¦— j» United States Prevention, Pesticides EPA 740-C-09-007
Environmental Protection and Toxic Substances October 2009
%#Crri A9ency
Endocrine Disruptor
Screening Program
Test Guidelines
OPPTS 890.1350:
Fish Short-Term
Reproduction Assay
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NOTICE
This guideline is one of a series of test guidelines established by the Office of
Prevention, Pesticides and Toxic Substances (OPPTS), United States Environmental Protection
Agency for use in testing pesticides and chemical substances to develop data for submission to
the Agency under the Toxic Substances Control Act (TSCA) (15 U.S.C. 2601, et seq.), the
Federal Insecticide, Fungicide and Rodenticide Act (FIFRA) (7 U.S.C. 136, etseq.), and section
408 of the Federal Food, Drug and Cosmetic (FFDCA) (21 U.S.C. 346a).
The OPPTS test guidelines serve as a compendium of accepted scientific
methodologies and protocols that are intended to provide data to inform regulatory decisions
under TSCA, FIFRA, and/or FFDCA. This document provides guidance for conducting the test,
and is also used by EPA, the public, and the companies that are subject to data submission
requirements under TSCA, FIFRA and/or the FFDCA. As a guidance document, these
guidelines are not binding on either EPA or any outside parties, and the EPA may depart from
the guidelines where circumstances warrant and without prior notice. The procedures contained
in this guideline are strongly recommended for generating the data that are the subject of the
guideline, but EPA recognizes that departures may be appropriate in specific situations. You
may propose alternatives to the recommendations described in these guidelines, and the
Agency will assess them for appropriateness on a case-by-case basis.
For additional information about OPPTS harmonized test guidelines and to access the
guidelines electronically, please go to http://www.epa.gov/oppts and select "Test Methods &
Guidelines" on the left side navigation menu. You may also access the guidelines in
http://www.regulations.gov grouped by Series under Docket ID #s: EPA-HQ-OPPT-2009-0150
through EPA-HQ-OPPT-2009-0159, and EPA-HQ-OPPT-2009-0576.
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OPPTS 890.1350: Fish Short-Term Reproduction Assay
(a) Scope.
(1) Applicability. This guideline is intended to meet testing requirements of
the Toxic Substances Control Act (TSCA) (15 U.S.C. 2601, et seq.), the
Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) (7 U.S.C.
136, et seq.), and the Federal Food, Drug, and Cosmetic Act (FFDCA) (21
U.S.C. 346a).
(2) Background. The Endocrine Disruptor Screening Program (EDSP)
reflects a two-tiered approach to implement the statutory testing
requirements of FFDCA section 408(p) (21 U.S.C. 346a). In general, EPA
intends to use the data collected under the EDSP, along with other
information, to determine if a pesticide chemical, or other substances, may
pose a risk to human health or the environment due to disruption of the
endocrine system.
This test guideline is intended to be used in conjunction with other
guidelines in the OPPTS 890 series that make up the full screening
battery under the EDSP to identify substances that have the potential to
interact with the estrogen, androgen, or thyroid hormone (Tier 1
"screening"). The determination will be made on a weight-of-evidence
basis taking into account data from the Tier 1 assays and other
scientifically relevant information available. The fact that a substance may
interact with a hormone system, however, does not mean that when the
substance is used, it will cause adverse effects in humans or ecological
systems.
Chemicals that go through Tier 1 screening and are found to have the
potential to interact with the estrogen, androgen, or thyroid hormone
systems will proceed to the next stage of the EDSP where EPA will
determine which, if any, of the Tier 2 tests are necessary based on the
available data. Tier 2 testing is designed to identify any adverse
endocrine-related effects caused by the substance, and establish a
quantitative relationship between the dose and that endocrine effect.
(3) Source. The source material used in developing this harmonized OPPTS
guideline is US-EPA (2002) (Ref. 15). This guideline is consistent with
OECD Test Guideline 229 (OECD 2009) published by the Organization for
Economic Cooperation and Development (OECD) (Ref. 9).
(b) Introduction. The Agency has described a short-term reproduction assay with
the fathead minnow (Pimephales promelas) that considers reproductive fitness
as an integrated measure of toxicant effects, and measures of a suite of
histological and biochemical endpoints that reflect effects associated with
disturbance to the hypothalamus-pituitary-gonadal (HPG) endocrine axis (Ref.
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15). The assay is initiated with mature male and female fish. During a 21-day
chemical exposure, survival, reproductive behavior, secondary sexual
characteristics, fecundity, and fertility are monitored (Ankley et al. 2001) (Ref. 2).
Assessments of F1 development can be made, if desired. At the end of the test,
measurements are made that are reflective of the status of the reproductive
endocrine system, including male secondary sex characteristics, gonadal
histopathology, gonado-somatic index, and plasma concentrations of vitellogenin
(VTG).
Principle of the Assay. The experimental protocol for the short-term
reproduction assay is based upon the protocol developed by Ankley et al. (2001)
(Ref. 2) using the fathead minnow (Pimephales promelas). A more detailed
description of the method is presented in US-EPA (2002) (Ref. 15), which can be
consulted for further methodological information. This assay will measure the
reproductive performance of groups of fathead minnows as the primary indicator
for potential endocrine disruption. Additional measurements of morphological,
histopathological, and biochemical endpoints should be performed to ensure that
potential toxicological and endocrine mechanisms of concern are detected for the
test chemical.
The assay should be initiated with sexually mature male and female fish. During
a 21-day chemical exposure, survival, reproductive behavior, and secondary sex
characteristics should be observed while fecundity and fertilization success
should be monitored daily. At termination of the assay, measurements should be
made of a number of endpoints reflective of the status of the reproductive
endocrine system, including the gonado-somatic index (GSI), gonadal histology,
male secondary sex characteristics, and plasma concentrations of VTG. Also,
plasma sex steroid concentrations may also be determined.
The assays should be initiated with newly mature spawning adults of the same
age. Spawning performance should be established during a pre-exposure period
of at least 14 days.
The pre-exposure observations should occur in separate but identical
system/tanks as will be utilized for the chemical test (exposure period). Once
target test concentrations are reached and verified in exposure tanks, fish will be
transferred between tanks from the pre-exposure and exposure periods. An
overview of the relevant test conditions are provided in Table 1.
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Table 1. Experimental
Design for the Assay Method.
Parameter
Assay Protocol
Test species:
Reproductively active fathead minnows Pimephales promelas (4.5-6 months old)
Pre-exposure evaluation
Duration: minimum of 14 days; Data Collected: fecundity (daily)
Dilution water
Clean, surface, well, or reconstituted water
Total alkalinity > 20 mg/L (as CaCC>3)
Total organic carbon < 2 mg/L
Unionized Ammonia < 1 |_ig/L
Residual chlorine < 10 i_ig/L
Test material
NS
Test chamber size
18 L (40x20x20 cm)
Test volume:
10 L
# Exchanges/day
at least 6 tank volume exchanges
Flow rate:
2.7 L/hr
# Concentration / chemical
3
# Replicates:
4
Weight of each fish
NS. If possible, ± 20% arithmetic mean weight of same sex
# Fish/tank
4 females and 2 males
Total # fish/concentration
4 adult females and 2 adults males per replicate = 24 fish total per experimental
level. (96 fish per test chemical)
Feeding regime
Frozen brine shrimp, twice a day
# Controls
1 Dilution water control and a solvent control added if a solvent used
# Fish/control
4 adult females and 2 adults males per control
Photo period:
16 h light: 8 h dark
Temperature:
25°C ± 1°C
Light intensity
540- 1080 lux
Aeration:
None unless D.O. <4.9 mg/L
PH
O
CD
I
LO
CD
Biological endpoints:
Fecundity, fertilization success, adult survival, secondary sexual characteristics,
VTG, plasma sex steroids (optional), GSI, and gonadal histology,
Test validity criteria:
D.O. > 4.9 mg/L (> 60% saturation); mean temp. 25°C ± 1°C; 90% survival in the
controls and successful egg production in controls (e.g., spawning occurs at least
every 4 days in each control replicate or approximately 15 eggs/female/day/
replicate). Fertility > 95%. Measured exposure concentration CV < 20% for all
replicates.
NS = Not specified in procedure.
(d) Description of the Method.
(1) Selection of Test Organisms. The exposure phase should be started
with sexually dimorphic adult fish from a laboratory supply of
reproductively mature animals cultured at 25 ± 2°C. The fish must be
capable of spawning. For the whole group of fish used in the test, the
range in individual weights by sex at the start of the test should be kept
within ± 20% of the arithmetic mean weight of the same sex. A subsample
of fish should be weighed before the test to estimate the mean weight.
The fish selected should be the same age within the range of 4.5 to 6
months and reproductively mature.
Test fish should be selected from a single laboratory stock which has been
acclimated for at least two weeks prior to the test under conditions of
water quality and illumination similar to those used in the test. (Note, this
acclimation period is not an in situ pre-exposure period). It is
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recommended that test fish be obtained from an in-house culture, as
shipping of adult fish is stressful and will interfere with reliable spawning.
Fish should be fed brine shrimp twice per day throughout the holding
period and during the exposure phase.
Mortalities in the culture fish should be recorded and the following criteria
applied:
~ Mortalities of greater than 10% of culture population in seven days
preceding transfer to the test system: reject the entire batch;
~ Mortalities of between 5% and 10% of population: acclimation for
seven additional days; if more than 5% mortality during second
seven days, reject the entire batch;
~ Mortalities of less than 5% of population in seven days: accept the
batch.
Fish should not receive treatment for disease in the two-week acclimation
period preceding the test, during pre-exposure, or during the exposure
period. Moribund fish or fish with clinical signs of disease should not be
used.
Water. It is well-established that fathead minnow can reproduce
successfully over a wide range of water quality parameters. Therefore, no
specific water type is required for this test. Any uncontaminated surface,
well, or reconstituted water which meets the dilution water criteria (Table
1), and in which the fish can be cultured successfully should be
acceptable. The animals should be tested using a flow-through water
renewal system that maintains adequate water quality (temperature,
dissolved oxygen, low ammonia, etc.) and ensures a consistent exposure
of the test organisms to the parent chemical.
Assay System. Eighteen-liter glass exposure tanks are used for the test
system. As recommended by Ankley et at. (2001) (Ref. 2), dimensions of
the test tanks must be such that the animals can interact in a fashion
conducive to successful spawning. Each tank should contain three semi-
circular spawning substrates {e.g., PVC pipe 10-20 cm in length split
lengthwise, US-EPA 1987) (Ref. 14). The test tank contains 10 L of test
solution, which is renewed at least once every 4 hours (6 volume
exchanges per day). This particular animal loading/water renewal rate is
within recommended guidelines and in studies conducted according to this
method, has maintained acceptable water quality while not utilizing an
excessive amount of test material.
A randomized complete block design (4 blocks with one replicate tank of
each treatment) should be used for the reproductive assay. This design is
intended to randomize for effects associated with the local environment
(e.g., light and water) and possible trends associated with diluter function
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during testing. All fish should be impartially assigned to tanks before pre-
exposure, and then tanks should be randomly assigned to treatments
within a block after spawning is established in the pre-exposure period.
The blocks are filled in a random order, with the four tanks with the highest
per-female fecundity (established during pre-exposure) being assigned
first, followed by the second-highest spawners, etc. Thus, when one
evaluates the difference between treatment means, the variability
associated with experimental environment, experimental containers, and
organisms being treated is removed and only the effect(s) of the treatment
remains.
Preparation and Testing of Chemical Exposure Water. Test chemicals
will possess varied physicochemical properties that will require different
approaches for preparation of chemical exposure water. When possible,
direct addition to water should be utilized if the test chemical has
sufficiently high water solubility. For test chemicals with limited water
solubility, the use of a saturator column is recommended to prepare the
concentrated stock solution (US-EPA 2002) (Ref. 15). For poorly soluble
or otherwise difficult to test substances, a solvent may be necessary, but
only as a last resort (see OECD 2000, Guidance Document 23: Guidance
Document on Aquatic Toxicity Testing of Difficult Substances and
Mixtures) (Ref. 8). All aqueous stock solutions should be encased in
black tarpaulin during agitation and subsequent storage to prevent photo-
oxidation.
Analytical Determinations. After preparation of the stock solution,
determinations of the concentration should be made using appropriate
methods. The concentrations of the test chemical in the exposure tanks
should be measured prior to adding fish to verify that target concentrations
are reached. Additionally, water samples should be collected weekly
thereafter and analyzed for the test chemical, testing two of the four tanks
per exposure level each time (High, Medium, Low, and Control). More
frequent sampling is required for chemicals with known lability, if the
chemical concentrations are not maintained between sampling intervals,
or if disturbances to the delivery system occur.
Chemicals should be directly measured using methods such as
spectrophotometry, gas chromatography - electron capture detection (GC-
ECD), gas chromatography-mass spectroscopy (GC-MS), HPLC (high
performance liquid chromatography) with an appropriate detector, or ion-
chromatography with conductivity detection.
~ 96 Hour Range-finding. A range-finding test may be necessary to
establish test chemical concentrations when 96-hour LC50 data for
fathead minnow are not available.
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The highest target test concentration for the range-finder may be
based upon toxicity data from other fish species (US-EPA 2002)
(Ref. 15). If such information is lacking, the highest concentration
should be the solubility limit of the chemical in water but no more
than 100 ppm. The test concentration should then be decreased by
a factor of 10 for each successively lower exposure. Range-finding
tests should be performed with fish of similar age and size to those
that should be utilized in the test using a 96-hour exposure to five
test concentrations plus a control (six total), with four females and
two males per exposure tank (36 fish total). The number of
mortalities that occur should be used to develop a dose response
curve. Based upon the results, the highest concentration that does
not result in increased mortality or signs of overt morbidity
compared to controls, or 1/3 the derived 96-hr LC50 will serve as the
highest exposure concentration in the 21-day test.
Biochemical Determinations.
(i) Vitellogenin (VTG). Enzyme-linked immunosorbent assay (ELISA)
tests should be conducted using commercially available test kits or
equivalents, preferably using homologous VTG standard and
homologous antibodies. If heterologous assays are used, these
should be well validated and meet all the relevant Q/A criteria. The
methods used for the bioanalytical measurements of VTG should
follow manufacturer's specifications. Any non-detect samples
should be recorded as % the limit of quantification (LOQ).
It is recommended to use a method capable of detecting VTG
levels as low as few ng/ml plasma, which is the background level in
unexposed male fish. Quality control of vitellogenin analysis will be
accomplished through the use of standards, blanks, and duplicate
analyses. Each ELISA plate used for VTG assays should include
the following quality control samples: at least 6 calibration
standards covering the range of expected vitellogenin
concentrations, and at least one non-specific binding assay blank
(analyzed in duplicate). Absorbance of these blanks should be less
than 0.06 absorbance units. Two aliquots (well-duplicates) of each
sample dilution should be analyzed. Well-duplicates that differ by
more than 20% should be reanalyzed.
The correlation coefficient (R2) for calibration curves must be
greater than 0.99. However, a high correlation is not sufficient to
guarantee adequate prediction of concentrations in all ranges. In
addition to having a sufficiently high correlation for the calibration
curve, the concentration of each standard, as calculated from the
calibration curve, should all fall between 70 and 120 % of its
nominal concentration. If the nominal concentrations trend away
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from the calibration regression line (e.g. at lower concentrations), it
may be necessary to split the calibration curve into low and high
ranges or to use a nonlinear model to adequately fit the absorbance
data. If the curve is split, both line segments should have R2 >
0.99.
On each day that vitellogenin assays are performed, a fortification
sample made using an inter-assay reference standard should be
analyzed. The vitellogenin used to make the inter-assay reference
standard will be from a batch different from the one used to prepare
calibration standards for the assay being performed.
The fortification sample should be made by adding a known
quantity of the inter-assay standard to a sample of control male
plasma. The sample should be fortified to achieve a vitellogenin
concentration between 10 and 100 times the expected vitellogenin
concentration of control male fish. The sample of control male
plasma that is fortified may be from an individual fish or may be a
composite from several fish.
A subsample of the unfortified control male plasma should be
analyzed in at least two duplicate wells. The fortified sample also
should be analyzed in at least two duplicate wells. The mean
quantity of vitellogenin measured in the two unfortified control male
plasma samples will be added to the calculated quantity of
vitellogenin added to the fortification samples to determine an
expected concentration. The ratio of this expected concentration to
the measured concentration will be reported along with the results
from each set of assays performed on that day.
Sex steroids. Plasma concentrations of 17(3-estradiol and
testosterone are recommended and should be determined using
radioimmunoassay (RIA) techniques optimized for the relatively
small sample volumes obtained from the fathead minnow (Jensen
et at. 2001) (Ref. 6). Additional guidance for the measurement of
fathead minnow plasma sex steroids using RIA are provided in US-
EPA (2002) (Ref. 15). Given that limited plasma volume may be
available, analysis priority should be given to (3-estradiol for females
and testosterone in males, then (3-estradiol in males and
testosterone in females if possible. Since plasma 11-
ketotestosterone concentrations typically are correlated with
testosterone concentrations in males, 11-ketotestosterone
measurements are not deemed mandatory. Any non-detect
samples should be recorded as % the limit of quantification (LOQ).
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Assay Initiation and Conduct.
(i) Duration of Exposure. The pre-exposure phase is at least 14
days; exposure duration is 21 days.
(ii) Feeding. The fish should be fed at least twice per day with brine
shrimp at a rate sufficient to promote active reproduction and
maintain body condition. Food should be withheld from the fish for
12 hours prior to the day of sampling to aid in histological
processing of small fish. Uneaten food and fecal material should
be removed from the test tanks at least twice weekly, e.g., by
carefully cleaning the bottom of each tank using suction.
(iii) Pre-exposure. The pre-exposure phase should last a minimum of
14 days. The assay should use fish previously maintained in
communal culture tanks that are of the same optimal size and
age— approximately 4.5-6 months —for reproduction. To avoid
mistaking immature males for females, fish should be sexually
mature when chosen, with identifiable secondary sex
characteristics. At 4.5 to 6 months of age, males are larger and
darker and start exhibiting nuptial tubercles, while females possess
an ovipositor. However, unless males are housed with females in
conditions suitable for spawning {e.g., spawning substrates
present) they may not fully express secondary sex characteristics
and one sex can be mistaken for the other.
Additional tanks set up at the beginning of pre-exposure will ensure
that sufficient replicates with the correct sex ratio are available.
Four females and two males should be randomly assigned to the
replicate exposure tanks, with additional tanks set up for pre-
exposure to account for a lack of spawning in some tanks, incorrect
sex ratio, and/or mortality during the pre-exposure phase. Any
specimens whose sex cannot be identified should be excluded from
the assay. The pre-exposure phase of the assay should be
conducted under conditions (temperature, photo period, feeding,
etc.) identical to those used during the chemical exposure. The
animals should be fed frozen brine shrimp twice daily. Fecundity
data should be collected daily.
For each assay, successful pre-exposure conditions (suitability for
testing) are established when regular spawning occurs in each
replicate test tank at least two times in the immediately preceding 7
days and egg production exceeds 15 eggs/female/day/replicate
tank. Tanks thus established as suitable for testing (correct sex
ratio, successful spawning, and no mortality) should then be
randomly assigned to treatments within a block for the exposure
phase (see section (d)(3)).
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Chemical Exposure. Prior to initiation of the exposure period,
proper function of the chemical delivery system should be
confirmed. After successful spawning is verified during pre-
exposure as per the requirements of the assay, the chemical
exposure should be initiated and continued for 21 days. The
exposures should be conducted at three chemical concentrations,
as well as a diluent water control and solvent control (if a solvent is
necessary), with four experimental units (replicates) per treatment.
Each replicate tank should contain four female and two male fish.
The test chemical should be delivered to the exposure tank using a
proportional diluter or other appropriate delivery system. The
exposure should be conducted for 21 days, during which time the
appearance of the fish, behavior, and fecundity should be assessed
daily. At termination of the exposure, blood samples are taken from
adults and analyzed for VTG and also sex steroids (optional).
Secondary sex characteristics should be recorded, and the gonads
should be removed after fixation for GSI determination and later
histological analysis.
Frequency of Analytical Determinations and Measurements.
All necessary analytical methods should be established before
initiation of the exposure period, including sufficient knowledge of
the substances' stability in the test system. During the test, the
concentrations of the test substance should be determined at
regular intervals, as follows: the flow rates of diluent and toxicant
stock solution should be checked at intervals, at least once per
week, and should not vary by more than 20% throughout the test.
Actual test chemical concentrations should be measured in all
tanks at the start of the test and at weekly intervals thereafter,
sampling two of the four replicates per group each time.
~ Results should be based on measured concentrations and
should be included in reporting.
~ Samples may need to be centrifuged or filtered (e.g., using a
0.45 |jm pore size). If needed, then centrifugation is the
recommended procedure; however, if the test material does
not adsorb to filters, filtration may also be acceptable.
~ During the test, dissolved oxygen, temperature, and pH should
be measured in all test tanks at least once per week. If
dissolved oxygen reaches 4.9 mg/L, aeration should be
started for all test tanks immediately. Total hardness and
alkalinity should be measured in the controls and one tank at
the highest concentration at least once per week.
Temperature should preferably be monitored continuously in
at least one test tank.
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Observations and Measurements.
(1) Endpoints. A number of endpoints will be assessed over the course of,
and/or at conclusion of the assay. For those endpoints measured at test
termination, evaluations should proceed as follows:
~ Anesthesia with MS-222 (see US-EPA 2002 (Ref. 15) for details).
~ Body weight and length measurements.
~ Collection of fresh tissues (e.g., blood; Appendix A).
~ Evaluation of secondary sex characteristics (Appendix B).
~ In situ fixation of gonads.
~ Excision and weighing of gonads.
~ Preparation of gonads for histological evaluation.
(2) Behavior of Adults. Abnormal behavior (relative to controls), such as
hyperventilation, loss of equilibrium, uncoordinated swimming, atypical
quiescence, or feeding abstinence, should be noted during the daily
observations. Alterations in reproductive behavior, particularly loss of
territorial aggressiveness by males, also should be noted. Behavior
parameters have not been established for measurement, as this is
primarily intended to inform interpretations of any alterations in core
endpoints such as fecundity and possibly fertility. Thus, although they are
not specifically measured quantitatively, proactive evaluation of such
behaviors should be made as informative indicators of condition of the
fish.
(3) Fecundity. Egg production should be determined daily. Because fathead
minnows spawn within a few hours after the lights are turned on, they
should not be disturbed (except for feeding) until late morning. This
should allow time for spawning and fertilization to be completed and for
eggs to water-harden. The spawning substrates should be removed from
the tanks to enumerate any eggs that are present. It is expected that one
spawn typically will be composed of 50 to 250 eggs (US-EPA, 2002) (Ref.
15). If no eggs are present, the substrate is left in the tank; new
substrates should be added to replace any that are removed. Fecundity
should be expressed on the basis of surviving females per reproductive
(test) day per replicate. Cumulative eggs laid over the test duration should
also be determined.
(4) Fertilization Success. After the spawning substrate has been removed
from the tank, the embryos should be carefully rolled off with a gentle
circular motion of an index finger and visually inspected under appropriate
magnification. If spawning occurred that morning, embryos typically
should be undergoing late cleavage, and determination of the fertility rate
(number embryos/number of eggs x 100%) should be easily achieved.
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Infertile eggs are opaque or clear with a white dot where the yolk has
precipitated; viable embryos remain clear for 36 to 48 hours until reaching
the eyed stage.
Appearance of Adults. Secondary sex characteristics are under
endocrine control; therefore, observations of physical appearance of the
fish should be made over the course of the test, and quantified at
conclusion of the study as described below in the section "Secondary sex
characteristics". Experience to date with fathead minnows suggests that
some endocrine active chemicals may initially induce changes in the
following external characteristics: body color (light or dark), coloration
patterns (presence of vertical bands), body shape (head and pectoral
region), and specialized secondary sex characteristics (size of dorsal nape
pad, number of nuptial tubercles in male fathead minnow, ovipositor size
in females). Notably, chemicals with certain modes of action may cause
abnormal occurrence of secondary sex characteristics in animals of the
opposite sex; for example, androgen receptor agonists, such as
methyltestosterone and dihydrotestosterone, can cause female fathead
minnows to develop pronounced nuptial tubercles (Ankley et al. 2001
(Ref. 2); Smith 1974 (Ref. 12); and Panter etal., 2004 (Ref. 10)). It also
has been reported that estrogen receptor agonists and androgen receptor
antagonists can decrease nuptial tubercle numbers and size of the dorsal
nape pad in adult males (Miles-Richardson et al., 1999 (Ref. 7); Holbech
etal., 2001 (Ref. 5)).
Survival. Daily assessment of survival should be made to provide a basis
for expression and interpretation of reproductive output, that is, number of
eggs/female/day. Fish should be examined daily during the test period
and any external abnormalities (such as hemorrhage, discoloration) noted.
Any mortality should be recorded and the dead fish removed as soon as
possible. Dead fish should not be replaced in either the control or
treatment tanks.
Sampling offish. At the conclusion of the exposure, the fish should be
anesthetized by transfer to an oxygenated solution of MS-222 (100 mg/L
buffered with 200 mg NaHC03/L) for sampling. If potency of the solution
is not adequate, additional MS-222 (< 10 mg) may be added to strengthen
effectiveness.
Body weight and length: Body weight is recorded to the nearest 0.01 g
at test termination, and standard length is recorded to the nearest 0.1 mm.
Blood Sampling. Blood should be collected from the caudal artery/vein
(Appendix A) with a heparinized microhematocrit capillary tubule.
Depending upon the size of the fathead minnow (which usually is sex-
dependent), blood volumes generally range from 20 to 60 |jL. Plasma
should be separated from the blood via centrifugation (approximately 3
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minutes at 15,000 x g) and stored with protease inhibitors at -75°C to -
85°C until analyzed for VTG and sex steroids. If limited plasma volume is
available, VTG should be analyzed first, followed by 17(3-estradiol for
females and testosterone in males, then by 17(3-estradiol in males and
testosterone in females if possible. Any non-detect samples should be
recorded as % the limit of quantification (LOQ).
Vitellogenin (VTG). The measurement of VTG in plasma samples should
be performed using an enzyme-linked immunosorbent assay (ELISA).
Polyclonal fathead minnow (Pimephales promelas) VTG antibody and
purified VTG protein, also from the fathead minnow, should be utilized.
Any non-detect samples should be recorded as % the limit of
quantification (LOQ).
Sex Steroids. Plasma concentrations of 17(3-estradiol and testosterone
are recommended and should be determined using radioimmunoassay
(RIA) techniques optimized for the relatively small sample volumes
obtained from the fathead minnow. Procedures for the measurement of
fathead minnow plasma sex steroids using RIA are provided in US-EPA
(2002) (Ref. 15). Given that limited plasma volume may be available,
analysis priority should be given to 17(3-estradiol for females and
testosterone in males, then 17(3-estradiol in males and testosterone in
females if possible. Any non-detect samples should be recorded as % the
limit of quantification (LOQ).
Secondary Sex Characteristics at Test Termination. In addition to
observations of external characteristics made throughout the test
(described above in the section "Appearance of adults"), secondary sexual
characteristics should be quantified directly at test termination (Appendix
B). Because some aspects of appearance (primarily color) can change
quickly with handling, it is important that qualitative observations be made
prior to removal of animals from the test system.
Nuptial tubercles should be counted, mapped, and scored. Methods for
the evaluation of secondary sex characteristics in fathead minnow are
provided in Appendix B.
Gonad Size and Histology. Necropsy should quickly follow other
evaluations at test termination (anesthetization, length and body weight
measurements, collection of fresh tissues (e.g., blood), and evaluation of
secondary sex characteristics). Rapid gonad fixation in Davidson's
fixative will prevent autolysis and cellular deterioration. Fixed gonads are
removed for weighing and histological analysis.
The following steps should be followed for gonad fixation (see Appendix C
for illustration):
Page 12
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Step 1: Using a syringe, approximately 0.5 mL of Davidson's fixative
should be gently applied to the gonads in situ. Approximately 90
seconds following the application of fixative, the liquid fixative
within the abdomen should be removed with a gauze sponge, by
gentle blotting. Abdominal viscera should be removed and the
gonads should be excised in a manner similar to the abdominal
viscera:
a. Using microdissection scissors, the spermatic ducts or
oviducts should be severed proximal to the genital pore.
b. Microdissection forceps should then be applied to the
spermatic ducts/oviducts. Using gentle traction, the gonads
should be dissected out of the abdominal cavity in a caudal to
cranial direction, severing the mesorchial/mesovarial
attachments as needed using the microdissection scissors.
The left and right gonads may be excised individually or they
may be excised simultaneously and subsequently divided at
their caudal attachment.
Step 2: The fixed gonads should be weighed (fixed weight to the nearest
0.1 mg) to determine the GSI (GSM00% x gonad wt/body wt).
Typical GSI values for reproductively active fathead minnows
range from 8 to 13% for females and from 1 to 2% for males.
Step 3: The gonads (right and left) should be placed into a pre-labeled
plastic tissue cassette which should then be placed into an
individual container of Davidson's fixative accompanied by the
abdominal viscera. The volume of fixative in the container should
be at least 20 times the approximated volume of the tissues. The
fixative container should be gently agitated for 5 seconds to
dislodge air bubbles from the cassette.
Routine histological procedures should be used to assess the condition of
testes and ovaries from the fish using procedures provided in
Histopathology Guidelines for the Fathead Minnow (Pimephales promelas)
21-day Reproduction Assay (Appendix E), and should be performed by a
toxicologic pathologist with experience with small fishes. The
histopathology guidelines detail the post mortem and histotechnical
procedures that should be used.
Table 2. Measured Endpoints and Associated Criteria.
Parameter
Units
Expected Results
Survival: Daily assessment of survival
should be made to provide a basis for
expression and interpretation of
reproductive output.
Percentage
90% or greater survival in controls.
Page 13
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Parameter
Units
Expected Results
Fecundity: Egg production should be
determined daily, but only during the
late morning.
Fecundity should be
expressed on the
basis of number of
eggs laid by surviving
females per
reproductive (test) day
It is expected that one spawn typically
will be composed of 50 to 250 eggs. If
no eggs are present, the substrate
should be left in the tank; new
substrates should be added to replace
any that are removed.
Fertilization Success: If spawning
occurred that morning, embryos
typically will be undergoing late
cleavage, and determination of the
fertility rate is easily achieved.
Number
embryos/number of
eggs x 100%
Fertilized eggs will be apparent within
a few hours of fertilization. Infertile
eggs will be opaque or clear with a
white dot where the yolk has
precipitated. Control fertilization
should be >95%.
Appearance of Adults: The external
appearance of the adults should be
assessed as part of the daily
observations, and any unusual
changes should be noted. These
observations are especially important
for assessing endocrine active agents
that are androgenic.
Not Applicable
External features of particular
importance include body color (light or
dark), coloration patterns (presence of
vertical bands), body shape (head and
pectoral region), and specialized
secondary sex characteristics (size of
dorsal nape pad, number of nuptial
tubercles in males; ovipositor size in
females).
Body Weights
Nearest 0.01 grams
Normal/increased/decreased relative
weights to control animals.
Blood Samples: Collected from the
caudal artery/vein with a heparinized
microhematocrit capillary tubule and
analyzed for VTG and sex steroids
Depending upon the
size of the fathead
minnow (which usually
is sex-dependent),
blood volumes
generally range from
20 to 60 |jl_.
Plasma should be separated from the
blood sample via centrifugation
(approx. 3 minutes at 15,000 xg) and
stored with protease inhibitors at -75°C
to -85°C until analysis.
Vitellogenin (VTG) Concentration
ng/mL
The measurement of VTG in plasma
samples should be performed using
an enzyme-linked immunosorbent
assay (ELISA). For the ELISA,
polyclonal Fathead minnow (FHM)
(Pimephales promelas) VTG antibody
and purified VTG protein, also from
the FHM, should be utilized.
Sex Steroid Concentration
ng/mL
Plasma concentrations of sex steroids
are recommended and should be
measured via radioimmunoassay
(RIA), with priority given to 17(3-
estradiol for females and testosterone
in males, then 17p-estradiol in males
and testosterone in females. Any non-
detect samples should be recorded as
1/2 the limit of quantification (LOQ).
Secondary Sex Characteristics:
Nuptial tubercles should be counted,
mapped and scored.
Various
See Appendix B.
Page 14
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Parameter
Units
Expected Results
Gonad Size: After sampling the blood,
the fixed gonads should be removed
and weighed (to the nearest 0.1 mg) to
determine the GSI (GSM 00% x gonad
wt/body wt).
Not Applicable
Typical GSI values for reproductively
active fathead minnows range from 8
to 13% for females and from 1 to 2%
for males.
Gonad Morphology: Routine
histological procedures should be used
to assess the condition of testes and
ovaries from the fish. Gonads should
be placed in fixative (Davidson's
fixative). A toxicologic pathologist with
experience with small fish should
perform histology procedures and
should refer to Histopathology
Guidelines for the Fathead Minnow
(Pimephales promelasj 21-Day
Reproduction Assay (Appendix E)
Not Applicable
Refer to Histopathology Guidelines for
the Fathead Minnow (Pimephales
promelasj 21-Day Reproduction Assay
(Appendix E)
Not Applicable: No unit can be defined for this parameter.
(f) Statistical Analysis.
Statistical analyses of the data should follow guidelines described in OECD
(2006) (Ref. 17).
The statistical unit for analysis is the replicate. Whereas some measurements
are made on a per-tank basis {e.g., fecundity, normalized to number of female
reproductive days), others are made on a per-fish basis (e.g., tubercle measures)
and should be averaged per tank.
For all continuous quantitative endpoints (fecundity, VTG, GSI, FPI) consistent
with a monotonic dose-response, the Jonckheere-Terpstra test should be applied
in step-down manner to establish a significant treatment effect.
For continuous endpoints that are not consistent with a monotonic dose-
response, the data should be assessed for normality (preferably using the
Shapiro-Wilk or Anderson-Darling test) and variance homogeneity (preferably
using the Levene test). Both tests are performed on the residuals from an
ANOVA. Expert judgment can be used in lieu of these formal tests for normality
and variance homogeneity, though formal tests are preferred. Where non-
normality or variance heterogeneity is found, a normalizing, variance stabilizing
transformation should be sought. If the data (perhaps after a transformation) are
normally distributed with homogeneous variance, a significant treatment effect is
determined from Dunnett's test. If the data (perhaps after a transformation) are
normally distributed with heterogeneous variance, a treatment effect is
determined from the Tamhane-Dunnett or T3 test or from the Mann-Whitney-
Wilcoxon U test. Where no normalizing transformation can be found, a
significant treatment effect is determined from the Mann-Whitney-Wilcoxon U test
using a Bonferroni-Holm adjustment to the p-values. The Dunnett test is applied
Page 15
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independently of any ANOVA F-test and the Mann-Whitney test is applied
independently of any overall Kruskall-Wallis test.
Significant mortality is not expected but should be assessed from the step-down
Cochran-Armitage test where the data are consistent with dose-response
monotonicity, and otherwise from Fisher's Exact test with a Bonferroni-Holm
adjustment.
A treatment effect for tubercle score is determined from the step-down
application of the Jonckheere-Terpstra test applied to the replicate medians.
Alternatively, and preferably, the multi-quantal Jonckheere test should be used
for effect determination.
The appropriate unit of analysis is the replicate so the data consist of replicate
medians if the Jonckheere-Terpstra or Mann-Whitney U test is used, or the
replicate means if Dunnett's test is used. Dose-response monotonicity can be
assessed visually from the replicate and treatment means or medians or from
formal tests such as previously described in OECD (2006) (Ref. 17). With fewer
than five replicates per treatment or control, the exact permutation versions of the
Jonckheere-Terpstra and Mann-Whitney tests should be used if available. All
statistical tests should be judged at the 0.05 significance level.
Analysis may be conducted both with and without suspected outliers (Chapman
et al. 1996) (Ref. 3). Potential outliers may be identified by values that exceed
the median plus three times the interquartile range {i.e., the difference between
the 75th and 25th percentiles). If an explanation cannot be made for the
divergence of data, then both analyses should be presented, assuming that the
results differ. If there are no changes to the results, then the analysis including
the outliers should be presented. If differences occur, then the implications of
removing the outliers should be carefully documented. This is particularly true for
vitellogenin measurements, as responses will span several orders of magnitude,
and males producing vitellogenin may appear to be statistical outliers, whereas
there may be a biological response indicated. If an explanation can be made for
the existence of outliers, the analysis excluding outliers may be sufficient.
Please see Figure 1 for a flow chart for performing statistical tests on continuous
data.
Page 16
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Figure 1. Flowchart for statistical approaches for continuous response data.
Flow-Chart for Continuous Response
Are data consistent with monotonic dose-response?
Yes
No
Apply step-down Jonckheere-Terpstra test to
determine effects. With <5 reps per concentration]
use exact version of test if available.
Are data normally distributed
(possibly after transform)?
Yes
No
Are variances homogeneous
(possibly after transform)?
Yes
No
Use Mann-Whitney test with
Bonferroni-Holm adjustment to
determine effects. With <5 reps per
concentration, use exact version of/
test if available.
Use Dunnett test to
determine effects.
Use Tamhane-Dunnett
(T3) test if available.
Otherwise follow arrow/
Page 17
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Special Data Analysis Considerations.
(i) Use of Compromised Treatment Levels. Several factors are
considered when determining whether a replicate or entire
treatment demonstrates overt toxicity and should be removed from
analysis. Overt toxicity is defined as >2 mortalities in any replicate
that can only be explained by toxicity rather than technical error.
Other signs of overt toxicity include hemorrhage, abnormal
behaviors, abnormal swimming patterns, anorexia, and any other
clinical signs of disease. For sub-lethal signs of toxicity, qualitative
evaluations may be necessary, and should always be made in
reference to the clean water control group.
(ii) Solvent controls. The use of a solvent should only be considered
as a last resort, when all other chemical delivery options have been
considered. If a solvent is used, then a clean water control must be
run in concert. At the termination of the test, an evaluation of the
potential effects of the solvent must be performed. This is done
through a statistical comparison of the solvent control group and
the clean water control group. If statistically significant differences
are detected in these endpoints between the clean water control
and solvent control groups, then best professional judgment should
be used to determine if the validity of the test is compromised.
Interpretation of Results.
(i) Data Interpretation. The fish short-term reproduction assay as
presented is intended to serve in a screening capacity to provide an
indication of potential endocrine activity, not to confirm any specific
mechanism, mode of action, or adverse effect. Therefore, any
statistically significant effect in one or more of the core endpoints of
this assay (fecundity, secondary sex characteristics, vitellogenin,
GSI, and histopathology) may be indicative of a potential of the test
material to disturb the HPG axis of fishes. Because the statistical
analysis methods recommended tend to favor detection of
monotonic responses, it is important to consider any significant
finding an indication of a positive response. Also, the suite of
endpoints included is deemed necessary to provide a fully
comprehensive assessment of the disrupting potential to the HPG-
axis in a representative fish.
Responses may have many forms other than monotonic, so even if
a general test of significance had p>0.05 but was low and the
response showed a trend, it should be considered biologically
relevant. Conversely, a significant global test indicates the need for
further examination even if no multiple comparison tests are
significant, to evaluate whether between-treatment differences
Page 18
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suggest a meaningful response. These evaluation approaches are
particularly appropriate for reducing false negatives.
It is important to note however that if a given exposure level results
in substantial mortality or other overt signs of toxicity, responses in
other endpoints may be due to general toxicity, not necessarily
mediated primarily via interaction with the endocrine system. The
lower treatment level(s) should be examined for effects outside of
the range of general toxicity. If all test concentrations exhibit
mortality, then the assay would need to be repeated with lower
concentrations before inferences about possible endocrine activity
can be made.
It is recognized that some endpoints may be responsive to non-
endocrine stresses in addition to endocrine-mediated pathways,
particularly fecundity. Although reductions in fecundity indicate
adverse organismal and, potentially, population level effects (i.e.,
reproductive toxicity), these cannot be definitively distinguished
from direct endocrine-mediated effects by this assay when changes
in other core endpoints are not present. Nevertheless, reductions
in fecundity are considered a positive effect in this assay because
they may be endocrine-mediated and should be considered in
concert with results of other relevant endocrine disruptor screening
assays. Similarly, responses in secondary measurements (e.g.,
length, weight) also should be considered in light of other results.
Results that would be considered equivocal for this single assay
should be considered indications of potential endocrine activity and
evaluated in light of the weight of the evidence from other assays.
Performance Criteria.
~ Water quality characteristics should remain within the limits of tolerance
described in Table 1 (water temperature did not differ by more than 1°C
between test tanks at any one time during the exposure period and was
maintained within + 1 °C of the 25°C temperature specified for the fathead
minnow).
~ There should be more than 90% survival of control animals over the
duration of the chemical exposure.
~ Evidence that fish are actively spawning in all replicates prior to initiating
chemical exposure and in control replicates during the test (e.g., spawning
occurs every 4 days or an average of at least 15
eggs/female/day/replicate).
~ There should be greater than 95% fertility of eggs from the control animals
during the exposure.
Page 19
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Contingencies. The three problems most likely to be encountered are related to
insufficient numbers of tanks with correct sex ratios and successfully
demonstrated spawning, unplanned mortality in control or exposure tanks, and
low or high measured concentrations relative to the nominal level of the test
chemicals. These problems should be dealt with in the following manner:
~ For each chemical, extra tanks (up to 8 additional tanks) should be
established to ensure an adequate supply of spawning fathead minnows
in groups with the correct sex ratio.
~ If there is excessive mortality in any tank during pre-exposure or in control
tanks during exposure, the cause should be investigated and the
experiment reinitiated.
~ Prior to initiation of the chemical exposure, each diluter should be tested
for at least 1 week for its ability to maintain the desired concentration. If,
during the exposures the measured concentration becomes unacceptably
low or high, adjustments should be made to the diluter to correct the
problem.
Data Reporting.
The test report should include the following information:
~ Test substance.
• Physical nature and relevant physical-chemical properties.
• Chemical identification data, including purity and analytical method(s)
for quantification of the test substance where appropriate.
• Source.
• CAS number.
• Lot number.
~ Test species. At a minimum, the scientific name, supplier, and any
pretreatment.
~ Test conditions.
• Test procedure used (test type, loading rate, stocking density, etc.);
• Method of preparation of stock solutions and flow-rate;
• Nominal test concentrations, means of the measured values and
standard deviations in test tanks and method by which these were
attained and evidence that the measurements refer to the
concentrations of test substance in true solution;
• Dilution water characteristics (including pH, hardness, alkalinity,
temperature, dissolved oxygen concentration, residual chlorine levels,
total organic carbon, suspended solids, and any other measurements
made);
• Water quality within test tanks: pH, hardness, temperature, and
dissolved oxygen concentration;
Page 20
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• Detailed information on feeding (e.g., type of food(s), source, amount
given and frequency, and analyses for relevant contaminants if
necessary, e.g., PCBs, PAHs and organochlorine pesticides);
• Source and treatment of dilution water, average and ranges of water
chemistry parameters, photo period, light intensity, tank size, numbers
of male and female fish per replicate, number and composition of
spawning substrate, lot number of feed, number of daily water volume
exchanges.
~ Results.
• Evidence that controls met the validity criterion for survival, and data
on mortalities occurring in any of the test concentrations;
• Statistical analytical techniques used, statistics based on fish,
treatment of data and justification of techniques used;
• Tabulated data (using suggested data template for fathead minnow
studies (Ref. 18)) on biological observations of gross morphology
(including secondary sex characteristics), GSI, vitellogenin and sex
steroids (optional);
• Detailed report on gonadal histology (using suggested data template
(Ref. 18));
• Results of the statistical analysis preferably in tabular and graphical
form;
• Incidence of any unusual reactions by the fish and any visible effects
produced by the test substance; and
• Average, standard deviation, and range for each test endpoint.
(i) References.
1. Ankley, G.T., K.M. Jensen, E.A. Makynen, M.D. Kahl, J.J. Korte, M.W. Hornung,
T.R. Henry, J.S. Denny, R.L. Leino, VS. Wilson, M.C. Cardon, P.C. Hartig and
L.E. Gray (2003). Effects of the androgenic growth promoter 17-beta-trenbolone
on fecundity and reproductive endocrinology of the fathead minnow (Pimephales
promelas). Environ. Toxicol. Chem. 22, 1350-1360.
2. Ankley GT, Jensen KJ, Kahl MD, Korte J J, and Makynen EA (2001). Description
and evaluation of a short-term reproduction test with the fathead minnow
(Pimephales promelas)." Environ Toxicol Chem 20: 1276-1290.
3. Chapman, P.F., M. Crane, J. Wiles, F. Noppert, and E. Mclndoe (1996). Improving
the quality of statistics in regulatory ecotoxicity tests. Ecotoxicology 5:169-186.
4. Harries JE, Runnalls T, Hill E, Harris C, Maddix S, Sumpter JP, Tyler CR (2000).
Development and validation of a reproductive performance test for endocrine
disrupting chemicals using pair-breeding fathead minnows (Pimephales promelas).
Environ Sci Technol 34:3003-3011.
Page 21
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5. Holbech H, Andersen L, Petersen Gl, Korsgaard B, Pedersen KL, Bjerregaard P
(2001). Development of an ELISA for vitellogenin in whole body homogenate of
zebrafish (Danio rerio). Comp. Biochem. Physiol. C. 130: 119-131. 15.
6. Jensen KM, Korte J J, Kahl MD, Pasha MS, Ankley GT (2001). Aspects of basic
reproductive biology and endocrinology in the fathead minnow (Pimephales
promelas). Comp Biochem Physiol 28C(1): 127-141.
7. Miles-Richardson SR, Kramer VJ, Fitzgerald SD, Render JA, Yamini B, Barbee SJ,
Giesy JP (1999). Effects of waterborne exposure of 17beta-estradiol on secondary
sex characteristics and gonads of fathead minnows (Pimephales promelas). Aquat
Toxicol 47:129-145.
8. OECD (2000). Guidance Document on Aquatic Toxicity Testing of Difficult
Substances and Mixtures. OECD Environmental Health and Safety Publications,
Series on Testing and Assessment No.23. Organisation for Economic Co-
operation and Development. Document# ENV/JM/MONO(2000)6. Paris, France.
53 pp.
9. OECD (2009). Fish Short Term Reproduction Assay. OECD Guideline for the
Testing of Chemicals: Test No. 229. Paris, France. 40 pp.
10. Panter GH, Hutchinson TH, Hurd KS, Sherren A, Stanley RD, Tyler CR (2004).
Successful detection of (anti-) androgenic and aromatase inhibitors in pre-
spawning adult fathead minnows (Pimephales promelas) using easily measured
endpoints of sexual development. Aquat Toxicol 70:11-21.
11. Panter GH, Thompson RS, Sumpter JP (1998). Adverse reproductive effects in
male fathead minnows (Pimephales promelas) exposed to environmentally
relevant concentrations of the natural oestrogens, oestradiol and oestrone. Aquat
Toxicol 42:243-253.
12. Smith, RJF (1974). Effects of 17a-methyltestosterone on the dorsal pad and
tubercles of fathead minnows (Pimephales promelas). Can J Zool 52:1031 -1038.
13. Snedecor, G.W., and W.G. Cochran (1980). Statistical Methods. The Iowa State
University Press, Ames, Iowa.
14. US-EPA (1987). Guidelines for the culture of fathead minnows Pimephales
promelas for use in toxicity tests. EPA/600/3-87/001.
15. US-EPA (2002). A Short-term Method for Assessing the Reproductive Toxicity of
Endocrine Disrupting Chemicals Using the Fathead Minnow (Pimephales
promelas). EPA/600/R-01/067.
16. US EPA (2006). Histopathology Guidelines for the Fathead Minnow (Pimephales
promelas) 21-day Reproduction Assay.
Page 22
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17.
OECD (2006). Current Approaches in the Statistical Analysis of Ecotoxicity Data:
A Guidance to Application. Environmental Health and Safety Publications. Series
on Testing and Assessment, No. 54. Paris, France.
18. EPA. OPPTS Harmonized Test Guidelines. Available on-line at:
http://www.epa.gov/oppts (select "Test Methods & Guidelines" on the left side
navigation menu). You may also access the guidelines in
httpV/www.regulations.gov grouped by Series under Docket ID #s: EPA-HQ-
OPPT-2009-0150 through EPA-HQ-OPPT-2009-0159, and EPA-HQ-OPPT-2009-
0576.
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APPENDIX A
SAMPLE COLLECTION PROCEDURES FOR VITELLOGENIN AND SEX STEROID
ANALYSES
Procedure 1A: Fathead Minnow, Blood Collection from the Caudal Vein/Artery
After anaesthetization and measurement of body weights, the caudal peduncle should
be partially severed with a scalpel blade and blood should be collected from the caudal
vein/artery with a heparinized microhematocrit capillary tube. After the blood has been
collected, the plasma should be quickly isolated by centrifugation for 3 min at 15,000 g.
If desired, percent hematocrit can be determined following centrifugation. The plasma
portion should then be removed from the microhematocrit tube and stored in a
centrifuge tube with 0.13 units of aprotinin (a protease inhibitor) at -75°C to -85°C until
determination of vitellogenin and sex steroid concentrations can be made. Depending
on the size of the fathead minnow (which is sex-dependent), collectable plasma
volumes generally range from 20 to 60 microliters per fish (Jensen et al. 2001).
Procedure 1B: Fathead Minnow, Blood Collection from Heart
Alternatively, blood may also be collected by cardiac puncture using a heparinized
syringe (1000 units of heparin per ml). The blood should be transferred into Eppendorf
tubes (held on ice) and then centrifuged (5 min, 7,000 g, room temperature). The
plasma should be transferred into clean Eppendorf tubes (in aliquots if the volume of
plasma makes this feasible) and promptly frozen at -75°C to -85°C until analyzed
(Panter et al., 1998).
A-1
-------
APPENDIX B
Assessment of Secondary Sex Characteristics (Nuptial Tubercle) in EDC Tests
with Fathead Minnows
Michael Kahl and Gerald Ankley
Nuptial Tubercle Scoring
Overview
Potentially important characteristics of physical appearance in adult fathead minnows in
endocrine disrupter testing include body color (i.e., light/dark), coloration patterns (i.e.,
presence or absence of vertical bands), body shape (i.e., shape of head and pectoral
region, distension of abdomen), and specialized secondary sex characteristics (i.e.,
number and size of nuptial tubercles).
Nuptial tubercles are located on the head (dorsal pad) of reproductively active male
fathead minnows, and are usually arranged in a bilaterally symmetric pattern (Jensen et
al. 2001). Control females and juvenile males and females exhibit no tubercle
development (Jensen et al. 2001). There can be up to eight individual tubercles around
the eyes and between the nares of the males. The greatest numbers and largest
tubercles are located in two parallel lines immediately below the nares and above the
mouth. In many fish there are groups of tubercles below the lower jaw; those closest to
the mouth generally occur as a single pair, while the more ventral set can be comprised
of up to four tubercles. The actual number of tubercles is seldom more than 30 (range,
18-28; Jensen et al. 2001). The predominant tubercles (in terms of numbers) are
present as a single, relatively round structure, with the height approximately equivalent
to the radius. Most reproductively-active males also have at least some tubercles which
are enlarged and pronounced such that they are indistinguishable as individual
structures.
Some types of endocrine-disrupting chemicals (EDCs) can cause the abnormal
occurrence of certain secondary sex characteristics in the opposite sex; for example,
androgen receptor agonists, such as 17a-methyltestosterone or 17(3-trenbolone, can
cause female fathead minnows to develop nuptial tubercles (Smith 1974; Ankley et al.
2001; 2003), while estrogen receptor agonists may decrease the number or size of
nuptial tubercles in males (Miles-Richardson et al. 1999; Harries et al. 2000).
This protocol describes characterization of nuptial tubercles in fathead minnows based
on procedures used at the U.S. Environmental Protection Agency lab in Duluth, MN.
Specific products and/or equipment listed can be substituted with comparable materials.
A - 2
-------
Procedures
1. Evaluation of secondary sex characteristics should be performed after blood sample
collection and body weight measurement of anesthetized fish.
2. Viewing is best accomplished using an illuminated magnifying glass or 3X
illuminated dissection scope. View fish dorsally and rostrum forward (head toward
viewer).
a. Place fish in small petri dish, rostrum forward, ventral side down. Focus
viewfinder to allow identification of tubercles. Gently and slowly roll fish from
side to side to identify tubercle areas. Count and score tubercles as
described below.
b. Repeat the observation on the ventral head surface by placing the fish
dorsally with the rostrum forward in the petri dish.
Tubercle Counting and Rating
Six specific areas have been identified for assessment of tubercle presence and
development in adult fathead minnows. A template was developed to map the location
and quantity of tubercles present (see below). The number of tubercles is recorded and
their size can be quantitatively ranked as: 1-present, 2-enlarged and 3-pronounced for
each organism (Figure 1C).
Figure 1C, The actual number of tubercles in some fish may be greater than the
template boxes (see template below) for a particular rating area. If this happens,
additional rating numbers may be marked within, to the right or to the left of the box.
The template therefore does not have to display symmetry. An additional technique for
mapping tubercles which are paired or joined vertically along the horizontal plane of the
mouth could be done by double-marking two tubercle rating points in a single box.
Rating f-present; identified as any tubercle having a single point whose height is nearly
equivalent to its radius.
A- 3
-------
Rating 2- enlarged; identified by tissue resembling an asterisk in appearance; usually
has a large radial base with grooves or furrows emerging from the center. Tubercle
height is often more jagged but can be somewhat rounded at times.
Rating 3- pronounced; usually quite large and rounded with less definition in structure.
At times these tubercles will run together forming a single mass along an individual or
combination of areas (B, C and D, described below). Coloration and design are similar
to rating 2 but at times are fairly indiscriminate. Using this rating system generally will
result in overall tubercle scores of <50 in a normal control male possessing a tubercle
count of 18 to 20 (Jensen et al. 2001).
Mapping Regions:
A - Tubercles located around eye. Mapped dorsal to ventral around anterior rim of eye.
Commonly multiple in mature control males, not present in control females, generally
paired (one near each eye) or single in females exposed to androgens.
B - Tubercles located between nares, (sensory canal pores). Normally in pairs for
control males at more elevated levels (2- enlarged or 3- pronounced) of development.
Not present in control females with some occurrence and development in females
exposed to androgens.
C - Tubercles located immediately anterior to nares, parallel to mouth. Generally
enlarged or pronounced in mature control males. Present or enlarged in less developed
males or androgen-treated females.
D - Tubercles located parallel along mouth line. Generally rated developed in control
males. Absent in control females but present in androgen-exposed females.
E - Tubercles located on lower jaw, close to mouth, usually small and commonly in
pairs. Varying in control or treated males, and treated females.
F - Tubercles located ventral to E. Commonly small and paired. Present in control
males and androgen-exposed females.
A - 4
-------
ID Tubercle Template Numerical Rating
Date 1 -present
Total Score 2-enlarged
3-pronounced
A
X1
X1
X1
X1
B
X1
X1
X1
X1
C
X1
X1
X1
X1
X1
X1
X1
X1
X1
X1
D
X1
X1
X1
X1
X1
X1
X1
X1
X1
X1
E
X1
X1
X1
X1
X1
X1
A- 5
-------
APPENDIX C
Removal of Gonads from Fathead Minnows
After other evaluations are made at test termination (body length and weight
measurements, blood sampling, and evaluation of secondary sex characteristics),
gonads should be perfused in situ with Davidson's fixative, then removed for weighing
and histological evaluation. Gonad weights should be recorded to the nearest tenth of a
milligram. Routine histological procedures should be used to assess the condition of
testes and ovaries from the fish. A toxicologic pathologist with experience with small
fish should perform histology procedures and should refer to Histopathology Guidelines
for the Fathead Minnow (Pimephales promelasj 21-Day Reproduction /Assay. The
Histopathology Guidelines detail the post mortem and histotechnical procedures that
should be used.
viscer;
Fathead Minnow, Male: Excision of the testes during necropsy.
A. The abdominal wall is pinned laterally. The cavity is flooded with fixative to perfuse
gonads. B. The terminal intestine is severed and retracted prior to removal. C. The
testes are grasped near the spermatic ducts. D. Removal of the testes is complete.
A - 6
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ovarie
ovari
Fathead Minnow, Female: Excision of the ovaries during necropsy. A. The abdominal
wall is pinned laterally. The cavity is flooded with fixative to perfuse gonads. B. The
terminal intestine is severed and retracted prior to removal. C. The ovaries are grasped
near the oviducts. D. Removal of the ovaries is complete.
A- 7
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APPENDIX D
Glossary
Acute toxicity: Effects observed in tests of <96 h in duration
ANOVA: analysis of variance
Chronic toxicity: Effects observed in tests > 28 d in duration
EDC: endocrine disrupting chemical
EDSP: EPA's Endocrine Disruptor Screening Program
EIA: enzyme immunoassay
ELISA: enzyme-linked immunosorbent assay; analytical method used for determining
plasma vitellogenin concentration
EPA: United States Environmental Protection Agency
Fat pad: soft enlargement of flesh on top of the head of sexually-mature male fathead
minnows that extends onto the back of the fish to, or near, the anterior margin of
the dorsal fin
Fecundity: measure of total egg production
Fertility: measure(s) of fertilization success as indicated for example by actively-
dividing embryonic cells or occurrence of eyed embryos
GC: gas chromatography
GSI: gonadosomatic index; gonad weight relative to total body weight ((gonad wt(mg)/
body wt(mg)) x 100)
HPG: Hypothalamus-pituitary-gonadal axis
HPLC: high performance liquid chromatography
LC: liquid chromatography
LOD: the concentration of the lowest analytical standard
LOQ: the concentration of the lowest analytical standard multiplied by the lowest
dilution factor
MS: mass spectrometry/ spectrometer
Nuptial tubercles: visible external horny outgrowths on the surface of the head of the
sexually-mature male fathead minnow in breeding condition
OECD: Organisation for Economic Co-operation and Development
Ovipositor: Urogenital structure present in sexually-mature females for egg deposition.
RIA: radioimmunoassay; analytical method used for determining plasma steroid
concentrations
Saturator: An apparatus capable of generating a saturated stock solution of a chemical
that is relatively insoluble in water
A- 8
-------
T: testosterone; androgenic sex steroid normally present in both sexes and necessary
for development and maintenance of reproductive function.
Tank: Chamber or vessel holding replicate group of fish (initiated as 4 females & 2
males)
TSH: thyroid stimulating hormone
Viability: Measure(s) of embryonic development subsequent to fertilization, including
hatching success and normal larval maturation
VTG: vitellogenin; precursor to egg yolk protein that occurs normally in the blood of
sexually-mature female fish; it can be induced by estrogen receptor agonists in
male fathead minnows
A- 9
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APPENDIX E
Histopathology guidelines for the Fathead Minnow
(Pimephales promelas) 21-day reproduction assay
6 July 2006
A-10
-------
ACKNOWLEDGMENTS
The following persons contributed time and effort toward the creation of this document;
Gerald Ankle}. l.'SKPA. USA
Christiana Grim. I "SI-PA. USA
Stephen Duficll. Svngenia. UK
John Fournie. USHPA. USA
Anne Gourmelon. OLCD
Rodne\ Johnson. I 'SLPA. USA
Christine Riihl-Fehlert. Bayer AG, German}
Christoph Schiifers. I'raunhol'er IMH. German)
Masanori Seki. CLR1 hnvironment. Inc.. Japan
Leo van der Yen. RIVM. I lie Netherlands
Pieter Wester. RIVM. The Netherlands
Jell re}' Wolt. Lxperiniental Pal ho logy 1 -aboratories, Inc., USA
Marilyn Wolfe, Experimental Pathology Laboratories, Inc., USA
INTRODUCTION
The purpose ot this document is to provide guidelines for the preparation and
histopathological evaluation of gonads Irom fathead minnow (Pwiepholes promelas).
The goals of these guidelines are to provide an updated source of direction for the
participating laboratories, to supply template text for laboratory protocols, arid most
important'}. to facilitate non-biased comparisons of inter-Iaborator\ results.
1 hroughout this document, the proposed procedures were derived from the consensus
opinions of various fish pathologists, recommendations from the Biltho\en 2002 and
Paris 2003 workshops, information distilled from previous guidelines, and the scientifie
literature.
This guidance document is divided into three sections: I) Post-mortem and Histotechnical
Procedures; II) Gonadal I listopathoiogy Glossary and Diagnostic Criteria; and III)
Gonadal Staging Criteria.
1
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I. POST-MORTEM AND HISTOTECHMCAL PROCEDURES
The purpose oi this section is to outline all of the post-mortem steps and procedures that
occur prior to the evaluation of histologic sections on glass slides, to include euthanasia,
necropsx, tissue fixation, decalcification, tissue trimming, processing, embedding,
microtomy, staining, eoverslipping, and slide labeling.
1. Substrate obtained for vitellogenin analysis.
Fathead Minnow {FHV1): blood sample from the caudal vein-artery or heart
2, Tissue specimen for .gonad histopathoIog\. Techniques were selected that
would most optimally: I) preserve the cellular structure of the gonads: 2)
maxiini/e the amount of gonad tissue available tor analysis: 3) sample the
gonads in a representative and consistent fashion: and 4) allow the pathologist
to examine at least three step sections ot both gonads on a single slass slide.
In FHV1. the gonads are excised from the fish.
Davidson's fixative is the recommended fixative. Compared to other common fixatives,
such as 10% neutral buffered formalin or Bouin's fixative, the advantages of Davidson's
fixative are: 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: 3) there is anecdotal information which suggests that Bouin's fixative may
be difficult to obtain in the near future: 4) specimens fixed in Bouin's fixative require
multiple rinses prior to transfer to alcohol or formalin. Please sec photographic
comparison, of specimens fixed in Davidson's versus Bouin's fixatives (Appendix A. '
). Please be aware that different recipes and products that are designated as "Davidson's
fixative" max actual 1\ be modifications of the original formula ( ): if a
modified I)a\ idson's fixative is used, this should be noted by the laboratory. If
necessary, a recommended decalcification solution is listed ( ). Factors that
may affect the need for decalcification include the si/e of the individual fish, Lhe length
of time that the carcass was immersed in fixative, and the extent to which the abdominal
cavity came into contact with the fixative.
2
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I. Fathead Minnow
1. Euthanasia, Necropsy, and Tissue Fixation
Objectives:
1. Provide for the humane sacrifice of llsh.
2. Obtain neccssar\ body weights and measurements.
3. Obtain specimens for vitellogenin analysis.
4. I-'xeise gonad specimens.
5. l.valuate secondary sex characteristics.
6. Prov ide for adequate fixation of the gonads and the carcass.
Materials:
1. Fish transport container (-500 ml, contains water from the experimental
tank or s\ stem reservoir).
2. Small dip net.
3. Futhanasia chamber (--500 ml vessel).
4. Euthanasia solution {j.>id:> >. or FA-100 [Japan]).
5. klecironic slide caliper (minimum displav; < O.lmml
6. IJeetronic analytical balance {minimum display: < 0.1 mj>) and tared
vessels.
7. Stereoscopic microscope.
8. Pins and cork board.
9. Small scissors (e.g.. iris scissors).
10. Small forceps.
1 I. Microdissection forceps.
12. Microdissection scissors.
1 3. (iau/.e sponges.
14. Davidson's fixative ( & S.
15.. Plastic syringe (3ml).
16. Standard plastic tissue cassettes (one per fish).
17. Fixation containers (100 ml, one per fish).
Procedures;
1. Fish should be sacrificed within one to two minutes prior to necropsy.
Therefore, unless multiple prosectors are available, multiple llsh should
not be sacrificed simultaneously.
2. I.sing the small dip net. a llsh is removed from the experimental chamber
and transported to the necropsy area in the transport container. For each
test chamber, all male fish are sacrificed prior to the sacrifice of female
fish; the sex of each fish is determined b_\ external bod\ characteristics
(e.g.. presence or absence of nuptial tubercles, dorsal pad. etc.).
3. The llsh is placed into buffered MS-222 solution. The fish is removed
Irom the solution when there is cessation of respiration and the fish is
unresponsive to external stimuli.
3
-------
4. 1 he fish is wet weighed, measured according to protocol, and a blood
sample is obtained from the caudal arterv'vein or heart.
5. The fish is placed on a corkboard on the stage of a dissecting microscope.
Using iris scissors and small forceps, the abdomen is opened via a
carefully made incision that extends along the \emral midline from the
pectoral girdle to a point just cranial to the anus.
6. i he l ish is placed in dorsal recumbencv and the opposing flaps of bodv
wall arc pinned laterally to expose the abdominal viscera (Appendix A,
& ).
7. I sing the small forceps and small scissors, the abdominal viscera {liver,
gastrointestinal tract, spleen, pancreatic tissue, and abdominal mesentery)
are carefully removed en masse in the following manner:
a. I'he intestine is severed proximal to the anus.
b. A forceps is applied to the terminal portion of the intestine. Using
gentle traction and taking care not to disturb the gonads, the viscera
are dissected out ol the abdominal cav it_\ in a caudal to cranial
direction.
c. The distal esophagus is severed just proximal to the liver.
8. I. sing a syringe, approximately 0.5 mi of Davidson's fixative is thai
gentlv applied to the uonads in sii_u. Approximately 90 seconds following
the application of fixative, the liquid ilxative within the abdomen is
removed with a gau/e sponge, and the gonads are excised in a manner
similar to the abdominal viscera:
a. I sing the microdissection scissors, the spermatic ducts or oviducts are
severed proximal to the genital pore.
b. Microdissection forceps are then applied to the spermatic
ducts/oviducts. Using gentle traction, the gonads are dissected out of
the abdominal cavity in a caudal to cranial direction, severing the
mesorchial'mesovarial attachments as needed using the
microdissection scissors. The left and right gonads mav he excised
individual!) or they may be excised simultaneous!}, and subsequently
divided at their caudal attachment.
9. The gonads (right and left) are placed into a pre-labcled plastic tissue
cassette which is then placed into an individual container of Davidson's
ilxative accompanied by the abdominal viscera. The volume of fixative in
the container should be at least 10 times the approximated volume of the
tissues. The Ilxative container is gentlv agitated for five seconds to
dislodge air bubbles from the cassette.
10. Lsing the carcass, the secondary sex characteristics are assessed (e.g..
dorsal nape pad. nuptial tubercles). The carcass is then added to the
fixative container.
11. All tissues remain in Davidson's fixative overnight, followed bv transfer
to indiVidual containers of 10% neutral buffered formalin the next dav.
Containers with cassettes are gently agitated for 5 seconds to ensure
4
-------
adequate penetration of formalin into cassettes (it is not necessary to rinse
with water or perform multiple changes in formalin).
2. Tissue Trimming
Tissue trimming is not required for FHVf.
3. Tissue Processing
Objectives:
1. Dehydrate tissue to provide for adequate penetration of paraffin.
2, Impregnate the tissue with paraffin to maintain tissue integrity and create a
firm surface for microtomy.
Materials:
1. Tissue processor.
2. Paraffin heating pots.
3. Processing unit oven.
4. Activates charcoal.
5. Paraffin (Paraplast". or equi\ulent, 1.
6. 10°o neutral buffered formalin.
7. I'thyl alcohol (absolute and dilutions as required).
8. Proprietary clearing agent (Clear Rjte-3'M or equhalenL ).
9. X\ iene.
Procedures;
1. Labeled tissue cassettes are removed from formalin storage and are
washed in tap water.
2. The cassettes are placed in the processing baskets> in a single layer. The
processing basket is loaded into the tissue processor.
3. '! he processing schedule is seleeted (see Appendix B. ). The
'"Gonad Program" or equivalent is selected for FI1M.
4. Alter the tissue processor has completed the processing e\cle. the
basket(s) may be transferred to the embedding station.
4. Embedding
Objective:
1. Properly orient the tissue in solidified paraffin for microtomy.
Materials:
1. Embedding station (thermal, dispensing and cryo consoles).
2. Paraffin heating pots.
3. Paraffin transfer pots.
4. Laboratory oven.
5
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5. 1 hermometer.
6. Embedding molds.
7. Block drawers.
8. Forceps,
9. Scraper.
10. Standard paraffin.
Procedures:
1. The cryo console of the embedding station is turned on. (Power to the
dispensing console and thermal console should remain on at all times.)
2. The basket(s) of cassettes is/are removed from the processor and
immersed in the paraffin-lllled front chamber of the embedding station
thermal console.
3. The first cassette to be embedded is removed from ihe front chamber of
the thermal console. The cassette lid is removed and discarded, and the
cassette label is checked against the animal records to resolve potential
discrepancies prior to embedding.
4. An appropriately sized embedding mold is .selected.
5. The mold is held under the spout of the dispensing console and filled with
molten paraffin.
6. The gonads are removed from the base of the cassette and are placed in the
molten paraffin in the mold. 1 he two gonads (left and right) are oriented
horizontal to their long axis in the mold to allow for longitudinal
sectioning.
7. The base of the cassette is placed on top of the mold. Additional paraffin
is added to cover the hotiom of the base.
8. The mold with the cassette base is placed on the cooling plate of the cryo
console.
9. After ihe paraffin has solidified, the block (i.e.. the hardened paraffin
containing the tissues and the cassette base) is removed from the mold.
10. Steps 3 through 10 are repeated for each cassette to be embedded.
5, Microtomy
Objective:
1. Create and mount histologic sections for staining.
Materials:
1. Microtome.
2. Disposable microtome knives.
3. Lipshavv Pike"' oil (or equivalent lightweight, machine oil).
4. Temperature-controlled water bath.
5. Ice.
6
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6. Microscope slides.
7. Staining racks.
8. Permanent slide marking pen.
9. forceps.
10. Fine-lipped paint brash.
1 !, Temporary labels.
12. Slide warmer/oven.
Procedures:
1, The temperature in the water bath is allow ed to stabilize so that ribbons
cut from the tissue blocks will spread out uniformly on (he surface without
melting. This temperature assessment is a qualitative judgment made by
the microtoniist before and during microtomy.
2. If necessary, a new blade is mounted onto the microtome and the
microtome is lubricated with oil.
3. fhe initial phase of microtomy is termed "facing" the block and is
conducted as follows:
a. The block is placed in the chuck of the microtome.
b. i he chuck is advanced by rotating the microtome wheel and thick
sections are cut from the paraffin surface of the block until the knife
reaches the embedded tissues, fhis process is referred to as "rough
trimming" of the block,
c. The section thickness on the microtome is set between 4-10 microns.
The chuck is advanced and multiple sections are cut from the block to
remove any artifacts created on the cut surface of the tissue during
rough trimming. This process is termed "fast trimming" of the block.
d. The block is removed from the chuck and placed facedown on ice to
soak the tissue.
e. Steps a. through d. are repeated until all blocks to be microtomed have
been faced.
f. If it is determined during facing that any block is not of acceptable
quality for microtomy, it is returned for re-embedding before
proceeding with microtomy.
g. Any extraneous pieces of paraffin are removed from the microtome
and workstation periodically during facing and before proceeding with
the next phase of microtomy.
4, The next phase of microtomy is final sectioning and mounting of tissue
sections on slides. These procedures arc conducted as follows:
a. Macroscopic lesions (if any) that are reported in the records are noted.
Care is taken to include any macroscopic lesion?, in the sections
collected during final sectioning.
b. The block is removed from the ice and placed in the chuck of the
microtome.
7
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c. With the section thickness on the microtome set to 4-5 microns, the
chuck is advanced by rotating the microtome wheel. Sections are cut
from the block until a "ribbon" containing at least one acceptable
section has been produced. As necessary during sectioning, the block
may be removed from the chuck, placed on ice to soak the tissue, and
replaced in the chuck.
d. irach ribbon is floated Hat on the surface of the water in the water bath.
An attempt is made to obtain at least one section in the ribbon that
contains no wrinkles and has no air bubbles trapped beneath it.
e. A microscope slide is immersed beneath the best section in the floating
ribbon. The section is lifted out of the water using the slide. This
process is referred to as ""mounting" the section on the slide.
f. A sinule slide is prepared for each fish. A total of three step sect jojis
teach section consisting of both the right and left szonad) are mounted
on each slide. The first section is obtained at the point where
approximately half of the gonad has been remp\ cd and the size of the
section is maximized. For both the testis and the ovary. the second
and third sections arc taken at 50 micron internals following the llrst
section.
g. With a slide-marking pen. the block number from which the slide was
produced is recorded on the slide.
h. The slide is placed in a staining rack.
i. The block is removed from the chuck and placed facedown for storage,
j. Steps a. through h, are repeated for all blocks to be microtomed.
6- Staining, Cover-slipping, and Slide Labeling
Objectives:
1. Differential staining of intra- and inter-cellular components of the gonads
to facilitate diagnostic examination by brightfickl microscopy.
2. Permanently seal mounted and stained tissues.
3. Permanently identify stained sections in a manner that allows complete
traceability.
Materials:
1. Automated slide stainer (optional).
2. Robot covers lipping machine (optional).
3. Claritler solution (Richard Allen or equivalent).
4. Bluing reagent (Richard Allen or equivalent).
5. f 'osin-Y (Richard Allen or equivalent. & ).
6. Hemalox\tin-2 (Richard Allen or equivalent. & )
7. Xylene.
8. Absolute elhy I alcohol (100% ETOH).
9. lb%t:TOH.
8
-------
10. 80% ETOH.
1 1. Co\erslipping mountain (Permount or equhalent. ).
12. Glass coverslips. No. i, 24 x 50 (or 60} mm ( ).
13. Slide Hats.
Procedures:
1. Staining
a. Slides are routinely air-dried cnernight before staining.
b. An example ! 1&I\ staining schedule for automated stainers is in
Appendix B. . A similar schedule can be adapted for
manual staining.
2. Coverslipping
a. Coverslips can be applied manually or automatically.
b. A slide is dipped in xylene, and the excess xylene is gently knocked
off the slide.
c. Approximately 0.1 ml of mounting medium is applied near the end of
the slide opposite to the frosted end.
d. A coversfip is tilted at a shallow angle as it is applied to the slide.
3. Labeling
a. Each slide label should contain the following information:
i. Laboratory name
ii. Species
iii. Specimen No. / Slide No.
iv. Chemical / Treatment group
v. Date (optional)
9
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II. GONADAL HISTOPATHOLOGY GLOSSARY AND DIAGNOSTIC CRITERIA
The purposes of this section are:
!) 'I o prcn idc general guidance tor the light microscopic evaluation of tissue
sections:
2 ) Tu promote a common awareness of various pathological findings that ma\ be
ohsersed; and
3) 'fo foster consistency in the use of diagnostic terminology.
General approach to reading studies
Studies are to be read In 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 1 oncologic Pathology, or other certify ing 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 . 1 echnicians 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 21-day
reproduction assay. If an individual has toxicological pathology experience and is
familiar w ith 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.
Pathologists are to read the studies non-blinded (i.e. with knowledge of the treatment
group status of individual fish). However, it is expected that an\ 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 he read in a blinded manner due to the
diagnostic dependence on control gonads. As a rule, treatment groups should be
evaluated in the following order: Control. Sligh-dose. Intermediate-dose, and Low-dose.
It is suggested that the pathologists be provided with all available information related to
the stud\ prior to conducting their readings. Information regarding gross morphologic
abnormalities, mortality rates, and general lest population performance and health are
useful for pathologists to provide comprehensive reports and to aid in the interpretation
of findings. Tor a more comprehensive discussion of standard reading approaches for
toxicologic pathology' studies, please refer to the Society of i oxicoiogic Pathology Best
Practices for reading toxicologic histopathology studies (Crissman J\V et al. 126-3 1).
10
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Diagnostic Criteria
Histopathology is a descriptive and interpretse science, and therefore somewhat
subjective. However, histopathologic evaluations of the same study b\ any qualified
pathologist should identify the same treatment-related findings (Crissman AY et al. 126-
3!}. Therefore, we aim to define the diagnostic criteria that w ill likely be encountered
during the histopathologic analysis of the 21-day reproduction assa> in fathead minnow .
A consolidated set of diagnostic criteria follow, fhese criteria are based on pathologists'
experience with certain consistent histopathologic changes that occur in fathead minnow
gonads in response to chemical exposure, however novel findings that are exposure-
related shall also be reported.
The criteria below have been divided into two sections: I. Primarv criteria, and 2.
Additional criteria. The criteria are graded for severity on a numerical scale. Any novel
findings arc either graded on a numerical scale, or are quaiitati\eh described.
Primary Criteria - Males:
1. Increased proportion of spermatogonia: Increases in the proportion of
spermatogonia are consequent of changes of the relative ratios of spermatogenic
cells. This could be due to an increase in the number of spermatogonia, or a
decrease in the number of other cell types, such as spermatocytes, spermatids, and
spermatozoa. Because the diagnosis of increased proportion of spermatogonia is
dependant on a comparison to controls, it is necessan. to establish the normal
range of the ratios of spermatogenic cells in control male fish testes prior to
making determinations on relative proportions in dose groups,
2. Frcsemc at testis-ovo: I'he presence of one or more individualized or clustered
oogenic cells within the testis. Oocytes within the testis ma\ be determined to be
perinucleolar, cortical aheolar. \ itellogenic. or atretic. There is iiule or no
e\ idence of o\arian architecture. Whenever applicable, the term testis-ova should
be used in preference to less precise terms such as "intersex" or ¦'hermaphrodite".
3. Increased testicular degeneration: Testicular degeneration is characterized by 1)
indi\ idual or clustered apoptotic germ ceils: 2} vacuolated germ cells; and/or 3)
multinucleated (s\neytial) cells in the germinal epithelium or testicular lumen.
Apoptotic germ cells are characterized by cell shrinkage, nuclear condensation,
and fragmentation into spherical, membrane-bound bodies, which are often
phagoc>ti/ed by neighboring cells. There is no inflammation associated with
tiiese cells. If possible, testicular degeneration should be differentiated from
necrosis, which is characterized morphological!) by cytoplasmic coagulation or
swelling, nuclear karyorrhexis or pyknosis. associated inflammation, a locally
extensive pattern of tissue involvement, and/or the involvement of different local
II
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tissue elements (e.g.. both germinal and stromal tissues), i.xtensive testicular
degeneration may lead to localized or generalized loss of the germinal epithelium.
4. Interstitial cell fLeydig cell) hyperplasia'hypertrophy: An increase in number
and or size of the interstitial cells responsible lor producing androgens.
Interstitial cells may have larger, more rounded nuclei, and interstitial ceil
aggregates may occupy and expand some interstitial spaces.
Primary Criteria - Females:
1, Increased oocyte atresia-. An increase in degradation and resorption of oocytes at
any point in development. Atresia is characterized by clumping and perforation
of the chorion, fragmentation of the nucleus, disorganization of the ooplasm,
and/or the uptake of yolk materials by perifollicular cells.
2. Perifollicular cell hyperplasia'hy^rtrophy. Increase in the size or number of
granulosa, theca. and'or surface epithelium cells involved in a developing follicle.
Abnormal perifollicular cell hypertrophy must be distinguished from the normally
enlarged granulosa and theca cells of a post-ovulatory follicle.
3, Decreased yolk formation: A decrease in the amount of vitcilogenic yolk nmei j -tl
that is deposited in the developing oocyte. Decreased \ itcllogcncsis is
characterized by the presence of oocytes in which yolk material is not present
despite their relatively large size. Note that oocytes may be affected to varying
degrees. Some affected oocytes have extremely fine vitcilogenic granules, and
this is interpreted as ineffective vitellogenesis.
4. Change in gonadal staging: Gonadal staging results are \irtualh meaningless in
terms of individual fish (versus treatment groups). This is because considerable
animal-to-animal variation in gonad cell proportions is to be expected, even
among fish of the control groups, as a consequence of spawning cycle
asynchrony. Consequently, following the gonadal staging of individual fish, each
treatment group is assessed as a whole and compared to the appropriate control
group to determine if a compound-related effect has occurred. Hence, gonadal
staging cannot be performed in a blinded manner. Because the cell distribution
pattern is J ike K to vary throughout a given tissue section, the gonad should be
staged according to the predominant pattern in that section. Similarly, 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 I TS
tunable to stage).
12
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Criteria for Staging Ovaries
• 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 I - Early development: vast majority {e.g., >90° o) are pre-\ itellogenie
follicles, predominantly perinucleolar through cortical alveolar.
• Stage 2 — Mid-development: at least half of observed follicles are early and mid-
vitellogenic.
• Stage 3 - Late development: majority of developing follicles are late
vitellogenin
• Stage 4 — Late development/hvd rated: majority of follicles are late vitellogenic
and mature spawning follicles; follicles are larger as compared to Stage .v
• Stage 5 - Post-ovuiatorv: predominately spent follicles, remnants of theca
externa and granulosa.
Secondary criteria - males:
1. Decreased proportion of spermatogonia'. Decreased relative proportion of
spermatogonia to other spcrmatogcnic cell types. This can be due to a decrease in
the siumber of spermatogonia, or an increase in the number of other cell types,
such as spermatocytes, spermatids, and spermatozoa. Because the diagnosis of
decreased proportion of spermatogonia is dependant on a comparison to controls,
it is necessary to establish the normal range of the ratios of spcrmatogcnic cells in
control male fish testes prior to making determinations on relative proportions in
dose groups,
2. Increased vascular or interstitial proteinaceous fluid: Homogenous dark pink
translucent material, presumably vitellogenin, within the testicular interstitium or
blood vessels, fhe presence of this Iliad may cause a thickening of interstit ial
areas that might be misinterpreted as "stromal proliferation".
3. Asynchronous gonad development: The presence of more than one developmental
phase of spcrmatogcnic cell within a single spermatocyst enclosed by a Sertoli
cell. For example, this term may be applied to a spermatocyst that contains a
mixture of spermatocy tes and spermatids, or a spermatocyst that contains more
than one meiotic phase of primary spermatocyte (i.e.. leptotene. pachytene, and/or
zygotene i. It also refers to the presence of distinctly different populations (i.e.
developmental phases) of gametogenic cells in the right and left gonads.
4. Altered proportions- of spermatocytes or spermatidA change in the relative
proportions of spermatocytes or spermatids to other spermatogenic cell types.
Changes in relative ratios could be due to an increase in the number of
13
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spermatocytes or spermatids, or to a decrease in the number of other cell types.
Relative changes may also occur between spermatocytes and spermatids.
5. Gonadal staging: Gonadal staging results are virtual!} meaningless in terms of
individual fish (versus treatment groups). This is because considerable animal-to-
animal \ariation in gonad cell proportions is to be expected, even among llsh of
the control groups, as a consequence of spawning cycle asynehrony.
Consequent!), following the gonadal staging of individual llsh. each treatment
group is assessed as a whole and compared to the appropriate control group to
determine if a compound-related effect has occurred. Hence, gonadal staging
cannot be performed in a blinded manner. Because the cell distribution pattern is
likeh to vary throughout a given tissue section, the gonad should be staged
according to the predominant pattern in that section. Similar!}, both gonads
should be staged as a single organ according to the predominant pattern. Cum ads
that cannot be reasonably staged for various reasons (e.g.. insufficient tissue, or
extensive necrosis, inflammation. or artifact! should be recorded as 11 I S (unable
to stage).
Criteria for Staging Testes
• Juvenile: gonad consists of spermatogonia exclusively; it may be difficult or
impossible to confirm the se.x of these individuals.
• Stage 0 - In developed: entirely immature phases (spermatogonia to spermatids)
with no spermatozoa.
• Stage i - £arlv spermatogenic: immature phases predominate, but spermatozoa
ma} also be observed: the germinal epithelium is thinner than it is during Stage 2.
• Stage 2 - \lid-sperniatogenic: spermatocytes, spermatids, and spermatozoa are
present in rough!} equal proportions: the germinal epithelium is thinner than
Stage I but thicker than Stage 3.
• Stage 3 - Late sperroatogenie: all stages may be observed, however, mature
sperm predominate: the germinal epithelium is thinner than it is during Stage 2,
• Stage 4 - Spent: loose connective tissue w ith some remnant sperm.
6. Granulomatous inflammation: This process is characterized by the presence of
epithelioid macrophages that typically form sheets or nodules (granulomasI due to
dcsmosome-like cytoplasmic attachments (Noga et al.. 1^8^i. W hen compared to
histioeyiic-type macrophages, epithelioid macrophages have larger, more open-
faced. centralized nuclei and less abundant cytoplasm. During resolution of
inflammation, the epithelioid macrophages may become flattened into ilbrocytc-like
cells. Lymphoc}tes. granulocytes, and multinucleated giant cells may also be
components of granulomatous inflammation. Granulomatous inflammation is
intrinsically a pathologic process that is often associated with reactions to infectious
agents, foreign materials or the aftermath of necrosis: therefore, it is important to
distinguish this, if possible, from the presence of histiocy tic cells in the lumen of
the testis.
14
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Secondary criteria -females:
1. Interstitial fibrosis: The presence of increased fibrous connective tissue (collagenous
fibers and librocytes or fibroblasts) within the ovarian interstitium (stroma). L ollagen
may be difficult to appreciate in early phases of fibrosis.
2. Egg debris in the oviduct: The presence of inspissated-appearing. homogenous,
irregular, dense pink material, presumed to be yolk, within ihe oviduct.
3. Granulomatous inflammation: This process is characterized by the presence of
epithelioid macrophages that typically form sheets or nodules (granulomas) due to
desmosome-Iike cytoplasmic attachments {Noga et ul., I()8l)}. When compared to
histiocylic-iype macrophages, epithelioid macrophages have larger, more open-faced,
centralized nuclei and less abundant cytoplasm. During resolution of inflammation,
the epithelioid macrophages may become flattened into tlbroeyte-like ceils.
Lymphocytes, granulocytes, and multinucleated giant cells max also be components
of granulomatous inflammation. Granulomatous inflammation is intrinsically a
pathologic process that is often associated with reactions to infectious agents, foreign
materials or the aftermath of necrosis; therefore, it is important to distinguish this, if
possible, from the presence of macrophage aggregates in the ovary.
4. Decreasedpost-ovulatory follicles: A decrease in the number of collapsed
perifollicular sheaths, or membranous structures lined by granulosa cells, theca cells
and surface epithelium, following release of oocytes, in comparison to control tish,
1 he granulosa cells are often hypertrophic, although this appears to be species
dependent (Saidapur, 1982).
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 lo
which a particular histomorphologic change is present in a tissue section (Shackelford et
af. 2002'i. fhe 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)
15
-------
Grade 3 (moderate)
Grade 4 (severe)
A grading s_\ stem needs to be flexible enough to encompass a variety of different tissue
changes. In iheon . there are three broad categories of changes based on the intuitive
manner in which people tend to quantify observations in tissue sections:
Discrete: these arc changes that can be readiK counted. Hx am pies include atretic
follicles, ooc\ tcs in the testis, and clusters of apoptotic cells.
Spatial: these arc changes that can be quantified by area measurements. 1 his
includes lesions that are typically classified as focal, multifocal, coalescing, or
diffuse. Specific examples include granulomatous inflammation and tissue necrosis,
Olohal: these are generalized changes that would usualh require more sophisticated
measurement techniques for quantification. Hxamples include increased hepatocyte
basophilia. Sertoli cell'interstitial cell hypertrophy, or quantitative alterations in cell
populations.
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 bareh noticeable but so
minor, small, or infrequent as to warrant no more than the least assignable grade,
For discrete changes, grade I is used w hen there are fewer than 2 occurances per
microscopic field, or 1-2 occurances per section, f or multifocal or diffusely-
distributed alterations, this grade is used for processes where less than 20% of the
tissue in the section is involved.
• Grade 2: Mihl. 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 30-50% of the tissue in the section is 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 60-80% of the tissue in the section is 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 greater than 80% of the tissue in the section is involved.
At the discretion of the pathologist the severity of a given change should be scored
16
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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 animal?
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 (see liistopathologv
Report Format). B\ convention, severity grading should not he influenced by the
estimated physiologic importance of the change. For example, the presence of two
oocytes in the testis should not be graded as "se\erc", even if the pathologist considers
this finding to be highh significant in terms of endocrine modulation.
Pata recording
An i¦ \cei worksheet form has been created that includes worksheets for primary,
secondare. and additional diagnoses to facilitate histopathoiogy data collection. In this
worksheet, each data entry cell represents an individual fish. Additional sheets are
available for comments and additional findings. Fur each fish, the pathologist records a
severity score associated with the diagnosis (see Severity Grading). 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 Lhat
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 M l (missing tissue).
Adding a Modifier term to a diagnosis may help to further describe or categorize a
finding in terms of chronicity. spatial distribution, color, etc. In mam instances,
modifiers are superfluous or redundant (e.g.. fibrosis is always chronic); therefore, the
use of modifiers should be kept to a minimum. An occasionally important modifier for
evaluating paired gonads is unilateral (UNI): unless specified in this manner, all gonad
diagnoses are assumed to be bilateral. Other modifier codes can be created as needed by
the pathologist.
17
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Histopathology Report Format
Each histopathology narrative report should contain the following five sections:
Introduction. Materials and Methods. Results. Discussion. Summary Conclusions.
A References section can also be included if applicable. The Introduction sec lion
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
Ilistotcchnical Procedures. As applicable, specific severily grading criteria (see Severity
Grading) should also be listed in this section. The Results section should report findings
that are: 1) compound-related: 2) potentially compound-related: 3) nose! or unusual.
Detailed histomorphologic descriptions need only he included for findings that differ
substantially from diagnoses presented in Section JIB. 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.
Glossary / Diagnostic Criteria
The purposes of this section are: 1) to provide photomicrographs of normal gonadal
structure in fathead minnow. 2) to provide a common technical "language" and to
create a reference atlas of both microanatomieal structures and potential pathological
findings, ["he information in this section is derived from a number of sources including
scientific articles, conference proceedings, related guidelines, toxicologic pathology
textbooks, medical dictionaries, and the personal experience of various fish pathologists.
Regarding the last, opinions were solicited via a questionnaire that was circulated among
conference participants follow ing the October 20().i meeting of the histopathology
subcommittee of the F ish Discussion Group in Paris. Consensus replies to this
questionnaire form the basis for naming main of these terms. Other considerations
include traditional usage and scientific precedence, and attributes such as clarity and
brevity.
The section is arranged as follows:
1.
2.
3.
4,
>.-females
-------
Normal Testicular Architecture in Fathead Minnow
Spermatogenic Cell Types:
Spermatogonia. Spermatogonia A in a male
FHM (GMA, H&E).
Spermatogonia: The largest of the spermatogenic
cells (- 5-10 um), spermatogonia generally have pale
vesicular nuclei, prominent nucleoli, variably distinct
nuclear membranes, perinuclear cytoplasmic granules,
and moderate amounts of granular cytoplasm.
Spermatogonia B are smaller than spermatogonia A,
and spermatogonia B are usually present in larger
clusters (e.g., > 4 cells). If at all possible, an attempt
should be made to classify these cells as
spermatogonia rather than to label them with a non-
specific term such as "pale cells" or "light ceils".
Spermatocytes: Derived from spermatogonia,
spermatocytes are of intermediate cell size (~ 4-6 j^m), and
have comparatively dense nuclei and minimal to moderate
amounts of indistinct cytoplasm. Spermatocyte nuclei are
usually evident in one of three meiosis phases: pachytene,
leptotene, or zygotene. Primary spermatocytes are larger
than secondary spermatocytes, and the latter are derived
from primary spermatocytes following the first meiotic
division. Spermatocytes are usually one of the most
abundant spermatogenic cells, and they tend to contribute
to the largest spermatocysts.
Spermatocytes. FHM testis (GMA, H&E).
Spermatids. Spermatids in a male FHM. intercellular
attachments are lost just prior to rupture of the spermatocyst
and release of these cells as spermatozoa (GMA, H&E).
Spermatids: Derived from spermatocytes
following the second meiotic division,
these cells have dense nuclei and narrow
rims of eosinophilic cytoplasm. They are
the smallest cells within the germinal
epithelium (~ 2-3 fim), and the cells lose
their cytoplasmic attachments to one
another during spermiogenesis.
19
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Spermatozoa: These cells have dark, round nuclei and
minimal or no apparent cytoplasm. Tails are generally not
apparent in histologic sections. Spermatozoa are the
smallest spermatogenic cells (~ 2 um), and they exist as
scattered individual cells within tubular lumen.
Spermatozoa. Spermatozoa in a
male FHM (GMA, H&E).
Sertoli cells: These cells tend to have sharply-defined
elongated or triangular nuclei, variably evident
nucleoli, and cytoplasm that is often indistinct. The
cytoplasmic arms of a Sertoli cell encircle a clonal
group of spermatogenic cells, forming a spermatocyst.
Compared to germinal cells. Sertoli cells are usually
present in low numbers, usually as single cells located
adjacent to lobular septa. In some instances,
hypertrophic (enlarged, swollen) Sertoli cells may
resemble spermatogonia.
Sertoli cells (FHM, GMA, H&E).
20
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Interstitial (Levdis) cells-. These
cells have dense, dark round or
oval nuclei with little detail and
moderate amounts of variably-
evident, faintly vacuolated
cytoplasm. Compared to
germinal cells, interstitial cells are
usually present in low numbers,
usually as single cells or small
aggregates, within the interlobular
interstitium. Although they may-
resemble spermatocytes,
interstitial cells are only present
in interlobular areas.
Interstitial cells (FHM, H&E). Interstitial cells (small arrows)
are only found in interlobular areas. Note the resemblance
between these cells and spermatocytes (large arrow).
Spermatocvst: The functional unit of the testis, this
structure consists of a clonal group of spermatogenic
cells (spermatogonia, spermatocytes, or spermatids)
that are surrounded by the cytoplasmic arms of
(usually) one Sertoli cell. Cells within spermatocysts
exist as syncytia, maintained by intercellular
attachments (cytoplasmic bridges), until final
maturation and release of spermatozoa occurs
(spermiogenesis) (Grier, 1976).
Spermatocyst (FHM, adult male, plastic, H&E).
A group of dissociated spermatids are surrounded
by the cytoplasmic "arms" of a single Sertoli
cell (arrow). This arrangement is usually not
as obvious as it is in this photograph. The
nucleus of this particular Sertoli cell appears
enlarged (hy pertrophic).
21
-------
Spermatocysts (FHM, adult male, GMA, H&E). Spermatocysts outlined in red and green contain
spermatocytes and spermatids, respectively. Spermatogonia (black arrows) and spermatozoa within tubular
lumina (blue arrows) are also indicated.
Germinal epithelium (male): The
germinative intratubular (intralobular)
parenchyma of the testis, this
membrane-bound structure consists of
multiple spermatocysts in various
phases of development. For FHM,
boundaries of the germinal epithelium
at various locations throughout the
testis include the interlobular
interstitium, the lobular lumina,
collecting ducts, and the tunica
albuginea.
Germinal epithelium, male. Normal testis from an adult FHM.
Double arrow indicates width of germinal epithelium, which
extends from the interlobular interstitium to the lobular lumen
(GMA = glycol methacrylate, H&E, bar = 25 um).
22
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Normal Ovarian Architecture in Fathead Minnow
Oogenic Cell Types:
Ooeonia: These cells represent the replicative
pool of the ovary. Unlike mammalian oogonia
(although this dogma may soon change based
on recent data from rodent research), piscine
oogonia continue to divide in juveniles and
adults. The smallest of the oocytic cells,
oogonia reside within the ovarian germinal
epithelium, usually in comparatively low
numbers. Oogonia are characterized by a
relatively large nucleus with small or
inapparent nucleolus, and minimal amounts of
cytoplasm.
Oogonia (FHM, paraffin, H&E, bar= 10 pm). A small cluster
of oogonia reside within a portion of germinal epithelium; the
nucleus of only one oogonium is visible (small arrow). The
oogonia are dwarfed by a perinucleolar oocyte (large arrow).
Chromatin nucleolar oocytes: Slightly larger
than an oogonium, this oocyte is formed when
an oogonium becomes surrounded by
prefollicle cells (presumptive granulosa cells)
and the resulting complex buds from the
germinal nest as a primordial follicle. The
chromatin nucleolar oocyte has a relatively
large nucleus that contains a single, large
nucleolus. Compared to an oogonium, there is
more cytoplasm which is slightly more dense
and finely granular.
Chromatin nucleolar oocyte (FHM, paraffin, H&E, bar = 10 pm).
A single chromatin nucleolar oocyte protrudes from the germinal
epithelium (arrow).
23
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Perinucleolar oocytes: Concomitant with
oocyte growth, the nucleus (germinal vesicle)
increases in size and multiple nucleoli appear,
generally at the periphery of the nucleus. The
cytoplasm stains uniformly dark, although late
perinucleolar oocytes may have small clear or
amphophilic vacuoles in the cytoplasm. These
cells tend to be abundant in normal adult
ovaries.
Perinucleolar oocytes. Several perinucleolar oocytes
in the ovary of a FHM. Arrows indicate nucleoli at the
periphery of the germinal vesicle (paraffin, H&E, bar
= 25 urn).
Cortical alveolar oocytes: Generally larger
than perinucleolar oocytes, this phase is
characterized by the appearance of cortical
alveoli (yolk vesicles) within the ooplasm. The
cortical alveoli are technically not yolk, as they
do not provide nourishment for the embry o
(Selman and Wallace, 1989), The chorion
becomes distinctly evident in this phase, and
the perifollicular cells are more easily
visualized.
Cortical alveolar oocytes. FHM ovary demonstrating
multiple cortical alveolar oocytes. The cytoplasm is
predominately filled by numerous cortical alveoli, which
are amphophilic with this preparation. Evident are oocytes
in transition from the perinucleolar to cortical alveolar
phase (small arrow), and from the cortical alveolar to early
vitellogenic phase (large arrow) (paraffin, H&E, bar
= 100 (im).
24
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Early vitellosenic oocytes: Larger than
cortical alveolar oocytes, these cells are
characterized by the centralized appearance of
spherical, eosinophilic, vitellogenic yolk
granules / globules. In H&E sections,
accumulations of fine yolk granules in the
central region of the oocyte may somewhat
resemble (and thus be confused with) the
reddish nucleus.
Early vitellogenic oocytes. In this FHM ovary,
numerous fine pale pink granules (large arrow), and a
few larger dark red granules (small arrow), are evident
in the central region of an early vitellogenic oocyte.
Although nuclei are present they are not apparent in
every oocyte due to the comparatively vast amount of
cytoplasm (paraffin, H&E, bar = 100 fxm).
Late vitellosenic oocytes: These cells are
characterized by an increased accumulation of
vitellogenic granules that displace the cortical
alveolar material to the periphery of the
cytoplasm. It is during this stage that the
nucleus begins to migrate toward the periphery
of the cell.
Late vitellogenic oocytes. Late vitellogenic oocyte
in a FHM ovary. The yolk granules almost fill the
ooplasm. The nucleus has not yet begun to migrate
peripherally (paraffin, H&E, bar = 100 urn).
25
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Mature/spawning oocytes. Two mature/spawning
oocytes in a FHM ovary. The oocytes and the yolk
granules have attained their maximum size just prior
to spawning, and the nucleus is not evident (paraffin,
H&E, bar = 100 urn).
Mature/spawning oocytes: In this phase of
development, vitellogenesis has reached its peak,
the cell has become larger and more hydrated,
and the nucleus has migrated toward the
periphery of the cell and is in the process of
dissolution. The loss of nucleus is not a very
helpful diagnostic feature, however, as the
nucleus is often not visible in larger oocytes due
to the plane sectioning. Because of the transient
nature of these cells in fractional spawning fish,
mature/spawning oocytes are uncommonly
observed.
Zona radiala
Su rises
epsthelium
Ttieca
Vitelline
envelope
Ooplasm
Oocyle
Diagram of an ovarian follicle. From Tyler and
Sumpter, 1996.
Ovarian follicle: The functional unit of the ovary,
this term generally refers to an oocyte plus its
surrounding sheath of perifollicular cells (granulosa
cells, theca cells, and surface epithelium cells)
(Tyler and Sumpter, 1996). However, there are
subtypes of follicles in which the oocyte is not
present or may be difficult to appreciate; these
include post-ovulatory (spent), empty, and atretic
follicles. A post-ovulatory follicle (the follicle has
ruptured to release an oocyte during spawning) is
collapsed and often has enlarged (hypertrophic)
granulosa and theca cells. Conversely, an empty
follicle (in which the oocyte has been dislodged
from the histologic section as a post-mortem
artifact) generally retains the shape of the oocyte
and may or may not have enlarged granulosa and
theca cells. An atretic follicle must be distinguished
from both spent follicles and empty follicles; the
presence of at least some ooplasm ic material (often
heterochromatic) within a follicle indicates that it
contains an atretic oocyte.
26
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Perifollicular Cells
Ovarian Wall Epithelium
Perifollicular cells-. These cells form a three-
layered sheath around each oocyte, and combined
with the oocyte itself comprise the ovarian
follicle. These layers are more easily visualized
as the oocyte matures. The innermost layer
consists of the granulosa cells, the middle layer
consists of the theca cells, and the outermost layer
consists of the surface epithelial cells. The
granulosa cells especially may become enlarged
and vacuolated following ovulation or during
oocyte atresia. The perifollicular sheath should
not be confused with folds of the ovarian wall
epithelium.
Perifollicular cells. In this photomicrograph, the perifollicular cells
are compared to the cells of the ovarian wall epithelium, which
contains dark brown (melanin) pigment (arrow) and is comprised of
ciliated columnar cells in FHM. FHM, adult female, paraffin, H&E,
bar = 25 pm)
Chorion: Usually pale to dark eosinophilic and
refractile, the chorion is the thick external layer of
an oocyte that surrounds the ooplasm. The terms
zona radiata and vitelline envelope have been
used synonymously. In mature, unspent follicles,
the chorion is noticeably surrounded by
perifollicular cells (granulosa cells, theca cells,
and surface epithelial cells). As viewed by light
microscope, the chorion is often minimally
apparent or inapparent prior to the cortical
alveolar phase of oocyte development.
Chorion. The chorions of two oocytes are indicated by large arrows.
A smaller arrow denotes a post-ovulatory follicle. (FHM, paraffin,
H&E, bar = 25 nm).
27
-------
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Post-ovulatory follicle: A collapsed
perifollicular sheath following release of the
oocyte; this is a membranous structure lined by
granulosa cells, theca cells, and surface
epithelium. The granulosa cells are often
hypertrophic, although this appears to be species
dependent (Saidapur, 1982). Mammalian terms
such as "corpus lutea" and "Graafian follicles",
are probably less desirable, due to structural and
functional differences between these entities and
piscine post-ovulatory follicles. Whenever
possible, post-ovulatory follicles should be
differentiated from atretic follicles, the latter of
which contains oocyte debris.
Post-ovulatory follicle. Situated between three oocyte-containing
follicles is a collapsed follicle that does not contain oocyte remnants
(arrows) (FHM, adult female, paraffin, H&E, bar =. 25 urn).
28
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Primary Diagnoses - males:
Increased proportion of spermatogonia:
Increased proportion of spermatogonia: It is
recognized that endocrine active compounds may
alter the proportional distribution of gametogenic
cell types in the testis or ovary. Certain types of
alterations (for example, the proliferation or
absence of single cell population) may not be
adequately documented by gonadal staging. This
diagnostic term provides a mechanism for
documenting such changes. Quantitative
alterations are: 1) relative to other cell types in
the gonad; 2) relative to cell numbers in control
animals; and 3) estimates only, versus actual cell
counts.
Increased cells, spermatogonia. Testis from adult male
FHM negative control (GMA, H&E, bar = 25 jitn).
Ifc Spermatogonia dominate the germinal epithelium in this
testis from adult male FHM exposed to 10 nM E2 for 10 days.
Other diagnoses for this section include "Decreased cells,
spermatocytes", "Decreased cells, spermatids" (GMA, H&E,
bar = 25 p).
29
-------
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Testis from an adult male FHM. There is a minimal
(Grade 1) increase in the proportion of spermatogonia
(arrows). H&E.
Testis from an adult male FHM. There is a slight/mild
(Grade 2) increase in the proportion of spermatogonia
throughout the germinal epithelium. H&E.
Testis from an adult male FHM. There is a moderate
(Grade 3) increase in the proportion of spermatogonia.
H&E.
Testis from an adult male FHM. There is a severe (Grade
4) increase in the proportion of spermatogonia. H&E.
30
-------
Presence of testis-ova: An example of testicular oocytes is not available for FHM.
Increased testicular degeneration:
Testicular degeneration. Multiple clusters of apoptotic germ
cells (black arrows) and vacuolated germ cells (red arrow) within
the germinal epithelium. (FHM, adult male, GMA,H&F., bar =
25 (im).
Testicular, degeneration: Examples of
degenerative findings in the testis include: 1)
individual or clustered apoptotic germ cells; 2)
vacuolated germ cells; 3) multinucleated
(syncytial) cells in the germinal epithelium or
testicular lumen. These diagnoses may be
"lumped" together under the term testicular
degeneration. Apoptotic germ cells are
characterized by cell shrinkage, nuclear
condensation, and fragmentation into spherical,
membrane-bound bodies, which are often
phagocytized by neighboring cells. There is no
inflammation associated with these cells. If
possible, testicular degeneration should be
differentiated from necrosis, which is
characterized morphologically by cytoplasmic
coagulation or swelling, nuclear karyorrhexis or
pyknosis, associated inflammation, a locally
extensive pattern of tissue involvement, and/or
the involvement of different local tissue elements
(e.g., both germinal and stromal tissues).
Extensive testicular degeneration may lead to
localized or generalized loss of the germinal
epithelium.
Interstitial cell (Leydig cell) hyperplasia/hypertrophy:
Non-Remarkable
Non - remarkable testis from males FHM. Interstitial
areas contain small aggregates of interstitial (Leydig) cells
(arrows). Most interstitial cells have wispy, pale
cytoplasm. H&E.
31
-------
Grade 1
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Grade 2
Testis from an adult male FHM. Interstitial cell
aggregates (arrows) in the testis of this fish are larger than
in control fish, and the cytoplasm of these cells is slightly
more dense. This was diagnosed as Increased Cells,
Interstitial Cells, Grade 1 (minimal) severity. M&E.
Testis from an adult male FHM. Interstitial cell aggregates
(arrows) in the testis of this fish are larger than in control
fish, and the cells tend to fill and expand the interstitial
spaces. This was diagnosed as Increased Cells, Interstitial
Cells, Grade 2 (mild) severity. H&E.
32
-------
Primary diagnoses - females:
Increased oocyte atresia:
Oocyte atresia, increased, immature / mature:
Degradation and resorption of an oocyte at any
point in development. Histopafhologically,
atresia is often characterized by clumping and
perforation of the chorion, fragmentation of the
nucleus, disorganization of the ooplasm, and/or
the uptake of yolk materials by perifollicular
cells (FHM, e.g.). Separate diagnoses and
severity grades can be given to atretic oocytes
that are mature ("Oocyte atresia, increased,
mature") versus immature ("Oocyte atresia,
increased, immature"). In this context, oocytes
will be considered "mature" if they are appear
to have been interrupted in either the late
vitellogenic oocyte phase or mature / spawning
phase of development.
Non Remarkable
Ovary from a control group female. H&E.
LV = late vitellogenic
EA = early atretic
LA = late atretic
ve = vitelline envelope
Oocyte atresia, mature oocytes. Note clumping and pore-formation
in the vitelline envelope (chorion) of the early atretic oocyte (large
arrow), and the vacuolar hypertrophy of its surrounding granulosa
cells (small arrows). Compare these with granulosa cells that
surround a non-atretic late vitellogenic oocyte (arrowheads). In FHM,
granulosa cells of atretic oocytes often appear to contain phagocytized
material, whereas the granulosa cells of non-atretic oocytes do not.
(FHM, adult female, paraffin, H&E).
33
-------
Grade 3
Ovary from an adult female FHM. Numerous atretic
oocytes are evident (arrows). H&E.
Stage 4 ovary from an adult female FHM. This
ovary is characterized by severe oocyte atresia.
Asterisks indicate the relatively few non-atretic
oocytes. H&E.
Perifollicular cell hyperplasia/hypertrophy: An example of perifollicular cell
hyperplasia/hypertrophy is not currently available for FHM.
Decreased yolk formation:
Not Remarkable
Ovary (Stage 3) from a control group female. A single
atretic ovary is evident (arrow). H&E, bar = 250 microns.
Grade 4
34
-------
Grade 3
m
^ M °
Ovary from an adult female FHM, Decreased yolk
formation is characterized by the presence of oocytes in
which yolk material is not present despite their relatively
large size (large arrows). Note that oocytes are affected to
varying degrees. Some affected oocytes have extremely
fine vitellogenic granules (small arrow), and this is
interpreted as ineffective yolk formation and deposition.
H&E, bar = 250 microns.
Change in ovarian staging: Photographic examples are not currently available that
demonstrate changes in ovarian staging.
35
-------
Additional diagnostic criteria and an illustrated glossary of microanatomicat and
diagnostic terms
Asterisks denote "secondary" diagnoses for male and female FHM
**Asynchronous development, gonad (male or female): The presence of distinctly
different populations (i.e., range of developmental phases) of gametogenic cells in
different regions of a gonad.
Asynchronous development, spermatocyst (male): The presence of more than one
developmental phase of spermatogenic cell within a single spermatocyst. For example,
this term may be applied to a spermatocyst that contains a mixture of spermatocytes and
spermatids, or a spermatocyst that contains more than one meiotic phase of primary
spermatocyte (i.e., leptotene, pachytene, and/or zygotene).
Asynchronous development, right and left gonads (male or female): The presence of
distinctly different populations (i.e., developmental phases) of gametogenic cells in the
right and left gonads.
**Egg debris, oviduct: The presence of inspissated-appearing, homogenous, irregular,
dense pink material, presumed to be yolk, within the oviduct.
D = debris
Ov = oviduct
Egg debris, oviduct. (FHM. adult female, paraffin, H&E).
Gender: Because the genetic sex of a fish cannot be determined within the context of a
screening assay, and because the externa! phenotypic sex may be an unreliable indicator
and/or is not easily determined in some species, by convention the gender of a fish will
be assigned according to the most abundant mature cell type that is present in the gonad.
36
-------
Germinal epithelium (female): The germinative parenchyma of the ovary is a
membrane bound structure constitutively contains oogonia, prefollicular and prethecai
cells, epithelial cells, and occasionally small chromatin nucleolar (primary growth)
oocytes (Norberg et al., 2000: Parent! and Grier, 2003). The germinal epithelium
separates the ovarian lumen from the stroma, the latter of which often contains
perinucleolar, cortical alveolar, and vitellogenic follicles within a variably-apparent
extravascular space.
Germinal epithelium, atrophy / hypoplasia (male): Indicating loss or
underdevelopment of germinal epithelium, respectively, this condition may be associated
with interstitial fibrosis and increased prominence of interstitial cells in affected areas of
the testis. It may be difficult to distinguish atrophy from hypoplasia. Care should be
taken to avoid mistaking areas of collecting ducts for atrophy. Severity of this finding can
vary from Grade 1 (minimum, focal) to Grade 4 (severe, diffuse). If thinning of the
epithelium appears to be caused by degenerative changes that are obvious in the section,
the diagnostic term testicular degeneration should be used instead.
Germinal epithelium, atrophy. A: Atrophy
of germinal epithelium in an adult male
FHM. Also note the prominence of
interstitial (Leydig) cells (red circles) and
interstitial fibrosis. (GMA, H&E. bar = 25
m). B: Normal FHM testis (GMA, H&E,
bar = 25 m). C: Normal collecting duct
region in an adult male FHM (GMA, H&E,
bar = 25 m). The presence of pigment in
the duct walls and the lack of interstitial cells
are distinguishing features.
37
-------
**Granulomatous inflammation: In the early stages of inflammation, this process is
characterized by the presence of epithelioid macrophages that typically form sheets or
nodules (granulomas) due to desmosome-Iike cytoplasmic attachments (Noga et al.,
1989). When compared to histiocytic-type macrophages, epithelioid macrophages have
larger, more open-faced, centralized nuclei and less abundant cytoplasm. During
resolution of inflammation, the epithelioid macrophages may become flattened into
fibrocyte-like cells. Lymphocytes, granulocytes, and multinucleated giant cells may also
be components of granulomatous inflammation. Granulomatous inflammation is
intrinsically a pathologic process that is often associated with reactions to infectious
agents, foreign materials, or the aftermath of necrosis; therefore, it is important to
distinguish this, if possible, from the presence of macrophage aggregates in the ovary or
histiocytic cells in the lumen of the testis.
Granulomatous inflammation. A: Sheets of macrophages and other inflammatory cells eclipse much of
the germinative tissue in this testis (FHM, adult male, paraffin, H&E). B: Relatively few viable-appearing
oocytes remain in this ovary. As in the testis photo, the inciting cause of the inflammation is not evident at
this magnification (FHM. adult female, paraffin, H&E).
Hepatocvte basophilia, increased / decreased: A generally diffuse increase in
hepatocyte cytoplasmic basophilia has been observed in male fish that have been exposed
to compounds that are able to interact with hepatic estrogen receptors, including E2 and
17a-methyldihydrotestosterone (Wester et al., 2003). This increase in basophilia, which
is correlated with increased vitellogenin production, presumably mimics the heightened
metabolic state (e.g., increased endoplasmic reticulum) that is required for the production
of vitellogenin in the reproductively-active female fish.
38
-------
75 microns
Hepatocyte basophilia A: The liver from an adult male FHM
control. In addition to the overall coloration, note the
hepatocyte cytoplasmic vacuolization as indicated by the arrows.
B: Liver from an adult male FHM that was exposed to a compound
with estrogenic activity. There is a diffuse increase in hepatocyte
basophilia, a loss of cytoplasmic vacuolization, and hepatic blood
vessels contain proteinaceous fluid (arrows). (FHM, paraffin, H&E).
39
-------
Histiocytic cells (male): The presence of individual or clustered cells with small
eccentric nuclei and moderate to abundant, pale or vacuolated cytoplasm within the
testicular lumen, germinal epithelium, efferent ducts and/or ductus deferens. Such cells
may contain intracytoplasmic cellular debris (presumably phagocytized). The origin of
the histiocytic cells in each particular case may not be clear; for example, they may be
hematogenous macrophages or Sertoli cells. Histiocytic cells should be differentiated
from macrophage aggregates (these variably pigmented cells are primarily interstitial)
and granulomatous inflammation (which is predominately comprised of "epithelioid"
macrophages and/or flattened, fibrocytic cells).
Histiocytic cells. (FHM, adult male, GMA, H&E). Ai Cells with small
peripheral nuclei and abundant vacuolated cytoplasm are present within
the germinal epithelium and are scattered throughout the tubule lumen
(arrows). Some of these cells contain phagocytized cellular debris.
B: Similar cells are evident within the lumen of the collecting duct.
40
-------
Interstitial fibrosis (male or female): The presence of increased fibrous connective
tissue (collagenous fibers and fibrocytes or fibroblasts) within the testicular or ovarian
interstitium (stroma). Collagen may be difficult to appreciate in early phases of fibrosis.
In most cases, this term should be used in preference to terms such as "stromal
hyperplasia."
Macrophage aggregates: These cell clusters are constitutively present in the interstitium
of the ovary primarily, although they may also be found in the fish testis (unusual for
tank-raised FHM). These phagocytes usually have small condensed eccentric or
peripheralized nuclei and various brown, yellow, red, or gold pigmented granules
(lipofuscin, ceroid, hemosiderin, and/or melanin) that often impart a slightly cry stalline
appearance to their comparatively abundant pale cytoplasm. In the normal ovary,
macrophage aggregates are thought to be involved in the processing of breakdown
products associated with atresia of unspawned oocytes. It has been demonstrated that
macrophage aggregates may become larger and/or more numerous following exposure to
certain toxicants or infectious agents (Blazer et al., 1987). Whenever possible,
macrophage aggregates should be distinguished from granulomatous inflammation,
which is characterized by the presence of epithelioid macrophages. This is not always
easy, as macrophage aggregates often proliferate with, and become incorporated into,
granulomatous inflammation.
Macrophage aggregates. The arrows indicate multiple aggregates within
the ovarian interstitium. (FHM, adult female, paraffin, H&E).
41
-------
Nephropathy: Degenerative renal disease has been observed in a variety of fishes that
have been exposed to compounds with estrogenic activity (Herman & Kincaid, 1988;
Zillioux et al., 2001; Palace et al, 2002). Renal impairment presumably occurs due to
increased production of vitellogenin (especially in males) that stresses the kidney via
protein overload. Microscopic lesions may include swelling of tubular epithelial cells,
tubular necrosis, dilation of Bowman's capsule, interstitial fibrosis, casts, and hyaline
droplets in tubules or glomeruli.
-/¦ ' '
L 'r , '
[r>.
V V-
f?
7v.. v
\9
"\V
, - — v rfr:
r- c* • . .* \ .•* r *
I » » • - Vr—, -
Nephropathy. A: Kidney from an untreated aduit male FHM. B: Kidney from an adult male FHM
exposed to a compound with estrogenic activity. Changes include glomerular epithelial cell hypertrophy,
vacuolar swelling and necrosis of the tubular epithelium, and hyaline droplets within glomerular and
tubular epithelia (paraffin, H&E, bar = 25 ^m).
Ovarian spermatogenesis: The presence of non-neoplastic spermatogenic cells, usually
immature, within the ovary. There is little or no evidence of lobular or tubular testicular
architecture. Care should be taken to distinguish ovarian spermatogenesis from
mitotically dividing oogonia; a key feature of ovarian spermatogenesis is the presence of
multiple spermatogenic phases.
42
-------
Mitotically dividing oogonia. Packets of cells that resemble spermatocytes (arrow)
are situated between perinucleolar and cortical alveolar oocytes. This should not be
mistaken for spermatogenesis. (FHM, adult female, paraffin, H&E).
**Proteinaceous fluid, interstitial (male or female): Homogenous dark pink
translucent material, presumably vitellogenin, within the testicular or ovarian interstitium.
In male fish especially, this finding has been associated with exposure to estrogenic
substances. The presence of this fluid may cause a thickening of interstitial areas that
might be misinterpreted as "stromal proliferation".
% O ^
Proteinaceous fluid, interstitial. There is homogenous
dark pink material in interstitial spaces (arrows). (FHM, adult
female, paraffin, H&E, bar = 50 ^m).
43
-------
**Proteinaceous fluid, intravascular (male or female): Homogenous dark pink
translucent material, presumably vitellogenin, within testicular or ovarian blood vessels.
In male fish especially, this finding has been associated with exposure to estrogenic
substances.
Proteinaceous fluid, intravascular. There is
homogenous dark pink material within large and small
blood vessels (arrows). Also note the increased
proportion of spermatogonia in the testis. (FHM,
adult male, GMA, H&E, bar = 25 (im).
Sertoli cell hypertrophy: Exposure of male fish to estrogen-active compounds has been
reported to cause enlargement of Sertoli cells, with or without Sertoli cell proliferation
(Miles-Richardson et al., 1999a; Miles-Richardson et al., 1999b; Kinnberg et al., 2000;
van der Ven et al., 2003). In the scientific literature, the light microscopic appearance of
hypertrophic Sertoli cells tends to be ambiguous, as Sertoli cells resemble spermatogonia
in some descriptions and images.
Vitellogenic oocyte: An oocyte that contains microscopically visible yolk material.
Generally, such material is strongly eosinophilic and slightly refractile in hematoxylin-
and eosin-stained sections. This material may be present in the form of spherical,
globular, yolk granules. In some scholarly sources (e.g., Iwamatsu, et al., 1998), the term
"vitellogenic" has been applied to cortical alveolar oocytes, which lack eosinophilic yolk
granules/globules (although their amphophilic or clear cortical alveoli are also known as
yolk vesicles).
GONDAL 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
fathead minnows. 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-stained histologic
sections.
44
-------
Semi-quantitative gonadal staging has been proposed for. or employed in. studies
involving fathead minnows (Ankley et al., 2002; Jensen et at.. 2001: Miles-Richardson et
al.. 1999a; Nichols et al.. 2001; US EPA. 2002). among other fishes. Although such
studies general!} included excellent descriptions of the different gamelogenie 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 sludies. 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 s\stem can improve the consistency and objectivity of reported
observations within and among experiments; consequent!}. 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. t_\pes 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 even type of observation (either
predicted or unforeseen). To address tins last limitation, gonadal staging is accompanied
by a complete histopathologic^! evaluation of the gonads; in this manner, the loss or
overabundance of a specific gamelogenie cell type; for example, can be documented.
The semi-quantitative gonadal staging scheme selected for analysis of FHM gonads is a
modification of a s} stern 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 (REST) Program" (McDonald et al.. 2000). The
authors of the BEST system credit previous work by Treasurer and 1 loliday {1981).
Nagahama (1983 ). Rodriquez et al. (1995). and Goodbred et al. {1997). The foremost
benefits of this system arc speed and ease of use. especially when compared to fully-
quantitative staging, fhe basis of the BEST system is a visual assessment of the density
of szametogenic precursors as compared to mature gameiocytes 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
RHST sy stem was initially developed to assess reproductive function in seasonal
spawners such as carp
-------
example, there is currently no provision in the system for gonads that are comprised
entirely of spermatogonia or oogonia. Although it is intended that rcproduc lively mature
fish are used, it is possible thai 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, as one modification of the
BHST system, a pre-staging category called "juvenile" has been added lor both male and
female fish. Another modification to the system involves an apparent discrepancy
between the BEST system and Goodbred et al. concerning the thickness of the testicular
germinal epithelium as a slaging criterion. As indicated by Goodbred et al.. the germinal
epithelium becomes thinner as the testis stage increases, whereas, the reverse occurs
according to the BKST system (as presented in McDonald et al.). Although it is difficult
to find corroborating statements in the scientific literature, empirical evidence indicates
that Goodbred et al. is correct on this point. A third modification to the system is the
option to subdh ide a stage into two subordinate stages (e.g.. Stages ,v\ 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 var\ 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 reasonabh staged for various reasons (e.g.. insufficient tissue, or extensive
necrosis, inflammation, or artifact) should be recorded as IIS (unable to stage).
Criteria for Staging Testes
The following are morphologic criteria for slaging male fish:
• Juvenile: gonad consists of spermatogonia exclusively: it may be difficult or
impossible to confirm the sex of these individuals.
Stage 0 - Undeveloped: entirely immature phases (spermatogonia to
spermatids') with no spermatozoa.
0 Stage 1 - Early snermatogenic: immature phases predominate, but
spermatozoa may also be observed: the germinal epithelium is thinner than it
is during Stage 2.
• Stage 2 - Mid-spermatogenic: spermatocytes, spermatids, and spermatozoa
are present in roughly equal proportions: the germinal epithelium is thinner
than Stage 1 but thicker than Stage 3.
• Stage 3 - Late sperroatogeniei all stages may be observed, however, mature
sperm predominate: the germinal epithelium is thinner than it is during Stage
2.
• Stage 4 - Spent: loose connective tissue with some remnant sperm.
46
-------
Examples of staging system applied to the FHM testis. Testes from four different adult male FHM.
There is progressive thinning of the germinal epithelium and expansion of the lobular lumen with each
increase in stage. Note that no spermatozoa are present at in the Stage 0 image (GMA, H&E).
47
-------
Criteria for Staging Ovaries
The following are morphologic criteria for staging female fish:
• 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 pre-vitellogenic
follicles, predominantly perinucleolar through cortical alveolar.
• Stage 2 - Mid-development: at least half of observed follicles are early and mid-
vitellogenic.
• Stage 3 - I,ate development: majority of developing follicles are late
vitellogenic.
• Stage 4 - Late development/hydrated: majority are late vitellogenic and mature
/ spawning follicles; follicles are larger as compared to Stage 3.
• Stage 5 - Post-ovulatory: predominately spent follicles, remnants of theca
externa and granulosa.
its
„ oO' /o-
§o°.%< •«
Examples of staging system applied to the FHM ovary. Ovaries from four adult female FHM. Due
transient nature in FHM, Stage 4 is not often observed (paraffin, H&E).
48
-------
APPENDIX A: HISTOLOGY FIGURES
Bouin's Fixative Modified Davidson's Fixative
Fig. 1. Fathead Minnows, Testis (A&B) and Ovary (C&D): Gonads fixed in Bouin's fixative (A&C)
and modified Davidson's fixative (B&D). Color contrast was slightly superior in testes fixed with
Davidson's fixative and was clearly superior in ovaries fixed with Bouin's fixative. Either fixative is
satisfactory for diagnostic purposes; however. Davidson's fixative was selected for the Phase IB assay.
49
-------
Fig. 2. Fathead Minnow, Male: Excision of the testes during necropsy. A. The abdominal wall is
pinned laterally. B. The terminal intestine is severed and retracted prior to removal. C. The testes are
grasped near the spermatic ducts. D. Removal of the testes is complete.
50
-------
ovaries
viscera
ovaries
Fig. 3. Fathead Minnow, Female: Excision of the ovaries during necropsy. A. The abdominal wall is
pinned laterally. B. The terminal intestine is severed and retracted prior to removal. C. The ovaries are
grasped near the oviducts. D. Removal of the ovaries is complete.
51
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APPENDIX B: SCHEDULES
Schedule 1. Tissue Processing
Station
No,
Reagent
Pressure/ !
V"T IK'au°Ci
GONAD
PROGRAM
(minutes)
WHOLE-FISH
PROGRAM
(minutes)
1
io°.o\'Br
On
Ambient
40
60
2
70°"o ethy 1 alcohol
On
Ambient
40
60
3
80° o ethyl alcohol
On
Ambient
40
60
4
95% ethyl alcohol
On
Ambient
40
60
5
95% ethyl alcohol
On
Ambient
40
60
6
100% ethyl alcohol
"6n~
Ambient
40
60 ~ ~
7
100% ethyl alcohol
On
Ambient
40
60
8
100% ethyl alcohol
On
Ambient
40
60
9
Clear Rife 3
On
Ambient
60
80
10
Clear Rite 3
On
Ambient
60
80
11 j Paraffin
On
60
45(60=)
' 75(100b)
12
Paraffin
On
60
45(60")
75(1 OOS
13
Paraffin
On
60
45(60")
75(100b|
14
Paraffin
On
60
45
75
Drain and Clean Cycle1"
' Ncutia' buffered formalin
h Times ate increased for processors that have three (versus four) final stations
1 Automatic cleaning c> clc so be run after removal of tissues from the processor f imt, temperature. and vacuum art* preset by the
manufacturer.
52
-------
Schedule 2. Hematoxylin and Eosin Staining
| Reagent Maintenance
Reagent
Minutes in Reagent
After Ist Run
After 2nrt Run
Xylene
4
Remove
Remove
Absolute Alcohol
2
Remove
Remove
80% Alcohol
1
Renew
Water
1
...
—
Hematoxylin
3
.
Remove
Water
2
—
...
Clarifier
1
Renew
Renew
Water
1
...
...
Bluing
1
Renew
Renew
Water
2
—
95% Alcohol
1
Renew
Renew
Eosin
1
—
Renew
Absolute Alcohol
*
4
Remove
Remove
Xylene
3
Remove
Remove
53
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APPENDIX C: FORMULARY
Euthanasia Solution
Tricaine methanesulfonate 100 mg
Sodium bicarbonate 200 mg
Tank or reservoir water 1 L
Davidson's Fixative (Fournie et al., 2000)
Formaldehyde (37-40%) 200 ml
Glycerol 100 ml
Glacial acetic acid 100 ml
Absolute alcohol 300 ml
Distilled water 300 ml
Modified Davidson's Fixative
Formaldehyde (37-40%) 220 ml
Glacial acetic acid 115 mi
95% Ethyl alcohol 330 ml
Distilled water 335 ml
It is recommended that hematoxylin and eosin be purchased as premixed solutions,
examples arc the I Iematoxylin-2 (Gill hematoxylin) and Fosin Y solutions that are
manufactured by Richard-Allan Medical Industries (Appendix D).
G ill Hematoxylin Solution (Gill et at. 1974)
Distilled water 730 ml
Ethylene glycol 250 ml
Hematoxylin, anhydrous 2 g
Sodium iodate 0.2 g
Aluminum sulfate 17.6 g
Glacial acetic acid 20 ml
Eosin Solution
Eosin Y (1 % aqueous solution) 100 ml
Ethyl alcohol 95% 600 ml
Glacial acetic acid 4 ml
C-l
-------
APPENDIX D: EXAMPLE PRODUCT GUIDE
Example Product
Catalogue #
Supplier/Manufacturer
Clear Rite-3IM
6901
Richard Allen Medical Industries
8850 M89 Box 351
Richland, MI 49083
800-522-7270
http://www.rallansci.com.
Covergiass. 24x50 premier nonstick
Thinness: 0.13mm-0,17mm
00145-ACS
Suraipath Medical Industries, Inc.
H. 6. Box 528
Richmond, 11. 60071
800-225-3035
Davidson's Fixative
S2250
Pol> Scientific RtV: 1 > Corp.
70 Cleveland Avenue
Bay Shore, NY 11706
631-586-0400
Decalcificr: Formicai-2000*
1354
Decal Chemical Corp.
PO Box 916
Tall man, NY 10982-0916
800-428-5856
Losin Y (for H&E Stain)
Eosin-Y
Reagent Alcohol
Dcioni/.ed Waier
Glacial Acetic Acid
7111
Richard Allen Medical Industries
8850 M89 Box 351
Richland, MI 49083
800-522-7270
http:<7www rallansci.com.
Hematoxylin 2 (for H&E Stain)
Hematoxylin
Aluminum Sulfate
Sodium iodate
Ethylene Glycol
Deionized Water
Glacial Acetic Acid
7231
Richard Alien Medical Industries
8850 MS9 Box 351
Richland, Mi 49083
800-522-7270
http://wwvv.rallansci.com.
VIS-222 FenqueI'M C Tricaine
M ethanesulfonatc)
C-FINQ-UE
Argent Chemical Laboratories.
Redmond, WA 98052, USA.
Paraplast* (CSMP)
Kendall Paraplast Tissue Embedding
Medium 8889 501006
SHM8889-501006
Supplier: Laboratory Supply Co.
800-888*9004
Manufacturer:
Sherwood Serv ices AG
Tyco Healthcare Group L
15 Hampshire Street
Mansfield. MA 02048
Permount® Mounting Media
Toluene 55®o
HHT • ! "¦ «
Polymer Alpha pinene &
SP15-500
Fisher HealthCare
800-640-0640
Slide. Single Frosted, ground edge
Crystal Line Premier Brand
8105
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7241 Gabe Court
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703-330-1413
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