall be trimmed such that “cut 4”, which defines the sectioning plane, is 

parallel to the long axis of the gonad. Note that the gonad is “tilted” with respect to the spine so 

the trim-plane must traverse the spine in an oblique fashion. Cuts 1, 2, and 3 are made during 

necropsy. Cut 4 is done post-fixation, pre-paraffin processing (Figure 1-1).

Figure 1-1. Sequential cuts associated with trimming sub-adult (9 wpf) medaka in preparation for 

histopathology. Cut 4 should be performed by the Contractor prior to processing and embedding. 
MMT ISR Pathology Appendix DRAFT 5 March 29, 2013

b. Processing: The trimmed medaka shall be processed in an automatic tissue processor and 

infiltrated with paraffin.

c. Embedding: Each trimmed and processed medaka shall be embedded such that the dorsal cut 

surface of the fish is down and shall be sectioned first.

d. Microtomy: Section thickness for this protocol shall be set at 4-5 microns. The block shall be 

faced from the dorsum toward the ventrum of the fish until the kidney tissue appears the section. 

From this landmark, ~12 microns of tissue are microtomed from the block, and the first section 

with kidney shall be obtained. Another ~12 microns shall be microtomed and from there, obtain a 

second section with kidney.

 From here the microtomy protocol varies based on the gross sexual phenotype of the 

specimen (i.e., male or female).

 Phenotypic males: The intent is to obtain, as best as possible, longitudinal sections of the testis. 

To accomplish this, the microtomist shall be experienced and proficient in sectioning medaka of 

this age. Sectioning continues until the testis is reached. Once the testis is reached, the 

microtomist shall obtain 9 sections at 10 micron intervals to sample the middle half of the testis. 

The distance to the middle of the gonad will vary depending on the size of the fish and its gonads. 

Therefore the microtomist must estimate from experience and judgment where to begin taking 

sections. The microtomist may choose to obtain more than 9 sections if necessary to ensure 

sampling the central duct of the testis. 

 Phenotypic females: The intent is to obtain, as best as possible, longitudinal sections of the ovary. 

To accomplish this, the microtomist shall be experienced and proficient in sectioning medaka of 

this age. Sectioning continues until the ovary is reached whereupon the microtomist shall obtain 9 

sections at 40 micron intervals to sample near the middle of the ovary. The distance to the middle 

of the ovary will vary depending on the size of the fish and its gonads. Therefore the microtomist 

must estimate from experience and judgment where to begin taking sections. The microtomist 

may choose to obtain more than 9 sections if necessary to ensure adequate sampling of the ovary. 

 Following microtomy each paraffin block with residual tissue shall be sealed with paraffin.

e. Staining: The slides shall be stained with H&E on an automatic stainer. The tissue shall be 

covered with an appropriate mounting medium and glass cover slips.
MMT ISR Pathology Appendix DRAFT 6 March 29, 2013

Section 1.3 Preparation of slides from adult specimens (sagittal sectioning)

Typically adult fish shall be sagittally step-sectioned and evaluated for pathology in target 

tissues such as, the liver, gonad, kidney, inter-renals, pituitary, and spleen. Each specimen shall be 

paraffin embedded so that 5 slides with sagittal and parasagittal sections are produced for pathology 

evaluation. The detailed histotechnical protocol is illustrated below. 

a. Processing: The trimmed medaka shall be processed in an automatic tissue processor and 

infiltrated with paraffin. Due to the large size of some of the specimens, it assumed that 

decalcification and possibly longer processing times than usual will be required.

b. Embedding: Each sample shall be embedded with its left side down, oriented in a manner that the 

spine will be approximately parallel to the sectioning plane.

Figure 1-2. Embedded Adult Medaka in trimmed block.

c. Microtomy: Section thickness shall be 4-5 microns.

Slide 1. The block shall be faced starting from the left side of the fish. Trim until sections reveal 

the left eye; continue to trim midway into the lens. At this point the lens will be a ring within the 

eye. The ring can be seen in the sections and the block. The lens will be hard, the microtome 

blade will produce a scratch that can be heard and felt as the blade cuts through. At this point the 

sections should also reveal the visceral cavity. Collect a ribbon of 3-4 serial sections and mount on 

a glass slide (Figure 1-3).

Figure 1-3. Images of the blocks, ribbons and section showing the location of the sectioning plane in 

the lens and other tissues for slide 1.

Slide 2. Continue to trim through the left eye until it is no longer in the sections. The black pigment 

of the eye will diminish. Collect a ribbon of 3-4 serial section and mount on a glass slide (Figure 

1-4).
MMT ISR Pathology Appendix DRAFT 7 March 29, 2013

Figure 1-4. Image of the section indicating the location of the section plane for slide 2.

Slide 3. The target organ for this slide is the pituitary gland, which is located midway between the 

eyes. From the point where the sections for slide 2 were taken, trim 200 microns and take a 

section. Continue to take 2 or 3 more sections at 200 micron intervals and mount them in order 

(Figure 1-5).

Figure 1-5. An image of a mid-sagittal section of the medaka that includes the pituitary gland, 

which is the target organ for the sections on slide 3.

Slide 4. Trim to the medial portion of the right eye, the black pigment can be seen in the sections. 

Take a ribbon of 3-4 serial sections and mount on glass slide (Figure 1-6).

Figure 1-6. Image of a representative section showing the plane for collecting sections for slide 4. 

Slide 5. Continue trimming into the right eye until midway through the lens. At this point, light 

can be seen through the lens. Take a ribbon of 3-4 serial sections. Following microtomy, the

tissue blocks can be discarded (Figure 1-7).
MMT ISR Pathology Appendix DRAFT 8 March 29, 2013

Figure 1-7. Image of a representative section showing the plane for collecting sections for slide 5. 

d. Staining: The slides shall be stained with H&E on an automatic stainer. The tissue shall be 

covered with an appropriate mounting medium and glass cover slips.

Section 2: Pathology

Slides are evaluated for treatment-induced lesions in gonad, liver, kidney, and thyroid. The 

pathologist should also scan other tissues (e.g., pituitary, spleen, inter-renal tissue) for treatmentinduced lesions. Pathology is to be defined according to three general criteria: 

1) The pathologist’s judgment,

2) Pathology described in the Draft OECD Guidance Document for the Diagnosis of EndocrineRelated Histopathology of Fish Gonads (Johnson et al. 2010), and

3) Histomorphological effects observed in treated specimens compared to the controls.

The slides shall be evaluated by a pathologist for treatment-induced effects. The pathologist shall 

perform the evaluation according to the Best Practices Guideline: Toxicologic Histopathology 

(Crissman et al. 2004). It is recommended that evaluation begin with examining representative slides 

from specimens selected from control and the two highest treatments to determine likely treatmentrelated effects. Then, images of representative treatment-related effects and associated severity scores 

shall be prepared to be used as references for scoring the remainder of the specimens. Finally, after the 

initial evaluation, the study pathologist shall perform a masked re-evaluation of all the specimens, but 

for only the previously observed treatment-related lesions.

Each section shall be evaluated for gonad pathology, whereas kidney, reproductive duct, and liver 

pathologies shall be based on the assessment of one representative section. The prevalence and extent of 

the effects shall be assessed in the various treatments as compared to controls. Effects data containing 

diagnoses and associated severity grades shall be reported in an Excel spreadsheet similar to the 

example provided below. Diagnoses of treatment effects shall be based on the columns in the example 

pathology spreadsheet. However, for pathologies not listed in the spreadsheet, the pathologist shall 

provide new diagnostic criteria to the spreadsheet. Pathology not related to treatment shall also be 

noted as a comment in the spreadsheet. Create digital images of representative diagnostic features in an 

uncompressed file format. Images of representative control tissues shall be included for comparison. 

Each image shall be at an appropriate magnification to illustrate the features using the best judgment of 
MMT ISR Pathology Appendix DRAFT 9 March 29, 2013

the pathologist. An embedded calibrated scale bar shall be included in all pictures. The spreadsheet 

shall indicate imaged sections.

A report shall be prepared which includes a short summary of the protocols used in slide preparation 

and a description of treatment-related effects based on concentration responses and comparisons with 

control frogs. The narrative report shall include tables, figures, figure legends, and citations, as 

necessary to describe treatment effects. Citations, tables, figures, figure legends, shall be placed at the 

end of the narrative report. Other supporting histopathology data, images, and histology technical 

reports shall be provided as spreadsheets, images without legends or annotations, and documents. The 

titles and file names of reports and documentation shall include: laboratory; species name; 

developmental stage; test chemical; and tissue examined. The file names of digital images shall identify 

the test chemical, treatment, and specimen ID. A tabular cross-reference of image files and report figure 

numbers and titles shall be provided.

Section 2.1 General approach to reading studies

 Adapted from OECD guidance document for the Diagnosis of Endocrine-Related 

Histopathology of Fish Gonads. (2010)

Studies are to be read by individuals experienced in reading toxicologic pathology studies, and who are 

familiar with normal small fish gonad histology, with gonadal physiology, and with general responses 

of the gonads to toxicologic insult. Pathologists may be board certified (e.g. American College of 

Veterinary Pathologists, The European Centre of Toxicologic Pathology, or other certifying 

organizations); however, certification is not a requirement as long as the pathologist has obtained 

sufficient experience with, and knowledge of, fish histology and toxicologic pathology. Technicians 

should not be used to conduct readings due to the subtle nature of some changes and the need for 

subjective judgments based on past experience.

It is recognized that there is a limited pool of pathologists with the necessary training and experience 

that are available to read the gonadal histopathology for the Medaka Multigenerational Test (MMT). If 

an individual has toxicological pathology experience and is familiar with gonadal histology in small 

fish species, he/she may be trained to read the fish assay. If pathologists with little experience are used 

to conduct the histopathological analysis, informal peer review may be necessary.

It is preferable for pathologists to read the studies non-blinded (i.e. with knowledge of the treatment 

group status of individual fish). This is because endocrine effects on histomorphology tend to be 

incremental, and subtle differences between exposed and unexposed animals may not be recognizable 

unless tissue sections from high dose animals can be knowingly compared to those from controls. Thus, 

the aim of the initial evaluation is to ensure that diagnoses are not missed (i.e., to avoid false-negative 

results). However, it is expected that any potential compound-related findings will be re-evaluated by 

the pathologist in a blinded manner prior to reporting such findings, when appropriate. Certain 

diagnostic criteria, such as relative increases or decreases in cell populations, cannot be read in a 
MMT ISR Pathology Appendix DRAFT 10 March 29, 2013

blinded manner due to the diagnostic dependence on control gonads. As a rule, treatment groups should 

be evaluated in the following order: control, high-dose, intermediate-dose, and low-dose.

It is suggested that the pathologists be provided with all available information related to the study prior 

to conducting their readings. Information regarding gross morphologic abnormalities, mortality rates, 

and general test population performance and health are useful for pathologists to provide 

comprehensive reports and to aid in the interpretation of findings. For a more comprehensive discussion 

of standard reading approaches for toxicologic pathology studies, please refer to the Society of 

Toxicologic Pathology Best Practices for reading toxicologic histopathology studies (Crissman al., 

2004).

Severity Grading

In toxicologic pathology, it is recognized that compounds may exert subtle effects on tissues that are not 

adequately represented by simple binary (positive or negative) responses. Severity grading involves a 

semi-quantitative estimation of the degree to which a particular histomorphological change is present in 

a tissue section (Shackelford et al., 2002). The purpose of severity grading is to provide an efficient, 

semi-objective mechanism for comparing changes (including potential compound-related effects) 

among animals, treatment groups, and studies.

Severity grading will employ the following system:

Not remarkable

Grade 1 (minimal)

Grade 2 (mild)

Grade 3 (moderate)

Grade 4 (severe)

A grading system needs to be flexible enough to encompass a variety of different tissue changes. In 

theory, there are three broad categories of changes based on the intuitive manner in which people tend 

to quantify observations in tissue sections:

(1) Discrete: these are changes that can be readily counted. Examples include atretic follicles,

oocytes in the testis, and clusters of apoptotic cells.

(2) Spatial: these are changes that can be quantified by area measurements. This includes lesions

that are typically classified as focal, multifocal, coalescing, or diffuse. Specific examples 

include granulomatous inflammation and tissue necrosis.

(3) Global: these are generalized changes that would usually require more sophisticated 

measurement techniques for quantification. Examples include increased hepatocyte basophilia,

Sertoli cell/interstitial cell hypertrophy, or quantitative alterations in cell populations.
MMT ISR Pathology Appendix DRAFT 11 March 29, 2013

General severity grading scale

• Not Remarkable: This grade is used if there are no findings associated with a particular

diagnostic criterion.

• Grade 1: Minimal. Ranging from inconspicuous to barely noticeable but so minor, small, or

infrequent as to warrant no more than the least assignable grade. For discrete changes, grade 1 is 

used when there are fewer than 2 occurrences per microscopic field, or 1 - 2 occurrences per 

section. For multifocal or diffusely-distributed alterations, this grade is used for processes where 

≤ 20 % of the tissue in the section is involved.

• Grade 2: Mild. A noticeable feature of the tissue. For discrete changes, grade 2 is used when

there are 3 - 5 occurrences per microscopic field or per tissue section. For multifocal or 

diffusely-distributed alterations, this grade is used for processes where 20 - 50 % of the tissue in

the section are involved.

• Grade 3: Moderate. A dominant feature of the tissue. For discrete changes, grade 3 is used

when there are 6 - 8 occurrences per microscopic field or per tissue section. For multifocal or

diffusely-distributed alterations, this grade is used for processes where 50 - 80 % of the tissue in 

the section are involved.

• Grade 4: Severe. An overwhelming feature of the tissue. For discrete changes, grade 4 is

used when there are more than 9 occurrences per microscopic field or per tissue section. For

multifocal or diffusely-distributed alterations, this grade is used for processes where > 80 %

of the tissue in the section are involved.

At least some of the histomorphological changes that have been associated with EDCs in fish are 

considered to be exacerbations of “normal”, physiologic findings (e.g., oocyte atresia [Nagahama, 1983; 

Tyler and Sumpter, 1996]). At the discretion of the pathologist, the severity of a given change should

be scored according to one of the following two methods:

(1) score compound-exposed animals relative to the severity of the same change in control 

animals, or

(2) score all animals relative to “normal” as determined by the pathologist’s experience.

For each important (i.e., treatment-associated) finding, the method that was used should be stated in the 

Materials and Methods section of the pathology narrative report. By convention, severity grading 

should not be influenced by the estimated physiologic importance of the change, since this would add a 

further layer of subjectivity to the findings that complicates inter-laboratory results comparisons. For 

example, the presence of two oocytes in the testis should not be graded as “severe”, even if the 

pathologist considers this finding to be highly significant in terms of endocrine modulation.
MMT ISR Pathology Appendix DRAFT 12 March 29, 2013

Section 2.2 Data Recording.

An Excel worksheet form has been created that includes worksheets for primary, secondary, and 

additional diagnoses to facilitate histopathology data collection. In this worksheet, each data entry cell 

represents an individual fish. Additional sheets are available for comments and additional findings. For 

each fish, the pathologist records a severity score associated with the diagnosis. Diagnostic criteria with 

non-remarkable findings shall be denoted using (-). If there is no reasonably appropriate diagnostic term 

for a particular finding, the pathologist can create a term that can be recorded in the “Additional 

diagnoses” worksheet. If insufficient tissue is available for diagnosis, this should be recorded as IT 

(insufficient tissue). If a target tissue is missing, this should be recorded as MT (missing tissue).

(Attach sample spreadsheets: Medaka pathology .xls)

Section 2.3 Histopathology Report Format.

Each histopathology narrative report should contain the following five sections:

Introduction

Materials and Methods

Results

Discussion

Summary/Conclusions

(References)

The Introduction section briefly outlines the experimental design. The Materials and Methods section 

describes any items or procedures that are essentially different from Section 1: Post-mortem and 

Histotechnical Procedures. As applicable, specific severity grading criteria (see Severity Grading) 

should also be listed in this section. The Results section should report findings that are: (1) compoundrelated; (2) potentially compound-related; (3) novel or unusual. Detailed histomorphological 

descriptions need only be included for findings that differ substantially from diagnoses presented in 

Section 4 (Glossary and diagnostic criteria). It is intended that the Results section should be as objective 

as possible (i.e., opinions and theories should be reserved for the Discussion section). The Discussion 

section, which contains subjective information, should address relevant findings that were reported in 

the Results section. Opinions and theories can be included in this section, preferably backed by 

references from peer-reviewed sources, but unsupported speculation should be avoided. The 

Summary/Conclusions section should encapsulate the most important information from the Results and 

Discussion sections.
MMT ISR Pathology Appendix DRAFT 13 March 29, 2013

Section 2.4 Gonadal staging criteria

The goal of gonadal staging is to determine if the administration of a particular endocrine-active

substance affects the reproductive cycle status of adult male and female fish. The purpose of this

section is to describe a rapid, semi-quantitative method for assessing the proportions of various

gametogenic cell types (gonadal staging) based on the light microscopic examination of hematoxylin

and eosin- (H&E-)stained histological sections.

Semi-quantitative gonadal staging has been proposed for, or employed in, studies involving 

fathead minnow and other fish species (Ankley et al., 2002; Jensen et al., 2001; Miles-Richardson et

al., 1999a; Nichols et al., 2001; US Environmental Protection Agency, 2002). Although such 

studies generally included excellent descriptions of the different gametogenic maturation stages (e.g.,

spermatogonium through spermatozoa for the testis), they did not incorporate pre-defined categorical

guidelines for evaluating and reporting the reproductive cycle status of an individual fish. To maintain

scientific integrity across the board in a program that involves multiple studies, multiple laboratories,

and large numbers of animals, it is essential that observations are recorded on a fish-by-fish basis. The

use of a categorization system can improve the consistency and objectivity of reported 

observations within and among experiments; consequently, comparisons of the results are more 

meaningful.

Categorization systems also have some drawbacks and limitations, the most significant of which are (1)

the potential loss of discriminatory data when similar, but not identical, types of observations are

combined (binned) into a single class; (2) the questionable biological relevance of the classification

criteria in some cases; and (3) the inability of any single classification system to address every type of

observation (either predicted or unforeseen).

To address this last limitation, gonadal staging is accompanied by a complete histopathological

evaluation of the gonads; in this manner, the loss or overabundance of a specific gametogenic cell type,

for example, can be documented.

The semi-quantitative gonadal staging scheme selected for analysis of fish gonads is a modification of 

a system adopted by the United States Department of the Interior, U.S. Geological Survey, Biological 

Resources Division as part of the “U.S. Biomonitoring of Environmental Status and Trends (BEST) 

Program” (McDonald et al., 2000). The authors of the BEST system credit previous work by Treasurer

and Holiday (1981), Nagahama (1983), Rodriquez et al. (1995), and Goodbred et al. (1997). The

foremost benefits of this system are speed and ease of use, especially when compared to fullyquantitative staging. The basis of the BEST system is a visual assessment of the density of

gametogenic precursors as compared to mature gametocytes in one or more gonad sections.

Accordingly, the stage numbers (testis: Stages 0 to 4; ovary: Stages 0 to 5) increase in direct

relationship to the relative proportion of mature cells.

Although the BEST system was initially developed to assess reproductive function in seasonal

spawners such as carp (Cyprinidae) and black basses (Centrarchidae), the same stage categories can be

applied to fractional spawners such as fathead minnow. This was demonstrated at the October 2003

meeting of the histopathology subcommittee of the Fish Drafting Group at the Paris meeting, at

which the participants were asked to briefly evaluate the applicability of a modified BEST system 

using actual histological specimens of the three species fathead minnow, medaka and zebrafish. In
MMT ISR Pathology Appendix DRAFT 14 March 29, 2013

general, the participants agreed that they could readily recognize the various gonadal stages as

defined by modified BEST criteria. The participants acknowledged that the terminal stages of the

system (testis: Stage 4, “Spent”; ovary: Stage 5, “Post-ovulatory”) would be minimally present (or

possibly not present at all) among reproductively-active adult fathead minnow, medaka and zebrafish,

because they are not seasonal spawners. Similarly, it was thought that Stage 4, “Late

development/hydrated”, would be rarely observed in the test fishes, due to the

very transient nature of this stage in fractional spawners.

A few modifications have been made to the BEST system to adapt it for use. For example, there

is currently no provision in the system for gonads that are comprised entirely of spermatogonia or

oogonia. Although it is intended that reproductively mature fish are used, it is possible that an

occasional animal may not attain sexual maturity by the time the experiment is terminated, or that

certain test compounds might cause reversion of the gonads to a juvenile phenotype. Therefore, a prestaging category called “juvenile” has been added for both male and female fish as one modification of 

the BEST system. Another modification to the system involves an apparent discrepancy between the

BEST system and Goodbred et al. (1997) concerning the thickness of the testicular germinal

epithelium as a staging criterion. As indicated by Goodbred et al. (1997), the germinal epithelium

becomes thinner as the testis stage increases, whereas the reverse occurs according to the BEST

system (as presented in McDonald et al., 2000). Although it is difficult to find corroborating statements

in the scientific literature, empirical evidence indicates that Goodbred et al. (1997) is correct on this 

point. A third modification to the system is the option to subdivide a stage into two subordinate stages

(e.g., Stages 3A and 3B), if the pathologist believes that this tactic would reveal a subtle,

compound-related effect that might otherwise be missed. Other modifications to the system are 

relatively minor and primarily involve rewording for clarification.

The cell distribution pattern is likely to vary throughout a given tissue section, the gonad should be

staged according to the predominant pattern in that section. Both gonads should be staged as a single

organ according to the predominant pattern. Gonads that cannot be reasonably staged for various

reasons (e.g., insufficient tissue, or extensive necrosis, inflammation, or artifact) should be recorded as

UTS (unable to stage).
MMT ISR Pathology Appendix DRAFT 15 March 29, 2013

Section 2.4.1 Criteria for staging testes in Medaka.

To derive each stage score, the estimated width of the germinal epithelium (EWG) can be

compared to the estimated width of the testis (EWT) in the following manner:

• Stage 1: EWG > 2⁄3 EWT

• Stage 2: EWG 2⁄3 to > 1⁄2 EWT

• Stage 3: EWG 1⁄2 to > 1⁄4 EWT

• Stage 4: EWG < 1⁄4 EWT

Figure 2-1.Examples of staging system applied to testes from Japanese medaka: Black arrows (↔) 

represent the estimated width of the testis (EWT), and red arrows (↔) represent the estimated width of the

germinal epithelium (EWG; measurements are illustrated unilaterally for simplicity). In order to obtain

comparable sections, it is imperative that each section contains a portion of the central duct (CD), 

preferably at its widest and longest extent (paraffin, H&E).
MMT ISR Pathology Appendix DRAFT 16 March 29, 2013

Section 2.4.2 Criteria for staging ovaries in Medaka.

The following are morphologic criteria for staging female Japanese medaka:

• Juvenile: gonad consists of oogonia exclusively; it may be difficult or impossible

to confirm the sex of these individuals.

• Stage 0 – undeveloped: entirely immature phases (oogonia to perinucleolar 

oocytes); no cortical alveoli

• Stage 1 – early development: vast majority (e.g., > 90%) are previtellogenic follicles, predominantly perinucleolar through cortical alveolar.

• Stage 2 – mid-development: at least half of observed follicles are early and midvitellogenic.

• Stage 3 – late development: majority of developing follicles are late vitellogenic.

• Stage 4 – late development/hydrated: majority of follicles are late 

vitellogenic and mature/spawning follicles; follicles are larger as compared to 

Stage 3.

Stage 0 Stage 1

Stage 2 Stage 4

Figure 2-2. Examples of the staging system applied to the ovaries of adult Japanese medaka (paraffin, H&E)
MMT ISR Pathology Appendix DRAFT 17 March 29, 2013

Section 4: WebPath

The valid and consistent evaluation of pathology effects among pathologists and labs requires 

that each pathologist be familiar with the diagnostic features and the appropriate severity scores 

of EDC-induced pathology. Accurate and consistent pathology is necessary to perform valid 

statistical analyses of EDC and chemical induced treatment effects. To ensure that 

histopathology is performed to the highest standards of quality and consistency, it is imperative 

that persons performing the microscopic examinations are fully trained and that standardized 

diagnostic criteria and terminology are available for these evaluations. Toward this end, EPA is 

creating an interactive online website (figure 1) for viewing virtual images of scanned whole 

histologic sections (figure 2) from different animal species. These sections have been selected to 

demonstrate various types of findings that have been associated with endocrine disruption 

activity, and annotations have been added to the images to describe the changes and provide 

context. As time permits and the knowledge base expands, further images may be added to this 

site. Although these images are intended primarily for use by professional scientists who are 

actively involved in EDSP research, EPA is currently maintaining this as an open site.

Figure 1. Beta version of the homepage for the Online Pathology Reference Atlas currently under 

development by EPA.

Section 5: References cited in text

• Johnson R, Wolf, J, and Braunbeck T, 2009. OECD Guidance Document for the Diagnosis of 

Endocrine-Related Histopathology of Fish Gonads. OECD Environmental Health and Safety 

Monograph Series on Testing and Assessment. 

• Crissman, J.W., Goodman, D.G., Hildebrandt, P.K., Maronpot, R. R., Prater, D. A., Riley, J. H., 

Seaman, W. J., and Thake, D. C. (2004). “Best Practices Guideline: Toxicologic Pathology.” Toxicol 

Pathol 32:126-131.

• MMT pathology summary octylphenol

a. Gray MA, Metcalfe CD, 1997. Induction of testis-ova in Japanese medaka (Oryzias latipes) 

exposed to p-nonylphenol. Environ Toxicol Chem 16: 1082-1086.

b. Karels AA, Manning S, Brouwer TH, Brouwer M, 2003. Reproductive effects of estrogenic 

and antiestrogenic chemicals on sheepshead minnows (Cyprinodon variegates). Environ 

Toxicol Chem 22: 855-865.

c. Knörr S and Braunbeck T, 2002. Decline in reproductive success, sex reversal, and 

developmental alterations in Japanese medaka (Oryzias latipes) after continuous exposure to 

octylphenol. Ecotoxicol and Environ Safety 51: 187-195.

d. Vajda AM, Barber LB, Gray JL, Lopez EM, Woodling JD, Norris DO, 2008. Reproductive 

disruption in fish downstream from an estrogenic wastewater effluent. Environ Sci Technol 

42: 3407-3414.

e. Van den Belt K, Verheyen R, Witters H, 2001. Reproductive effects of ethynylestradiol and 

4t-octylphenol on the zebrafish (Danio rerio). Arch Environ Contam Toxicol 41: 458-467.

f. Yokota H, Seki M, Maeda M, Oshima Y, Tadokoro H, Honjo T, Kobayashi K, 2001. Lifecycle toxicity of 4-nonylphenol to medaka (Oryzias latipes). Environmental Toxicology and 

Chemistry, 20: 2552-2560. 

• MMT pathology summary trenbolone
MMT ISR Pathology Appendix DRAFT 18 March 29, 2013

a. Ankley GT, Jensen KM, Makynen EA, Kahl MD, Korte JJ, Hornung MW, Henry TR, Denny 

JS, Leino RL, Wilson VS, Cardon MC, Hartig PC, Gray LE, 2003. Effects of the androgenic 

growth promoter 17β-Trenbolone on fecundity and reproductive endocrinology of the fathead 

minnow. Environmental Toxicology and Chemistry 22, 1350-1360.

b. Örn S, Yamani S, Norrgren L, 2006. Comparison of Vitellogenin Induction, Sex Ratio, and 

Gonad Morphology Between Zebrafish and Japanese Medaka After Exposure to 17αEthinylestradiol and 17β-Trenbolone. Arch Environ Contam Toxicol 51, 237-243.

c. Seki M, Fujishima S, Nozaka T, Maeda M, Kobayashi K, 2006. Comparison of response to 

17β-estradiol and 17β-trenbolone among three small fish species. Environmental Toxicology 

and Chemistry 25, 2742-2752. 

• MMT pathology summary procholoraz

a. Andersen HR, Vinggaard AM, Rasmussen TH, Gjermandsen IM, Bonefeld-Jorgensen EC 

(2002) Effects of currently used pesticides in assays for estrogenicity, androgenicity, and 

aromatase activity in vitro. Toxicol Appl Pharmacol, 179(1):1-12.

b. EPA (2005) Phase 1-B Pathology Analysis in Fish Exposed to Endocrine Disruptors. Study 

No. WA 05-12, Test PROCHL-1, Medaka / Prochloraz. USEPA Mid-Continent Ecology 

Division, Duluth, MN.

c. Mason JI, Carr BR, Murry BA (1987) Imidazole antimycotics: selective inhibitors of steroid 

aromatization and progesterone hydroxylation. Steroids, 50(1-3):179-189.

d. Sanderson JT, Boerma J, Lansbergen GW, van den Berg M (2002) Induction and inhibition of 

aromatase (CYP19) activity by various classes of pesticides in H295R human adrenocortical 

carcinoma cells. Toxicol Appl Pharmacol, 182(1):44-54.

e. Vinggaard AM, Hnida C, Breinholt V, Larsen JC (2000) Screening of selected pesticides for 

inhibition of CYP19 aromatase activity in vitro. Toxicol In Vitro, 14(3):227-234.

f. Vinggaard AM, Nellemann C, Dalgaard M, Jorgensen EB, Andersen HR (2002) 

Antiandrogenic effects in vitro and in vivo of the fungicide prochloraz. Toxicol Sci., 

69(2):344-53.