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Validation of the
Larval Amphibian Growth and Development Assay:
Integrated Summary Report
U.S. Environmental Protection Agency
Endocrine Disruptor Screening Program
Washington, D.C.
28 May 2013
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46 TABLE OF CONTENTS
47
48 EXECUTIVE SUMMARY 3
49 1 OBJECTIVE 5
50 2 INTRODUCTION 6
51 2.1 Endocrine Disruptor Screening Program 6
52 2.2 Validation 8
53 3 OVERVIEW 01 THE LAGDA 10
54 3.1 Summary of the LAGDA test protocol 10
55 3.2 Purpose of the test and relevance of the LAGDA 11
56 3.3 Scientific basis for the test method 11
57 4 PROTOCOL DEVELOPMENT 12
58 4.1 Rationale for test species and exposure period 12
59 4.1.1 Species selection 13
60 4.1.2 Test duration 13
61 4.2 Test details 14
62 4.3 Core Endpoints 17
63 4.4 Statistical Analyses 18
64 5 INTER-LABORATORY EVALUATION OF THE LAGDA 20
65 5.1 Overview of approach 20
66 5.2 Inter-laboratory data analysis 20
67 5.3 Summary of results 21
68 5.3.1 Prochloraz 21
69 5.3.2 4-fert-octylphenol 27
70 5.3.3 17p-trenbolone 32
71 5.3.4 Benzophenone-2 35
72 6 DISCUSSION OF THE INTER-LABORATORY STUDIES 40
73 6.1 Comparison of control performance 40
74 6.2 Effectiveness of the LAGDA 42
75 6.3 Lessons learned from the inter-laboratory studies 46
76 6.4 Conclusions 47
77 7 REFERENCES 47
78 8 APPENDICES 54
79 8.1 LAGDA Guideline 54
80 8.2 Histological Development of the Gonad in Juvenile Xenopus laevis 87
81 8.3 Histopathology Guidance for Reading LAGDA Studies 91
82 8.4 Inter-laboratory validation study results 100
83 8.4.1 Prochloraz results 100
84 8.4.2 4-tert-Octylphenol results 114
85 8.4.3 17-P Trenbolone results 121
86 8.4.4 Benzophenone-2 results 125
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EXECUTIVE SUMMARY
This Integrated Summary Report (ISR) provides the validation record of the EDSP Tier 2 Larval
Amphibian Growth and Development Assay (LAGDA) aimed at characterizing potential
disruption of the endocrine system by putative Endocrine Disrupting Chemicals.
The LAGDA protocol describes a chronic toxicity test with an amphibian species that considers
growth and development from fertilization through the early juvenile period. It also enables
measurement of a suite of other endpoints that allows for diagnostic evaluation of endocrine
disrupting chemicals or other types of developmental and reproductive toxicants. The LAGDA
is a relatively long-term assay (normally 130 days or longer) that assesses early development,
growth, and partial reproductive maturation. The test is designed to detect both endocrine and
non-endocrine mechanisms by including diagnostic endpoints specific to key endocrine
mechanisms. Endpoints evaluated during the course of the exposure include those indicative of
generalized toxicity, mortality, abnormal behavior, and growth determinations (length and
weight), as well as endpoints designed to characterize endocrine-specific modes of action
targeting estrogen, androgen or thyroid-mediated physiological processes.
Individual and inter-laboratory evaluations of the LADGA were conducted to evaluate the
practical transferability of the assay protocol and quantitative reproducibility of the results. The
inter-laboratory validation evaluated the ability of four labs to conduct and evaluate the LAGDA
assay. The following chemicals were evaluated across individual or multiple laboratories:
prochloraz (aromatase inhibitor, AR agonist), 4-/e/7-octyl phenol (ER agonist), 17-P trenbolone
(AR agonist), and benzophenone-2 (ER agonist, TPO inhibitor). Prochloraz was tested in four
labs (A, B, C, and D), and 4-/£/7-octyl phenol was tested in three labs (A, B, and C). Trenbolone
and benzophenone-2 were tested in single labs (E and A, respectively), precluding their use in
the inter-laboratory comparisons. However, these studies serve to demonstrate the
responsiveness of the LAGDA to additional modes of action.
The results of the inter-laboratory validation were mixed. For larval growth and development,
two laboratories, B and C, reported growth rates where confidence intervals were outside of one
standard deviation from the overall mean weight for at least one chemical. Further investigation
indicated that these laboratories did not closely follow the feeding regimens recommended in the
LAGDA guidance, resulting in either excessive or inadequate growth of the developing larvae.
Similarly, for juvenile growth and development, two studies had control juvenile weights more
than one standard deviation form the overall mean in both juvenile males and females but in
different directions: lab D's prochloraz study had higher mean weights whereas the lab C's
prochloraz study had lower mean weights. Laboratory D used the recommended diet
formulation but fed at a higher rate than what was recommended in the LAGDA guideline, and
laboratory C used their own diet formulation but fed at a rate similar to that recommended in the
LAGDA guideline. This diet formulation was clearly inadequate for supporting growth rates
consistent with the other studies and led to confounding effects on liver-somatic index (LSI) in
their prochloraz study. The inconsistent findings related to growth and development observed
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can likely be corrected with more thorough guidelines regarding the dietary and feeding
regimens.
The LAGDA was an effective test model. All four chemicals produced endocrine-related effects.
Of the two chemicals available for inter-laboratory comparison, prochloraz resulted in thyroid
gland pathologies consistent with a hypothyroid condition in 3 of the 4 labs, and vitellogenin
(VTG) induction and gonad/reproductive duct pathologies were noted in all 4 laboratories. The
second chemical, 4-/e/7-octyl phenol, produced thyroid gland pathologies consistent with a
hypothyroid condition and delayed development in only 1 of the 3 laboratories. However, VTG
production and mild gonad/reproductive duct pathologies were observed in all laboratory studies.
17-P Trenbolone and benzopehone-2, although only tested in single laboratories, produced
endocrine-related effects involving the thyroid gland, delayed metamorphosis, VTG production
and reproductive tract pathologies.
There were several areas of inconsistency noted among the laboratories:
• The growth and developmental differences related to feeding and diet have already been
discussed. In addition, inconsistencies in pathology findings were observed for
prochloraz. In the prochloraz studies, A and D gonad, oviduct and kidney samples were
read by a different pathologist than the B and C samples. Interestingly, the A and D
studies shared consistencies as did the B and C studies. It is not possible to definitively
demonstrate this as a cause for the observed inconsistencies. However, it does point to
the need for adherence to the pathology guidance document. Similar anomalies were
observed with liver pathology for the prochloraz studies. The A, B and D studies were
read by the same contracted pathologist whereas the C study was read by a different
contracted pathologist. It is interesting to point out that similar treatment-related liver
pathologies were reported for A, B and D whereas a different spectrum of treatment-
related pathologies were reported for C. It is worth noting that the animals in the C study
showed feeding-related reduced juvenile growth rates as compared to the other studies. It
is possible that disease issues may have factored into the apparent inconsistencies in liver
findings. Regardless, developing a pathology guidance document for both the liver and
kidney is critical.
• Three laboratories used commercially available ELISA kits specific for human or canine
T4 and were subsequently unsuccessful in quantifying T4. Although a methods for
determining serum T4 in X. laevis using high pressure liquid chromatography and
inductively-coupled plasma mass spectrometry (HPLC/ICP-MS) are available, US EPA
Mid-Continent Ecology Division (MED ) has developed an extraction method which
allowed for the use of these commercially available ELISA kits.
• Absolute control VTG levels varied considerably between laboratories with coefficient of
variation (CV) values close to, or exceeding 100%, and control means differed across
labs by up to 74-fold. Although there were concentration-dependent increases in VTG
production in response to prochloraz, 4-/£/7-octyl phenol, and benzophenone-2, VTG data
from all other studies are likely background levels and the VTG induction as a
reproductive endpoint should be interpreted in the context of overall potential magnitude
of response.
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Overall, the authors conclude that the Tier 2 LAGDA is capable of sufficiently characterizing
potential disruption of the endocrine system by putative endocrine disrupting chemicals.
However, careful attention to recommendations in the Test Guideline is necessary to ensure
consistent and reliable findings across multiple testing laboratories.
1 OBJECTIVE
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The objective of this integrated summary report (ISR) is to provide a detailed account of the
validation process for the Larval Amphibian Growth and Development Assay (LAGDA) so that
FIFRA Scientific Advisory Panel members may address the attached charge questions.
2 INTRODUCTION
Section 408(p) of the Federal Food Drug and Cosmetic Act (FFDCA) requires the
U.S. Environmental Protection Agency (US EPA) to:
develop a screening program, using appropriate validated test systems and other
scientifically relevant information, to determine whether certain substances may have an
effect in humans that is similar to an effect produced by a naturally occurring estrogen,
or other such endocrine effect as the Administrator may designate [21 U.S.C. 346a(p)].
Subsequent to passage of the Food Quality Protection Act in 1996, which amended FFDCA and
FIFRA, the EPA formed the Endocrine Disruptor Screening and Testing Advisory Committee
(EDSTAC), a committee of scientists and stakeholders that was charged by the EPA to provide
recommendations on how to implement its Endocrine Disruptor Screening Program (EDSP). The
EDSP is described in detail at the following website: http://www.epa.gov/scipoly/oscpendo/.
Upon recommendations from the EDSTAC (EDSTAC, 1998), the EPA expanded the EDSP
using the Administrator's discretionary authority to include the androgen and thyroid hormonal
systems as well as the endocrine systems of wildlife. Following broader international concerns
and the creation of similar programs in other countries, the Organization for Economic Co-
operation and Development (OECD) established the Endocrine Disrupters Testing and
Assessment (EDTA) Task Force in 1998 within its Test Guidelines Program. EDTA is charged
with developing an internationally harmonized testing strategy for the screening and testing of
endocrine disrupting chemicals, taking into account the consequences of such a testing strategy
on the development and validation of Test Guidelines, and on existing regulatory systems for
new and existing substances.
The LAGDA is one of the assays proposed as part of the EDSP Tier 2 and is the subject of this
report. This report complements the attached supporting materials and is meant to provide the
peer reviewers with the necessary information, and/or direction to necessary information, to
address the peer review charge questions. It introduces the purpose of the LAGDA and how it
fits into the EDSP, the scientific rationale for the assay, and an historical account of the
development and optimization of the assay protocol. It also synthesizes the information gained
during the validation process and addresses the advantages and limitations of this amphibian test
method based on its strengths and weaknesses, practicality, reproducibility, reliability, and
protocol transferability.
2.1 Endocrine Disruptor Screening Program
To comply with its mandate, the US EPA chartered a Federal advisory committee, the Endocrine
Disruptor Screening and Testing Advisory Committee (EDSTAC), to provide advice and
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guidance on the development of a screening and testing program. In 1998, it recommended to
the EPA a conceptual two-tiered approach that involved screening and testing chemical
compounds for effects on the estrogen (E), androgen (A), and thyroid (T) hormone axes
(collectively referred to as EAT). The ultimate goal of these assays is to provide input into
hazard identification to assess risk of adverse consequences to humans and wildlife (EDSTAC,
1998).
The US EPA submitted a proposal of the EDSP for public review and comment (FRN, 1998) as
well as peer review by a joint subcommittee of the US EPA Science Advisory Board and FIFRA
Scientific Advisory Panel (SAB-SAP, 1999). A complete description of the program proposal
can be found in the Federal Register Notice (FRN, 1998). Briefly, the EDSP proposed by
EDSTAC allows for: 1) initial sorting and prioritization of chemical compounds, 2)
identification of chemicals for further testing using a Tier 1 screening battery that includes in
vitro and in vivo mammalian, amphibian and fish assays, and 3) characterization of adverse
consequences resulting from possible endocrine disruption and establishment of dose-response
relationships for hazard identification using Tier 2 testing.
In comparison to the more refined, detailed, and definitive tests in Tier 2, EDSTAC indicated
that the in vitro and in vivo screening assays in the Tier 1 battery should:
be relatively fast and efficient;
be standardized and validated;
be more sensitive than specific to minimize false negatives without an unreasonable
rate of false positives;
be comprised of multiple endpoints that reflect as many modes of endocrine action as
possible;
have a sufficient range of taxonomic groups among test organisms represented; and
yield data that can be interpreted as either negative or positive for determining the
necessity and manner in which to conduct Tier 2 tests.
Together, the suite of Tier 1 assays would become a battery in which some endocrine axis
redundancy is incorporated (e.g., two different assays may cover some similar aspects of the
estrogenic response). This redundancy will allow for a weight-of-evidence approach, as
recommended by EDSTAC, to determine whether a chemical shall undergo further, more
definitive, testing.
The following assays, recommended by EDSTAC, have undergone validation and have been
adopted as the EDSP Tier 1 screening battery (Table 2.1-1). These assays are meant to detect
chemicals that may affect the estrogen, androgen and thyroid hormone axes through any known
modes of action.
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Table 2.1-1. Assays recommended for consideration for the Tier 1 screening battery.
Assay
Reason for Inclusion
Estrogen receptor binding or transcriptional
activation assay
An in vitro test to detect chemicals that may affect the endocrine
system by binding to the estrogen receptor.
Estrogen receptor transcriptional activation
assay
An in vitro test to detect chemicals that may affect the endocrine
system by binding to the estrogen receptor.
Androgen receptor binding assay
An in vitro test to detect chemicals that may affect the endocrine
system by binding to the androgen receptor.
In vitro steroidogenesis assay
An in vitro test to detect chemicals that interfere with the synthesis of
the sex steroid hormones
Placental Aromatase Assay
An assay to detect interference with aromatase.
Uterotrophic Assay
An in vivo assay to detect estrogenic chemicals.
Hershberger Assay
An in vivo assay to detect androgenic and anti-androgenic chemicals.
Pubertal Male
An in vivo assay to detect chemicals that act on androgen or through
the HPG axis that controls the estrogen and androgen hormone
systems. It is also enhanced to detect chemicals that interfere with
the thyroid system. This assay could in part substitute for the female
pubertal assay.
Pubertal female assay
An in vivo assay to detect chemicals that act on estrogen or through
the HPG axis that controls the estrogen and androgen hormone
systems. It is also enhanced to detect chemicals that interfere with
the thyroid system.
Amphibian metamorphosis assay
An in vivo assay for detection of chemicals that interfere with the
thyroid hormone system.
Fish screening assay
An in vivo assay for detection chemicals that interfere with the HPG
axes.
A weight of evidence (WOE) review of the Tier 1 screening battery and other scientifically
relevant information (OSRI) will be made to ascertain if a chemical is deemed sufficiently
endocrine active to prompt requirements for Tier 2 testing. The following tests, as generally
recommended by EDSTAC, are being considered as part of the EDSP Tier 2:
• Two-generation Rat Reproduction Test
• Medaka Multi-generation Test
• Japanese Quail Two-generation Test
• Mysid Two-generation Toxicity Test
• Larval Amphibian Growth and Development Assay
The Two-generation Rat Reproduction Test (OCSPP 870.3800) has already been adopted. The
remaining four Tier 2 tests are the subject of the FIFRA Scientific Review Panel for which the
LAGDA is reviewed here.
2.2 Validation
Validation has been defined as "the process by which the reliability and relevance of a test
method are evaluated for a particular use" (NIEHS, 1997; OECD, 1996).
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Reliability is defined as the reproducibility of results from an assay within and between
laboratories.
Relevance describes whether a test is meaningful and useful for a particular purpose (OECD
996). For Tier IEDSP assays, relevance can be defined as the ability of an assay to detect
chemicals with the potential to interact with the endocrine system.
Validation is generally recognized as necessary for the regulatory acceptance of new and revised
test methods, and is now an integral component of the international development and acceptance
of these methods (OECD 2005). The criteria used to guide the validation process for the
LAGDA were based on the principles of validation developed by the U.S. Interagency
Coordinating Committee for the Validation of Alternative Methods (ICCVAM) (NIEHS 1997)
and the OECD (OECD 2005). These criteria as stated by ICCVAM (NIEHS 1997) are as
follows:
1. The scientific and regulatory rationale for the test method, including a clear statement
of its proposed use, should be available.
2. The relationship of the endpoints determined by the test method to the in vivo biologic
effect and toxicity of interest must be addressed.
3. A formal detailed protocol must be provided and must be available in the public
domain. It should be sufficiently detailed to enable the user to adhere to it and should
include data analysis and decision criteria.
4. Within-test, intra-laboratory and inter-laboratory variability and how these parameters
vary with time should have been evaluated.
5. The test method's performance must have been demonstrated using a series of
reference chemicals preferably coded to exclude bias.
6. Sufficient data should be provided to permit a comparison of the performance of a
proposed substitute test to that of the test it is designed to replace.
7. The limitations of the test method must be described (e.g., metabolic capability).
8. The data should be obtained in accordance with Good Laboratory Practices (GLPs).
9. All data supporting the assessment of the validity of the test methods including the full
data set collected during the validation studies must be publicly available and,
preferably, published in an independent, peer-reviewed publication.
The US EPA has adopted these various validation criteria for the EDSP as described in
attachment A (EDSP, 2007). Although attempts have been made to thoroughly comply with all
validation criteria, the in vitro and in vivo screening assays in the Tier 1 battery are not
replacement assays (Validation Criterion No. 6). Many of them are novel assays; consequently,
large data bases do not exist as a reference to establish their predictive capacity (e.g.,
determination of false positive and false negative rates).
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In general, the US EPA is following a five-part or stage validation process outlined by
ICCVAM (NIEHS, 1997). The US EPA believes that it is essential to recognize that this process
was specifically developed for in vitro assays intended to replace in vivo assays. A rudimentary
problem confronting the US EPA is how to adapt and work with this process for rodent and
ecological in vivo assays in Tiers 1 and 2 that have no suitable in vitro substitute. Nonetheless,
the stages of the process outlined by the ICCVAM are as follows:
The first stage of the process was test development, an applied research function which
culminated in an initial protocol. As part of this phase, EPA drafted a Detailed Review
Paper (DRP) to explain the purpose of the test method, the context in which it will be
used, and the scientific bases upon which the assay's protocol, endpoints, and relevance
rest (attachment B). The DRP reviewed the scientific literature for candidate protocols
and evaluates them with respect to a number of considerations, such as whether candidate
protocols meet the assay's intended purpose, costs, and other practical considerations.
The DRP also identified the developmental status and questions related to each protocol;
the information needed to answer the questions; and, when possible, recommends an
initial protocol for the initiation of the second stage of validation, standardization and
optimization.
During the second stage, standardization and optimization, studies were performed
geared toward refining, optimizing, and standardizing the protocol, and initially assessing
protocol transferability and performance.
In the third stage, inter-laboratory validation, studies were conducted in several
independent laboratories with the refined protocol. The results of these studies were used
to determine inter-laboratory variability and to set or cross-check performance criteria.
In the fourth stage, peer review, an independent scientific review by qualified experts.
EPA has developed extensive guidance on the conduct of peer reviews because the
Agency believes that peer review is an important step in ensuring the quality of science
that underlies its regulatory decisions (US EPA, 2006).
The final stage is regulatory acceptance, adoption for regulatory use by an agency after
the other stages are completed.
3 OVERVIEW OF THE LAGDA
3.1 Summary of the LAGDA test protocol
The LAGDA protocol describes a chronic toxicity test with an amphibian species that considers
growth and development from fertilization through the early juvenile period. It also enables
measurement of a suite of other endpoints that allows for diagnostic evaluation of endocrine
disrupting chemicals (EDCs) or other types of developmental and reproductive toxicants. The
LAGDA is a relatively long-term assay (normally 130 days or longer) that assesses early
development, growth, and partial reproductive maturation.
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The LAGDA is intended to serve as a higher tier test with an amphibian for collecting definitive
concentration-response information on adverse effects suitable for use in ecological risk
assessment. Specifically, the design enables the collection of amphibian hormone-regulated
endpoint data (e.g., metamorphosis, gonadal development) and information concerning various
aspects of reproductive biology and life-stage viability.
The general experimental design entails exposing NF stage 8 (see Nieuwkoop and Faber, 1994
for staging details) Xenopus laevis embryos to four different concentrations of a test chemical
and a control until 10 weeks after the median time to NF stage 62 in the control, with one interim
sub-sample at NF stage 62. There are four replicates in each test concentration with eight
replicates for the control. Endpoints evaluated during the course of the exposure (at the interim
sub-sample and final sample at the completion of the test) include those indicative of generalized
toxicity, mortality, abnormal behavior, and growth determinations (length and weight), as well as
endpoints designed to characterize endocrine-specific modes of action targeting estrogen,
androgen or thyroid-mediated physiological processes.
3.2 Purpose of the test and relevance of the LAGDA
The LAGDA is intended to identify and characterize the adverse consequences of aquatic
exposure to substances which interfere with the normal development and growth of amphibians
through larval development and metamorphosis. It is an important assay for evaluating effects
from exposure to contaminants during the sensitive larval stage, where effects on survival and
development, including normal development of reproductive organs, may adversely affect
populations. The test is designed to detect both endocrine and non-endocrine mechanisms by
including diagnostic endpoints specific to key endocrine mechanisms. It should be noted that, to
date, no validated assay exists that serves this function for amphibians.
3.3 Scientific basis for the test method
Much of our current understanding of amphibian biology has been obtained using the laboratory
model species X. laevis. This species can be routinely cultured in the laboratory; ovulation can
be induced using human chorionic gonadotropin (hCG); and animal stocks are readily available
from commercial breeders.
Like all vertebrates, reproduction in amphibians is under the control of the hypothalamic
pituitary gonadal (HPG) axis (Kloas and Lutz, 2006). Estrogens and androgens are mediators of
this endocrine system, directing the development and physiology of sexually-dimorphic tissues.
There are three distinct phases in the life cycle of amphibians when this axis is especially active:
(1) gonadal differentiation during larval development, (2) development of secondary sex
characteristics and gonadal maturation during the juvenile phase, and (3) functional reproduction
of adults. Each of these three developmental windows are likely susceptible to endocrine
perturbation by xenobiotics ultimately leading to a loss of reproductive fitness by the organisms.
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The gonads begin development at NF stage 43, when the bipotential genital ridge first develops
(Nieuwkoop and Faber, 1994). Differentiation of the gonads begins atNF stage 52 when
primordial germ cells either migrate to medullary tissue (males) or remain in the cortical region
(females) of the developing gonads (Nieuwkoop and Faber, 1994). This process of sexual
differentiation of the gonads was first reported to be susceptible to chemical alteration in
Xenopus in the 1950s (Chang and Witschi, 1956; Gallien, 1953). Exposure of tadpoles to
estradiol during this period of gonad differentiation results in sex reversal of males that when
raised to adulthood are fully functional females (Villalpando and Merchant-Larios, 1990; Miyata
et al., 1999). Functional sex reversal of females into males is also possible and has been reported
following implantation of testis tissue in tadpoles (Mikamo and Witschi, 1963) or exposure to an
aromatase inhibitor (Olmstead et al., 2009a). Historically, toxicant effects on gonadal
differentiation have been assessed by histological examination of the gonads at metamorphosis,
and sex reversal could only be determined by analysis of sex ratios. Until recently, there had
been no means by which to directly determine the genetic sex of Xenopus. However, recent
establishment of sex-linked markers mX. laevis (Yoshimoto et al., 2008) and X. tropicalis
(Olmstead et al., 2010) makes it possible to determine genetic sex and allows for the direct
identification of sex-reversed animals.
In males, juvenile development proceeds as testosterone blood levels increase corresponding
with the development of secondary sex characteristics as well as testis development (Olmstead et
al., 2009b). In females, estradiol is produced by the ovaries resulting in the appearance of
vitellogenin in the plasma, vitellogenic oocytes in the ovary, and the development of the oviducts
(Olmstead et al., 2009b). Oviducts are female secondary sex characteristics that function in
oocyte maturation during reproduction (Wake and Dickie, 1998). Jelly coats are applied to the
outside of oocytes as they pass through the oviduct and collect in the ovisac, ready for
fertilization. Oviduct development appears to be regulated by estrogens as development
correlates with blood estradiol levels in X. laevis (Tobias et al., 1998) and X. tropicalis
(Olmstead et al., 2009b). The development of oviducts in males following exposure to
polychlorinated biphenyl compounds (PCBs) (Qin et al., 2001) and 4-/t'/7-octylphenol (Porter et
al., 2011) has been reported.
4 PROTOCOL DEVELOPMENT
4.1 Rationale for test species and exposure period
Short-term partial life cycle testing methods have been developed for amphibian species that
evaluate chemical impacts on metamorphosis (OCSPP 890.1100: Amphibian Metamorphosis
(Frog) Assay [US EPA, 2009]). However, there are no standardized tests for evaluating chronic
effects on growth and development of amphibians. Ideally a test of this nature would be
developed using a species that can be genetically sexed and would incorporate an exposure
period encompassing embryonic development through reproduction. There are several factors
that limit the current development of such a test, with the result being the proposed LAGDA as a
compromise in terms of test species selection and test duration.
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4.1.1 Species selection
Ideally, the test species should be robust in the laboratory setting, be able to be genetically sexed,
and develop to maturity in a reasonable amount of time. Several reports demonstrate the
potential for the well known laboratory model species X. laevis (Fort et al., 2004a; Pickford and
Morris, 2003; Pickford et al., 2003) and X. tropicalis (Fort et al., 2004b; Porter et al., 2011;
Olmstead et al., 2009a) as species for evaluating developmental impacts of chronic chemical
exposure. Although closely related, there are significant differences between these two species
as they relate to use in a chronic testing method.
1) Both species are widely cultured in the laboratory, although X. laevis has proven to be
considerably more robust and more amenable to routine culture in most laboratory
settings.
2) Both species can be genetically sexed due to the recent development of sex-linked
markers. The marker for X. laevis is the DMW gene which is located on the chromosome
which determines the female phenotype (Yoshimoto et al., 2008). The marker for X.
tropicalis is a female-linked polymorphism derived from one female animal raised at the
US EPA Mid-Continent Ecology Division (MED) (Olmstead et al., 2010). Unfortunately,
because of the nature of this sex-linked polymorphism, a very limited population of
animals currently exists, and this strain has proven to lack the robustness necessary for
routine testing.
3) It has been reported that X. tropicalis reach sexual maturity within 6 months of complete
metamorphosis (Hirsch et al., 2002). However, work conducted at MED has shown that
a typical time to maturation is 8 months to 1 year under laboratory conditions (Olmstead
etal., 2009b). X. laevis have a considerably longer maturation phase reaching sexual
maturity within 1.5 to 2 years.
Given that a robust strain capable of being genetically sexed does not currently exist forX.
tropicalis, X. laevis was chosen as the representative amphibian species for development of the
LAGDA.
4.1.2 Test duration
Initially the US EPA proposed the development of a testing protocol capable of addressing the
impacts of chemical exposure on development, growth and reproduction in amphibians. Over
the intervening years, the Agency has determined that, given the current state of the science, such
a protocol could not be developed. Instead the Agency has developed a protocol that addresses
many aspects of the original proposal, is scientifically sound, and is executable given the
constraints of existing resources.
Due to the lack of a standardized methodology, fecundity cannot be directly assessed in Xenopus
or any other amphibian species without the aid of exogenous hormones. Surrogate
measurements that are likely to be related to fecundity (sperm count, oocyte count) were
assessed throughout the maturation process in X. tropicalis, and it was determined that these
endpoints were highly variable (Olmstead et al., 2009b). Studies conducted with 4-tert-
octylphenol using a 40-week exposure protocol showed that conducting the exposure until sexual
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maturation did not offer additional information due to the high variability of these surrogate
reproductive endpoints (Porter et al., 2011). Evidence indicates that X. laevis would not differ
greatly from X tropicalis in this regard. Further, given the selection of X laevis as the most
viable test species, the exposure period necessary to encompass sexual maturation would need to
be in excess of 78 weeks. The cost and effort required to conduct an assay of this length would
be exceptionally high. Another important reality of aquatic testing is that longer assays run
greater risks of catastrophic failures (e.g., disease, exposure system mechanical failures, etc.).
Since it is impractical to conduct the exposure up to sexual maturation, it was decided that the
exposure would continue from NF stage 8 until significant hallmarks of gonad development are
achieved. Toward this end, MED scientists have conducted studies to determine when the
gonads show an appropriate level of maturation to allow identification of abnormalities in
development of reproductive organs. From these studies it was determined that, by 8 weeks
post-metamorphosis, nearly complete maturation of oocytes and spermatocytes had occurred,
males had developed seminiferous tubules and associated ducts, the female oviduct system was
developing, and supporting cells and tissues such as granulosa cells, Sertoli and Leydig cells
were present (Appendix 8.2). Based on these findings, the US EPA designed a protocol for an
exposure period which is initiated shortly after fertilization and continues through 8 weeks post-
metamorphosis.
4.2 Test details
The general experimental design entails exposing de-jellied NF stage 8 X laevis embryos to four
different concentrations of a test substance along with a control until 10 weeks after the median
time to NF stage 62 (approximately two weeks before completion of metamorphosis in the
control) with one interim sub-sample at NF stage 62. There are four replicates in each test
concentration with eight replicates for the control. Endpoints evaluated during the course of the
exposure include those indicative of generalized toxicity, mortality, abnormal behavior, and
growth determinations (length and weight), as well as endpoints designed to characterize
endocrine-specific modes of action targeting estrogen, androgen or thyroid-mediated
physiological processes. An assessment of the gonads two months post-metamorphosis is
expected to yield histological changes indicative of alterations in differentiation or delayed
development. The test details and performance criteria are summarized in Tables 4.2-1 and 4.2-2.
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Table 4.2-1. Summary of method
Test animal
Xenopus laevis
Initial larval stage
Nieuwkoop and Faber (NF) stage 8
Exposure period
Until 10 weeks after the median time to NF stage 62 in
the control
Test concentrations
Minimum of 4 different concentrations
Dilution water / laboratory control
Any water that permits normal growth and development
ofX. laevis (e.g., spring water or charcoal-filtered tap
water)
Replication
4 replicate test vessels / test concentration
8 replicate test vessels / control
Exposure regime
Flow-through
Test system flow-rate
Constant, in consideration of both the maintenance of
biological conditions and chemical exposure (e.g., >10
tank turnovers/day)
Test solution / test vessel
4 - 10 L (10 - 15 cm minimum water depth) / glass or
stainless steel
Initial larval density
20 larvae / test vessel
Larval cull to adjust density after NF
stage 66
10 juveniles / test vessel(determined randomly)
Feeding
See LAGDA Appendix 2
Lighting
Photoperiod
12 hr light: 12 hr dark
Intensity
600 to 2000 lux (lumens/m2) at the water surface
Water temperature
21°± 1°C
pH
6.5 to 8.5
Dissolved oxygen (DO) concentration
>3.5 mg/L (>40% of the air saturation)
Analytical chemistry schedule
Initiation (day 0), and weekly thereafter for each
replicate tank at each concentration
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Table 4.2-2. Performance criteria for the LAG
DA.
Criterion
Acceptable limits
Test concentrations
Maintained at <20% CV(variability of measured
test concentration) over the entire test
Mortality in controls
<20% mortality in any one replicate in the controls
Development in controls
The mean time to NF stage 62 is < 45 days, and the
mean weight at NF stage 62 is 1.0 ± 0.2 g. The
mean weight at test termination is 11.5 3 g.
Dissolved Oxygen
>40% air saturation
pH
pH should be maintained between 6.5-8.5. The inter-
replicate/inter-treatment differentials should not
exceed 0.5.
Water temperature
Target temperature ± 1"C - the inter-replicate/inter-
treatment differentials should not exceed 1.0 °C
Test concentrations without overt toxicity
>2
Replicate performance
< 2 replicates across the test can be compromised
Special conditions for use of a solvent
If a carrier solvent is used, both a solvent control
and clean water control must be used.
Statistically significant differences between solvent
control and clean water control groups are treated
specially. See LAGDA guideline for more
information.
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4.3 Core Endpoints
The general experimental design entails exposing de-jellied NF stage 8 X. laevis embryos to four
different concentrations of a test substance and a control until 10 weeks after the median time to
NF stage 62 in the control, with one interim sub-sample at NF stage 62. Table 4.3-1 summarizes
the endpoints and timing of tissue/sample collection.
Table 4.3-1 Summary of endpoint and collection stages
Apical Endpoints
Daily
Interim
Sampling
Test
Termination
Mortality
X
Clinical signs of disease and/or toxicities
X
Growth
X
X
Time to NF stage 62
X
Thyroid histology
X
Serum T4 (thyroxine)
X
Vitellogenin
X
Genetic/histological sex comparison
X
Gonad histology
X
Liver histology
X
Liver-somatic index
X
Kidney histology
X
Growth and overt toxicity endpoints
Routine toxicity test endpoints of mortality, clinical signs of disease and/or toxicity, and growth
are measured. In addition, liver and kidney histopathology and liver-somatic index (LSI) have
been included as measures of overt toxicity to the organism. Given the importance of the liver in
metabolism and the kidney in clearance of xenobiotics, these tissues are often targets of overt
toxicity associated with a chemical. Overt toxicity can confound and confuse the interpretation
of endocrine studies if it is not clearly understood, so inclusion of these endpoints in the LAGDA
will ultimately assist in interpreting future data sets.
Thyroid endpoints
Three endpoints have been included which are intended to identify impacts on the thyroid axis:
time to NF stage 62, thyroid histology, and serum thyroxine (T4) measures. In the AMA, the rate
of development is recorded as an indication of impacts on the thyroid axis. In contrast to the
AMA, which determines median stage achieved in a given amount of time (21 days), the
LAGDA measures the time it takes for each individual to reach NF stage 62, a stage referred to
as metamorphic climax. However, this measure should be interpreted with caution because non-
specific toxicity experienced prior to metamorphosis may impact the length of time it takes to
reach NF stage 62. Thyroid histology, the hallmark for diagnosing thyroid disruption, is a focal
endpoint in the AMA and has been validated extensively (Grim et al., 2009). Serum T4 has also
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been included as an endpoint for characterizing thyroid axis disruption in the LAGDA. The
robustness of the diagnosis for thyroid impacts is enhanced with this three-pronged approach.
Reproductive endpoints
Vitellogenin
Vitellogenin (VTG) is a widely accepted biomarker of estrogenic chemicals. The measure is
made using enzyme-linked immunosorbent assay (ELISA) methodology. Currently there are no
commercially available antibodies. However, given the wealth of information for this protein
and the availability of cost-effective commercial antibody production services, it is reasonable
that laboratories can easily develop an ELISA to make this measure (Olmlstead et al., 2009b).
Details are provided in LAGDA Appendix 4.
Gonad Histology
Gonad histology has been a standard measure for the EDSP Tier 1 Fish Short Term
Reproduction Assay which exposes fathead minnows for 21 days. The value of histological
analysis has been reviewed extensively, and a detailed internationally harmonized guidance
document is available for fish (Johnson et al., 2009). This fish guidance has served as the basis
for development of a LAGDA histopathology guidance document (Appendix 8.3).
Genetic Sex
DMW is a gene that has been shown to be linked to the female phenotype (Yoshimoto et al.,
2008). The presence of this gene (marker) indicates that the individual is genetically female.
The presence or absence of the marker when compared back to the histological determination of
sex can be used to identify if sex reversal has occurred (LAGDA Appendix 3).
4.4 Statistical Analyses
The LAGDA generates two forms of data: 1) quantitative continuous data including growth and
biochemical concentrations, and 2) ordinal data in the form of severity scores or developmental
stages from histopathology evaluations. Data are typically collected in spreadsheets and then
transferred to and analyzed with a statistical software package. Statistical analyses of the data
should preferably follow procedures described in the document Current Approaches in the
Statistical Analysis of Ecotoxicity Data: A Guidance to Application (OECD, 2006). Data for
continuous endpoints should be assessed for normality (preferably using the Shapiro-Wilk or
Anderson-Darling test) and variance homogeneity (preferably using Levene's 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
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heterogeneous variance, a significant 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
independently of any ANOVA F-test, and the Mann-Whitney test is applied independently of
any overall Kruskall-Wallis test. In addition, regardless of the results of the previous tests, a
step-down Jonckheere-Terpstra (JT) trend test should also be applied to the data as long as a
monotonic response was observed.
Mortality data should be analyzed for time periods encompassing larval development and
juvenile development. Tadpoles that do not complete metamorphosis in the given time frame,
those tadpoles that are in the larval sub-sample cohort, those juvenile frogs that are culled, and
any animal that dies due to experimenter error should be treated as censured data. Mortality data
expressed as the percentage that died should be arcsine square root transformed prior to analysis.
Time to metamorphosis data should be treated as time to event data, with any mortalities treated
as censured data. Median time to completion of metamorphosis should be determined by
Kaplan-Meier product-limit estimators and used in the statistical analysis.
Body weight and snout-vent lengths should be analyzed using the means of a given tank. Males
and females are not sexually-dimorphic at the completion of metamorphosis, and data for the
sexes are combined when analyzing larval sub-sampling data. Male and female juvenile body
size data should be analyzed separately.
Liver weights should be expressed as a proportion of whole body weight (LSI), arcsine square
root transformed, and analyzed separately for each sex.
Histopathology data is in the form of severity scores or developmental stages. A new test termed
RSCABS (Rao-Scott Cochran-Armitage by Slices) uses a step-down Rao-Scott adjusted
Cochran-Armitage trend test on each level of severity in a histopathology response. The Rao-
Scott adjustment incorporates the replicate vessel experimental design into the test. The "by
slices" procedure incorporates the biological expectation that severity of effect tends to increase
with increasing doses or concentrations, while retaining the individual subject scores and
revealing the severity of any effect found. The RSCABS procedure not only determines which
treatments are statistically different, that is, have more severe pathology than controls, but it also
determines at which severity score the difference occurs providing much needed context to the
analysis. Currently, this test can only be implemented by StatCHARRMS (explained below)
which will soon be made freely available as an open-source R package. A detailed description of
this test can also be found in the EDSP Tier 2 Medaka Multi-generation Test (MMT) Integrated
Summary Report (ISR).
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5 INTER-LABORATORY EVALUATION OF THE LAGDA
5.1 Overview of approach
Inter-laboratory evaluations of the LADGA were conducted to evaluate the practical
transferability of the assay protocol and quantitative reproducibility of the results. The following
laboratories participated in the inter-laboratory validation study: US EPA MED; U.S. Army
Center for Environmental Health Research (ACEHR) in Fort Detrick, MD; IDEA Consultants,
Inc., a Japanese environmental research facility that is contracted to the Japanese government;
ABC Laboratories, Inc. in Columbus, MO; and, Fort Environmental Laboratories (FEL) in
Stillwater, OK. The latter two labs were sub-contractors to the US EPA contractor Battelle
Memorial Institute, a global research and development organization. Four known endocrine
active chemicals were selected for testing: prochloraz, 4-fert-octylphenol, 17-/? trenbolone and
benzophenone-2. Table 5.1-1 summarizes which chemicals were tested in each participating
laboratory. The participating laboratories were provided a draft protocol to follow. This
protocol, in its revised form, can be found in Appendix 8.1. In some cases, minor changes were
made, and any deviations that occurred are pointed out in the section below.
Table 5.1-1 Out
ine of inter-laboratory participation
Prochloraz
4-terf-octylphenol
17-P trenbolone
Benzophenone-2
Laboratory
(aromatase inhibitor;
AR antagonist)
(ER agonist)
(AR agonist)
(ER agonist; TPO inhibitor)
A
X
X
X
B
X
X
C
X
X
D
X
E
X
5.2 Inter-laboratory data analysis
Following the studies, data were provided to MED for analysis using StatCHARRMS (Statistical
analysis of Chemistry, Histopathology, And Reproduction endpoints including Repeated
measures and Multi-generation Studies), a SAS® (SAS Institute, Cary, NC)-based program
developed for MED by John Green (Dupont Applied Statistics Group, Newark, DE). For
continuous data measured once per subject, normality (via Shapiro-Wilk test) and variance
homogeneity (via Levene's test) were assessed, and appropriate transforms were applied by the
program to determine the appropriate statistic for hypothesis testing. In addition, regardless of
the results of those two tests, a step-down Jonckheere-Terpstra (JT) trend test was also applied to
the data as long as a monotonic response was observed. For consistency across all LAGDA
validation studies, the statistical test that indicated the lowest concentration significantly
different than control was reported. Table 5.2-1 summarizes the statistical tests used by
StatCHARRMS, while a thorough description of the StatCHARRMS package including
statistical rationale can be found in the EDSP Tier 2 MMT ISR.
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Table 5.2-1 StatCHARRMS decision logic for statistical tests on continuous data.
Shapiro-Wilk
Levene's
Statistical test
Pass
Pass
Dunnett; Jonckheere-Terpstra
Fail
Pass
Modified Dunn's; Jonckheere-Terpstra
Pass
Fail
Tamhane-Dunnett; Jonckheere-Terp stra
Fail
Fail
J onckheere-T erp stra
Histopathology data were analyzed using the RSCABS test explained above implemented in
StatCHARRMS. As with the continuous data, for consistency across all LAGDA validation
studies, the lowest concentration exhibiting a pathology that was significantly different than
control was reported.
5.3 Summary of results
5.3.1 Prochloraz
Introduction
Prochloraz is an imidazole fungicide shown to have multiple mechanisms of action within the
HPG axis. In vitro studies have shown that prochloraz has characteristics of both an antagonist
of the androgen receptor and inhibitor of aromatase activity (Noriega et al., 2005; Villeneuve et
al., 2007). In adult fish, in vivo exposure to prochloraz produces effects that are consistent with
inhibition of aromatase (Ankley et al., 2005). Further, developmental exposure in rats produces
a spectrum of male reproductive tract malformations which have been attributed to anti-
androgenic properties (Noriega et al., 2005; Blystone et al., 2001). Interestingly, females were
not affected by developmental exposure in the aforementioned two studies.
Results
Exposure levels and measured test concentrations
The high test concentration was set based on range finding experiments conducted by A. In
those studies conducted with X. tropicalis, concentrations in excess of 200 |ig/L resulted in
mortality in early larval organisms. The high test concentration for the inter-laboratory
prochloraz validation study was 180 |ig/L with a 0.33 dilution factor resulting in nominal test
concentrations of 180, 60, 20 and 6.7 |ig/L. All four labs met the criteria of maintaining
coefficients of variation (CVs) of < 20% over the entire test for all concentrations with the
exception of the 6.67 and 20 |ig/L nominal concentrations at C which had CVs of 38 and 23%
and were on average 84 and 25% above nominal, respectively (Appendix 8.4.1, Table 8.4.1-1).
Measured concentrations did not show any crossover among treatment levels in all labs except C
which had several overlaps in treatment concentrations through week five of exposure. CV
values between replicates could not be calculated on a weekly basis for B and C due to
measurements of only one replicate tank per week. Treatment levels are referred to as nominal
concentrations regardless of mean-measured concentrations reported by each of the labs.
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Measure of General Toxicity
The first prochloraz study was performed by lab A in the initial stages of protocol development.
The interim sampling to evaluate endpoints related to larval development and potential thyroid
active agents consisted of sample collection at NF stage 66, or at the completion of
metamorphosis. Following completion of the prochloraz study at lab A, it was decided that
thyroid related endpoints were better assessed at NF stage 62 due to maximal constitutive levels
of circulating thyroxine (T4) occurring at this stage. Therefore, interim assessments occurred at
NF stage 66 for lab A while the other laboratories performed interim assessments at NF stage 62
Larval and Juvenile Growth
Summary of inter-laboratory prochloraz growth endpoints.
All endpoints with lowest concentration statistically different than control. Concentrations listed as nominal
regardless of mean-measured concentrations. Dash: Not significant.
Lab
Larval sample
Weight
Length
20 |ig/L
180 jj.g/L
180 |ig/L
180 jj.g/L
60 |ig/L*
Juvenile Males
Weight
Length
LSI
60 |ig/L
60 |ig/L
60 jj.g/L
6.7 ng/L
180 |ig/L
Juvenile Females
Weight
Length
LSI
60 |ig/L
180 |ig/L
180 jj.g/L
20 i-ig/L
20 |ig/L
20 i-ig/L
60 |ig/L*
180 i-ig/L
* Non-monotonic (i.e., the only treatment significantly different than control)
Several measures often associated with general toxicity have been incorporated into the test
protocol. They include mortality, measures of growth (length and weight), LSI, and liver and
kidney pathology. The responses of these endpoints were generally consistent across the four
studies. When considered together these results demonstrate that prochloraz, at the higher end of
the concentration range, was overtly toxic to the developing organism (Appendix 8.4.1, Table
8.4.1-5).
Two laboratories (B, D) demonstrated impacts on growth in the larval and juvenile organisms in
a concentration-dependent fashion; the other two did not. It is noteworthy that control length and
weight were greatest in these two studies that showed growth effects (Appendix 8.4.1, Tables
8.4.1-3 and 8.4.1-5).
Three laboratories (A, D and B) showed significant decreases in both juvenile male and female
LSI (Appendix 8.4.1, Table 8.4.1-5). This is consistent with the liver histopathology findings
which indicated significant pathologies produced by high concentrations of prochloraz for these
three laboratories (described below). In contrast, females in the C study showed an increase in
LSI at the highest test concentration. The increase in LSI at C may be due to an overall lack of
juvenile growth across the entire study performed by C. Further, increased liver weight relative
to body weight is reasonable given the unique spectrum of liver pathologies in this laboratory.
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Liver and Kidney pathology
Summary of inter-laboratory prochloraz liver and kidney pathology.
All endpoints with lowest concentration statistically different than control. Concentrations listed as nominal
regardless of mean-measured concentrations.
Lab A D B C
Juvenile Males
Liver pathology 60 j_ig/L 20 jj.g/L 20 jj.g/L 20 jj.g/L
Kidney pathology 180 jj.g/L 180 jag/L 6.7 jag/L 20 jag/L
Juvenile Females
Liver pathology
Kidney pathology
60 |ig/L
60 ug/L
20 |ig/L
60 ug/L
60 |ig/L
6.7 ug/L
20 |ig/L
20 ug/L
Consistencies in treatment-related findings were observed for hepatocellular degeneration and
hepatocellular atrophy in three of the four labs (Appendix 8.4.1, Tables 8.4.1-7 and 8.4.1-8). A
and B showed significant hepatocellular degeneration in the 180 |ig/L treatment in both male and
female organisms. Hepatocellular atrophy was observed in the D and B studies at the 20 |ig/L
treatment level and above in both male and female organisms.
The C study exhibited a different suite of treatment-related liver pathologies including basophilia,
increased hepatocellular vacuolization, and granulomatous inflammation, all of which were
significantly different than the control at 60 and 180 |ig/L (Appendix 8.4.1, Tables 8.4.1-7 and
8.4.1-8). Cellular hypertrophy was also observed in C liver samples and showed a significant
concentration-dependent effect. Prevalence, severity, sensitivity and pathologies were similar
between males and females with slight variation in severity and sensitivity. The stark difference
between findings at C compared to the other three labs is difficult to explain. It cannot be ruled
out that these organisms were impacted by an infectious disease. This would be consistent with
the observation that control organisms failed to thrive in terms of growth.
The most consistent treatment-related finding in the juvenile kidney was tubular amphophilic
intracytoplasmic inclusions, which were significantly present in the 180 |ig/L treatment at A, D
and C (Appendix 8.4.1, Tables 8.4.1-13 and 8.4.1-14). This response was consistent between
males and females. Other pathologies were observed but lacked consistency between studies and
often were not treatment related.
Thyroid Endpoints
Summary of inter-laboratory prochloraz thyroid endpoints.
All endpoints with lowest concentration statistically different than control. Concentrations listed as nominal
regardless of mean-measured concentrations. Dash: Not significant.
Lab A D B C
Thyroid endpoints
Time to NF stage 62 - 20 |ig/L*
Serum T4 60 j-ig/L - - 6.7 j-ig/L
Thyroid pathology 180 jJ-g/L 20 jJ-g/L - 6.7 ju.g/L
* Non-monotonic (i.e., the only treatment significantly different than control)
Time to NF stage 62
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880
881
882
883
884
885
886
887
888
889
890
891
892
893
894
895
896
897
898
899
900
901
902
903
904
905
906
907
908
909
910
911
912
913
914
915
24
There were no treatment-related effects on time to NF stage 62 (NF stage 66 in the case of A) in
all four participating laboratories (Appendix 8.4.1, Tables 8.4.1-2 and 8.4.1-3).
Larval Thyroid Pathology
A, D, and C studies showed mild but significant treatment-related effects in the thyroid gland
consistent with compensation commonly observed following thyroid axis disruption including
follicular cell hypertrophy and hyperplasia (Appendix 8.4.1, Table 8.4.1-6). Although these
pathologies were present in larvae at B, there were no clear concentration-dependent trends in
their study. This was an unexpected finding for prochloraz as there are no published reports of
thyroid axis disruption.
Larval blood thyroxine
The D, B and C laboratories used commercially available T4ELISA kits with specific antibodies
for human or canine T4. These laboratories failed to optimize these kits for use with X laevis
and consequently were unsuccessful in making these measurements; therefore, these data are not
included in this analysis. Laboratory A measured T4 levels in serum using a high pressure liquid
chromatography and inductively-coupled plasma mass spectrometry (HPLC/ICP-MS) method,
which has been previously optimized for use with X laevis (Tietge et al., 2010; Tietge el al.,
2012). In the A study, a significant increase in T4 was observed in the 60 and 180 |ig/L
treatments (Appendix 8.4.1, Table 8.4.1-4). It is important to note that the few days prior to NF
stage 66 are marked by a rapid and appreciable decrease in serum T4 levels. This, when
considered with the liver pathology findings, may suggest that compromised liver function may
have resulted in decreased metabolism and elimination of T4.
Reproductive Endpoints
Summary of inter-laboratory prochloraz reproductive endpoints.
All endpoints with lowest concentration statistically different than control. Concentrations listed as nominal
regardless of mean-measured concentrations. Dash: Not significant.
Lab A D B C
Juvenile Males
Plasma VTG 20 jj.g/L
Gonad pathology 6.7 j_ig/L
Duct pathology 20 j_ig/L
180 jJ-g/L - 6.7 ng/L
6.7 |ag/L 6.7 |ag/L 20 |ag/L
60 |ag/L 20 |ag/L 20 |ag/L
Juvenile Females
Plasma VTG 6.7 ng/L
Gonad pathology 180 j_ig/L
Duct pathology 180 |_ig/L
60 |-ig/L - 20 |-ig/L
60 |ag/L 20 |ag/L 6.7 |ag/L
- 180 i-ig/L -
Juvenile plasma vitellogenin
A, D and C showed significant increases in both juvenile male and female plasma vitellogenin
titers (Appendix 8.4.1, Table 8.4.1-5). B showed no significant difference between control and
treated organisms but also reported titer values that were much lower on average than the other
three laboratories. Although the VTG ELISA registered a measurable signal in control males
-------
916
917
918
919
920
921
922
923
924
925
926
927
928
929
930
931
932
933
934
935
936
937
938
939
940
941
942
943
944
945
946
947
948
949
950
951
952
953
954
955
25
and females in all labs, VTG production has been shown not to begin until later in development
(Olmstead, 2009b). Therefore, these control values likely reflect background noise within the
assay.
Gonad Histopathology
As with the liver pathology, two different contract pathologists were responsible for reading the
various studies. In terms of the testis and ovary, there appears to be some inconsistencies in the
results across the laboratories. However, this may be more a result of the differences in sample
processing and reading rather than a true difference in results among the four laboratories. Labs
A and D studies were processed and read by Integrated Laboratory Systems, Research Triangle
Park, NC whereas Labs B and C studies were processed and read by Experimental Pathology
Laboratory, Sterling, VA. In both the testis and ovary, the A and D studies showed many
consistencies in diagnosis whereas C and B share consistencies. In all cases, the genetic sex
matched the phenotype determined by the pathologist and indicated that treatment did not result
in sex reversal.
Testis
Of the ten observed pathologies showing significant concentration-dependent trends in at least
one of the four studies, several important findings showed a consistent treatment-related
response across the studies. A treatment-related increase in germ cell degeneration was observed
across all studies and showed significance in the A, B and C studies (Appendix 8.4.1, Table
8.4.1-9). Germ cell degeneration in the testis was characterized by the presence of apoptotic
germ cells (either as a single cell or small cluster of cells) or germ cell syncytia. Germinal
epithelium thinning was observed in the A, C and D studies, and its prevalence and severity
increased in a concentration-dependent fashion. Germinal epithelial thinning presented as
scattered enlargement of seminiferous tubule lumina, with irregular attenuation of the germinal
epithelium surrounding affected tubules (Figure 5.2-1). A significant increase in spermatogonia
was also observed in these same studies, and the pathologist noted that, in those cases with
moderate germinal epithelial thinning /loss, the increase in spermatogonia may reflect a relative
increase due to the loss of spermatocytes/spermatids rather than an absolute increase in numbers.
The degenerative testicular changes observed in these studies are consistent with the anti-
androgenic effects of prochloraz reported in species as divergent as fathead minnows (Ankley et
al., 2005) and rats (Blystone el al, 2007). In particular, germinal epithelium thinning/loss with
high doses of prochloraz has been described in rats following exposure during gonadal
development (Noriega et al., 2005).
-------
956
957
958
959
960
961
962
963
964
965
966
967
968
969
970
971
972
973
974
975
976
977
978
979
980
26
Figure 5.2-1 Control group male from prochloraz study, providedfor reference, 10X. B)
Prochloraz 180 ug L, treatment group male, I OX. Gonadal degeneration thinning/ loss grade 2.
The germinal epithelium lining of numerous spermatic tubules is thin to absent (black arrows)
with occasional exposure of the underlying basement membrane (red arrow). C) 180 fig/L
treatment group male, 10X. Gonadal degeneration thinning/loss, Grade 4. Diffusely, the
germinal epithelium is missing or thin with frequent exposure of the underlying basement
membranes (black arrows).
Ovary
Histopathological findings in the ovaries of prochloraz-exposed female frogs were inconsistent
and minimal (Appendix 8.4.1, Table 8.4.1-10). This finding is consistent with those findings in
rats in which only male gonads are affected following developmental exposure to prochloraz
(Noriega et al., 2005).
Reproductive Ducts
There was a very consistent response to prochloraz treatment across all four labs in oviduct
development (Appendix 8.4.1, Tables 8.4.1-11 and 8.4.1-12). In males, significant
concentration-dependent delays in oviduct involution were evident in all studies indicating
interference with normal oviduct regression (Kelley, 1996), a finding similar to the feminization
of the secondary sexual characteristics of male rats during perinatal prochloraz exposure
(Noriega et al., 2005; Vingaard et al, 2005). Female oviduct development was not affected by
treatment across all four studies. However, female Wolffian duct development was slightly
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981
982
983
984
985
986
987
988
989
990
991
992
993
994
995
996
997
998
999
1000
1001
1002
1003
1004
1005
1006
1007
1008
1009
1010
1011
1012
1013
1014
1015
1016
1017
1018
1019
1020
1021
1022
1023
27
advanced in the highest treatment at B, and minimal mononuclear inflammation of the Wolffian
duct was significantly present in the highest treatment at A.
5.3.2 4-ferf-octylphenol
Introduction
The weak estrogen, 4-/e/7-octyl phenol (OP), (Bonefeld-Jorgensen el al., 2007), is a degradation
product of octylphenol polyethoxylate used as non-ionic surfactants in industrial and household
settings (Ying el al., 2002). Vitellogenin has been shown to be elevated in male zebrafish (Ortiz-
Zarragoitia and Cajaraville, 2005), male eelpout (Rasmussen el al., 2005), male and female
medaka (Seki etal., 2003), and mX. laevis primary cultured hepatocytes (Mitsui et al., 2007)
exposed to OP. Decreased sperm count and the presence of testis-ova have been observed in
medaka exposed to OP (Seki etal. 2003); decreased gonadosomatic index and increased testis-
ova have been reported in eelpout exposed to OP (Rasmussen et al., 2005); and decreased
spermatozoa and increased testis anomalies have been documented in sheepshead minnows
exposed to OP (Karels et al., 2003). Female minnows exposed to OP have been shown to
produce a decreased percent of viable eggs (Karels et al., 2003). Kloas et al. (1999) reported
that exposure of larval X. laevis to OP resulted in a significant shift in sex ratio towards females.
These finding could not be reproduced in studies conducted with X. tropicalis (Porter etal.,
2011). The only significant findings in the Porter et al. (2011) studies were induction of VTG
and oviduct development in adult males. Based on these previously published studies, OP was
used as a representative weak estrogen for testing the transferability of the LAGDA.
Results
Exposure levels and measured test concentrations
Test concentrations were set based on the findings of Porter et al. (2011) and a short term
embryo/larval exposure conducted at MED. In these studies, concentrations above 60 |ig/L
resulted in significant mortality in embryos and early stage larvae. For this inter-laboratory
study, the high test concentration was set at 50 |ig/L with a 0.5 dilution factor resulting in
nominal test concentrations of 50, 25, 12.5 and 6.25 |ig/L. All test concentrations at A were
maintained with CVs < 20% (Appendix 8.4.2, Table 8.4.2-1). B maintained test concentrations
with CVs < 20% except for the lowest concentration. C had CVs < 20% in two of the four
concentrations and had measurable amounts of test chemical in control water on three different
occasions. Weekly CV values could not be calculated for B and C due to only one replicate tank
being measured per week. Lab C's concentrations were on average higher than nominal with the
lowest concentration >140% of nominal. Lab B's concentrations were on average lower than
nominal with all mean-measured concentrations being between 73 and 77% of nominal.
Measure of General Toxicity
-------
1024
1025
1026
1027
1028
1029
1030
1031
1032
1033
1034
1035
1036
1037
1038
1039
1040
1041
1042
1043
1044
1045
1046
1047
28
Mortality, measures of growth (length and weight), liver somatic index, and liver and kidney
pathology were generally consistent across the four studies. When considered together, results
demonstrate that OP had little impact on the general health of the organisms.
Survival
All labs met the criteria of < 20% mortality in any one control replicate that could not be
explained by technical error, and no treatment-related trends in survival were identified. Lab A
experienced technical difficulties (drain obstruction and tank overflow) late in the study and lost
one 12.5 |ig/L replicate and two 50 |ig/L replicates following the post-NF stage 66 cull (data not
shown).
Larval and Juvenile Growth
Summary of inter-laboratory octylphenol growth endpoints.
All endpoints with lowest concentration statistically different than control.
Concentrations listed as nominal regardless of mean-measured concentrations. Dash:
Not significant.
Lab A B
Larval sample
Weight
Length 6.25 |ag/L* 50 jag/L
Juvenile Males
Weight
Length
LSI
Juvenile Females
Weight
Length
LSI - -
* Non-monotonic (i.e. the only treatment significantly different than control)
There were no treatment-related effects on larval or juvenile growth in any of the three studies.
Juvenile LSI was significantly decreased in octylphenol-exposed males and females from C
whereas no treatment-related effects on LSI were measured at A or B (Appendix 8.4.2 Tables
8.4.2-2 and 8.4.2-4). However, it is again worth noting that lab C organisms were smaller on
average than the other labs and it is uncertain whether reduced growth factored into this finding.
Liver and Kidney pathology
Summary of inter-laboratory octylphenol liver and kidney pathology.
All endpoints with lowest concentration statistically different than control.
Concentrations listed as nominal regardless of mean-measured concentrations. Dash:
Not significant.
Lab A B C
Juvenile Males
Liver pathology ...
Kidney pathology - 25 j_ig/L
Juvenile Females
Liver pathology ...
Kidney pathology 50 j-ig/L 50 j-ig/L 6.25 j_ig/L
50 j_ig/L
12.5 |^g/L*
25 ug/L
-------
1048
1049
1050
1051
1052
1053
1054
1055
1056
1057
1058
1059
1060
1061
1062
1063
1064
1065
1066
1067
1068
1069
1070
1071
1072
1073
1074
1075
1076
1077
1078
1079
1080
1081
1082
1083
1084
1085
1086
1087
29
No treatment-related effects were present in any of the octylphenol-exposed livers from all three
studies. Granulomatous inflammation was observed in all three studies, but prevalence and
severity were comparable among treated and control samples. Cystic degeneration was observed
in one of the three studies (B), but prevalence and severity were also comparable among treated
and control samples (data not shown). These results are suggestive of response to an infectious
disease.
Male kidney tubule dilation and mineralization were observed throughout all three studies
(Appendix 8.4.2, Table 8.4.2-10). Prevalence and severity were comparable between treatment
and control samples in most cases. However, these pathologies were significantly attenuated
with octylphenol exposure at B. Minimal interstitial proteinaceous fluid was observed at B but
was essentially not remarkable.
Female kidney tubule dilation and mineralization were observed throughout all three studies
(Appendix 8.4.2, Table 8.4.2-11). Lab C male kidneys exhibited a similar pattern to B male
kidneys where these pathologies were attenuated with octylphenol treatment. Lab A showed a
significant, but slight increase in severity of tubule dilation in the 50 |ig/L treatment which was
inconsistent with the findings in the other studies. Lab B females did not show significant trends
in these pathologies but showed significant minimal to mild severity of edema, although
prevalence was very low.
Thyroid Endpoints
Summary of inter-laboratory octylphenol thyroid endpoints.
All endpoints with lowest concentration statistically different than control.
Concentrations listed as nominal regardless of mean-measured concentrations. Dash:
Not significant.
Lab A B C
Thyroid endpoints
Time to NF stage 62 50 jj.g/L
Serum T4 25 |ag/L
Thyroid pathology 12.5 |_ig/L - -
Time to NF stage 62
There was a significant delay in time to NF stage 62 in the A study at the highest test
concentration but not in studies conducted by B and C (Appendix 8.4.2, Table 8.4.2-2).
Larval thyroid pathology
In the B and C studies, there were no significant impacts on thyroid glands. The only
statistically significant findings were increases in follicular cell hypertrophy and hyperplasia in
the A study attest concentrations > 12.5 and 50 |ig/L, respectively (Appendix 8.4.2, Table 8.4.2-
5).
Larval blood thyroxine
-------
30
1088
1089
1090
1091
1092
1093
1094
1095
1096
1097
1098
1099
1100
1101
1102
1103
1104
As indicted above, failure to optimize the ELIS A kits by B and C resulted in no reliable T4
measurement data. Lab A elected to develop an extraction method which allowed for the use of
these commercially available ELISA kits (LAGDA Guideline Appendix 7) withX laevis serum.
Lab A also measured T4 levels in serum using an HPLC/ICP-MS method (Appendix 8.4.2, Table
8.4.2-3). OP exposure resulted in a concentration-dependent decrease in T4 (Figure 5.2.2-1).
The two methods were in agreement in terms of control levels and effects of OP.
con 6.25 12.5 25
Nominal 4-ferf-octylphenol concentration (ug/L)
Figure 5.2.2-1. The top panel (A) shows the results for the ELISA method, and the bottom panel
(B) shows results for the ICP-MS method. The detection limits were 2.5 ng ml and 0.15 ng ml for
the ELISA and ICP-MS methods, respectively. The measured T4 levels were consistent between
the two methods. * significant atp< 0.05
Reproductive Endpoints
Summary of inter-laboratory octylphenol reproductive endpoints.
All endpoints with lowest concentration statistically different than control.
Concentrations listed as nominal regardless of mean-measured concentrations. Dash:
Not significant.
Lab
A
Juvenile Males
Plasma VTG
Gonad pathology
Duct pathology
12.5 |ig/L
6.25 |ig/L
6.25 |ig/L
12.5 ng/L
25 |ig/L
Juvenile Females
-------
1105
1106
1107
1108
1109
1110
1111
1112
1113
1114
1115
1116
1117
1118
1119
1120
1121
1122
1123
1124
1125
1126
1127
1128
1129
1130
1131
1132
1133
1134
1135
1136
1137
1138
1139
1140
1141
1142
1143
1144
1145
1146
1147
31
Plasma VTG - 50 |ig/L 6.25 |ig/L
Gonad pathology 12.5 j_ig/L - 12.5 j_ig/L
Duct pathology 50 j_ig/L 12.5 j_ig/L 6.25 j_ig/L
Juvenile plasma vitellogenin
Vitellogenin titers showed a concentration-dependent increase in octylphenol-exposed males at
labs A and C (Appendix 8.4.2, Table 8.4.2-4). Female vitellogenin levels showed a
concentration- dependent increase at labs B and C.
Gonad histopathology
Genetic sex was determined using the DMW sex-linked marker. In the cases of laboratories B
and C, there were significant numbers of individuals in which genetic sex did not match
phenotypic sex as determined by histological analysis. There was no indication of a treatment-
related effect given that the mismatches occurred randomly throughout the control and treatment
groups. Further, both phenotypic males and females were misidentified. Upon further
investigation, it was determined that some, but not all, of the observed mismatches from
laboratory C could be explained by a labeling error that was identified and corrected. In order to
determine the nature of the remaining discrepancies, tissue samples from both lab B and C
studies were sent to MED for re-analysis of genetic sex. In all cases, the analyses performed by
MED resolved the apparent mismatching. Thus, OP did not produce sex reversal in any of the
studies.
Testis
Effects of octylphenol exposure on male gonads were minimal. Mononuclear cellular infiltrate
was observed throughout all three studies but was not treatment-related, although statistically,
severity was significantly less than controls at B (Appendix 8.4.2, Table 8.4.2-6). There were
very few observations of minimally increased spermatogonia at B and C, but the 50 |ig/L
treatment at B was significantly different than the control. Statistically, there was a slight
advancement of testicular development in the 50 |ig/L treatment at B, and this advancement
showed consistency across all three labs, although it was not statistically significant at labs A or
C.
Ovary
Unlike male gonads, female gonads experienced treatment-related increases in severity of
mononuclear cellular infiltrate which were significant (minimal to mild) in the 12.5, 25 and 50
|ig/L treatments at labs A and C (Appendix 8.4.2, Table 8.4.2-7). Granulomatous inflammation
was minimal at labs B and C in the low treatments and control, but was absent in the 25 and 50
|ig/L treatments.
Reproductive duct histopathology
-------
1148
1149
1150
1151
1152
1153
1154
1155
1156
1157
1158
1159
1160
1161
1162
1163
1164
1165
1166
1167
1168
1169
1170
1171
1172
1173
1174
1175
1176
1177
1178
1179
1180
1181
1182
1183
1184
1185
1186
1187
1188
1189
1190
1191
32
There were significant delays in male oviduct involution at lab A (6.25, 12.5, 25, 50 |ig/L) and
lab B (25, 50 |ig/L) and evidence of inappropriate advancement in male oviduct maturation at the
50 |ig/L treatment at B (Appendix 8.4.2, Table 8.4.2-8). Although there was a significant delay
in Wolffian duct development in the highest treatment at B, stage distributions were mostly
consistent across labs and similar between treatments and controls. Female oviduct development
was significantly accelerated with octylphenol exposure across all three labs (Appendix 8.4.2,
Table 8.4.2-9). However, sensitivity differed between labs: Lab A only showed a significant
effect in the 50 |ig/L treatment whereas significant effects were exhibited down to 12.5 and 6.25
|ig/L at B and C, respectively. Both effects on males and female ducts are consistent with an
estrogenic response, a commonly reported activity of OP.
5.3.3 17p-trenbolone
Introduction
Trenbolone is an androgen receptor agonist used in cattle production and has been measured in
aquatic systems near animal-feeding operations (e.g., stock yards). The effects of trenbolone
exposure on fish have been studied with respect to those physiological processes regulated via
the HPG axis mainly with respect to gonad differentiation and development in fish and
amphibian species (Orn et al., 2006; Holbech et al., 2006; Morthorst et al., 2010; Olmstead et al.,
2012). 17/?-trenbolone has been tested previously in the closely related amphibian species X.
tropicalis (Olmstead et al., 2012). In that study, the authors characterized the effects of aqueous
exposure to 17/?-trenbolone during larval X. tropicalis development using an exposure protocol
similar to the LAGDA. Concentrations >100 ng/L resulted in a spectrum of androgenic
responses which included hypertrophy of the larynx resulting in late larval mortality,
development of nuptial pads, a mixed-sex phenotype in the ovaries of females, and hypertrophy
of the Wolffian duct.
Results
Exposure levels and measured test concentrations
Given the observed mortality in the studies of Olmstead et al. (2012) the highest test
concentration was set at 100 ng/L, with a 0.5 dilution factor resulting in nominal test
concentrations of 100, 50, 25 and 12.5 ng/L. Mean ± SD (n = 40) measured concentrations of
the 12.5, 25, 50 and 100 ng/L 17/?-trenbolone treatments were 9.0 ± 2.8, 19.2 ± 5.8, 41.4 ± 9.7
and 79.9 ± 24.1 ng/L, respectively, over the duration of the experiment. CVs (averaged across
the duration of the exposure) were 31,30, 23 and 30% for the 12.5, 25, 50 and 100 ng/L
treatments, respectively. No 17/?-trenbolone was detected in control water.
Measure of General Toxicity
Survival
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1192
1193
1194
1195
1196
1197
1198
1199
1200
1201
1202
1203
1204
1205
1206
1207
1208
1209
1210
1211
1212
1213
1214
1215
1216
1217
1218
1219
1220
33
All control tanks met the guideline performance criteria of < 20% mortality and there were no
treatment-related effects on survival (data not shown).
Larval and Juvenile growth
There were no significant treatment-related effects on larval and juvenile growth as measured by
weight and length (Appendix 8.4.3, Tables 8.4.3-1 and 8.4.3-2). Further, there were no
significant treatment-related effects on LSI for either gender (Appendix 8.4.3, Table 8.4.3-2).
Liver and Kidney histopathology
Summary of trenbolone liver and kidney pathology.
All endpoints with lowest concentration statistically different
than control. Concentrations listed as nominal regardless of
mean-measured concentrations. Dash: Not significant.
Lab A
Nominal conc. 100, 50, 25, 12.5 ng/L
Juvenile Males
Liver pathology 50 ng/L
Kidney pathology 50 ng/L
Juvenile Females
Liver pathology 100 ng/L
Kidney pathology -
Treatment-related effects were present in the liver. The prevalence of minimal cellular
hypertrophy of hepatocytes was significantly different than the control in the 50 and 100 ng/L
treatments for juvenile males and females, respectively (Appendix 8.4.3, Table 8.4.3-4). The
prevalence and severity of the only other finding in the liver, granulomatous inflammation, were
comparable among treated and control samples.
Two common findings in male kidneys were renal tubule mineralization/casts (minimal to
moderate) and renal tubule dilation (minimal to mild). However, tubule dilation was
significantly attenuated in the 50 and 100 ng/L treatments whereas mineralization/casts were not
treatment-related (Appendix 8.4.3, Table 8.4.3-6). There was a low but statistically significant
occurrence of interstitial fibrosis (minimal to mild) in the 100 ng/L treatment when compared to
the control. There were no apparent effects of trenbolone exposure on the kidneys of juvenile
females (data not shown).
Thyroid Endpoints
Summary of trenbolone thyroid endpoints.
All endpoints with lowest concentration statistically
different than control. Concentrations listed as
nominal regardless of mean-measured concentrations.
Dash: Not significant.
Lab A
Nominal conc. 100, 50, 25, 12.5 ng/L
Larval sample
Time to NF stage 62
Serum T4
Thyroid pathology 25 ng/L
-------
1221
1222
1223
1224
1225
1226
1227
1228
1229
1230
1231
1232
1233
1234
1235
1236
1237
1238
1239
1240
1241
1242
1243
1244
1245
1246
1247
1248
1249
1250
1251
1252
1253
1254
1255
34
Time to NF stage 62
Trenbolone exposure did not affect the time it took for larvae to reach NF stage 62 (Appendix
8.4.3, Table 8.4.3-1).
Larval Thyroid histopathology
The two categories of morphologic findings in thyroid gland tissue, both of which were common,
were follicular cell hypertrophy (mild to moderate) and follicular cell hyperplasia (mild to
moderate). The severity of follicular cell hypertrophy was slightly decreased in the 50 and 100
ng/L trenbolone treatment groups as compared to the control, but those differences were not
statistically significant (Appendix 8.4.3, Table 8.4.3-3). The prevalence and severity of follicular
cell hyperplasia in trenbolone-treated frogs were mild but significantly different than the control
in the 25, 50 and 100 ng/L treatments.
Larval blood thyroxine
There were no treatment-related effects on larval serum T4 levels as measured by HPLC-ICP-MS
analysis (Appendix 8.4.3, Table 8.4.3-1).
Reproductive Endpoints
Summary of trenbolone reproductive endpoints.
All endpoints with lowest concentration statistically
different than control. Concentrations listed as nominal
regardless of mean-measured concentrations. Dash: Not
significant.
Lab A
Nominal conc. 100, 50, 25, 12.5 ng/L
Juvenile Males
Plasma VTG 50 ng/L
Gonad pathology 100 ng/L
Duct pathology 100 ng/L
Juvenile Females
Plasma VTG
Gonad pathology
Duct pathology 50 ng/L
Juvenile plasma vitellogenin
Trenbolone treatment did not affect vitellogenin titers in juvenile male or female frogs (data not
shown).
Gonad histopathology
In all cases, the genetic sex matched the phenotype determined by the pathologist and indicates
that treatment did not result in sex reversal. There were two significant treatment-related
findings in the testes of males: increased spermatogonia (minimal) and germ cell vacuolation
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1256
1257
1258
1259
1260
1261
1262
1263
1264
1265
1266
1267
1268
1269
1270
1271
1272
1273
1274
1275
1276
1277
1278
1279
1280
1281
1282
1283
1284
1285
1286
1287
1288
1289
1290
1291
35
(minimal to mild) in the 100 ng/L treatment (Appendix 8.4.3, Table 8.4.3-5). Germ cell
vacuolation was characterized by clear cytoplasmic ballooning in small clusters of pachytene-
phase (cell nuclei in prophase of meiosis) spermatocytes. There were no apparent effects of
trenbolone exposure on the ovaries of juvenile females (data not shown).
Reproductive duct histopatho/ogv
There were treatment-related effects on oviduct development in both genders. Males exhibited
accelerated regression of oviducts in the 100 ng/L treatment as compared to the control whereas
females experienced prodigious oviduct regression in the 50 and 100 ng/L treatments (Appendix
8.4.3, Table 8.4.3-7). There were no treatment-related effects on Wolffian duct development or
pathology in ei ther gender. Given the importance of the oviduct as a female reproductive organ
necessary for application of the gel coat to the oocyte and release of the oocyte, these effects are
expected to have considerable consequence on reproductive capacity (Figure 5.2.3-1).
Figure 5.2.3-1 A) Stage 2 oviduct (arrow) observed in control females. B) Stage 1 oviduct
(arrow) from a female exposed to 100 ng/L trenbolone. Stage 1 oviducts such as this were not
observed in any control females.
5.3.4 Benzophenone-2
Introduction
Benzophenone-2 (BP-2) is used as a uV filter in personal care products such as cosmetics and
sunscreens and in numerous other products for uV protection. BP-2 has been found to be
estrogenic in vitro and in vivo and is capable of impacting reproduction in fish (Weisbord el a!..
2007). In addition to this documented estrogenic activity, BP-2 has been shown to be a thyroid
peroxidase inhibitor, and exposure to BP-2 can result in disruption of the thyroid axis in aquatic
species (Thienpont et. al, 2011). BP-2 provided a unique study in the LAGDA evaluation of a
mixed mode of action chemical which included thyroid disruption. Laboratory E was the only
laboratory to conduct a test with BP-2. Due to limitations in exposure capabilities at this
laboratory, there were several deviations from the guidance provided. Instead of running eight
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control replicates and four treatment replicates at four test concentrations and a control, this
study was conducted using three test concentrations and a control, each with four replicates. Also,
the test was initiated with 30 embryos per tank rather than the recommended 25 embryos.
Although not completely consistent with the proposed test method, this study provides
significant value in terms of organism response and transferability of the approach.
Results
Exposure levels and measured test concentrations
The high test concentration was 6 mg/L with a 0.5 dilution factor resulting in nominal test
concentrations of 6.0, 3.0 and 1.5 mg/L. Mean ± SD (n = 36) measured concentrations of the 1.5,
3.0 and 6.0 mg/L benzophenone-2 treatments were 1.6 ± 0.1, 3.5 ± 0.2 and 6.1 ±0.1 mg/L,
respectively, over the duration of the experiment. CVs (averaged across the duration of the
exposure) were 4, 6 and 1% for the 1.5, 3.0 and 6.0 mg/L treatments, respectively. No
benzophenone-2 was detected in control water.
Measure of General Toxicity
Survival
BP-2 treatment resulted in significant mortality by NF stage 62 at 6.0 mg/L (Jonckeere-Terpstra,
Table 5.2.6-1).
Table 5.2.6-1
Mean percent mortality prior to completion of NF62 sample.
Highlighted cell is significantly different than control.
Nominal BP2 conc. (mg/L)
0
1.5
3
6
n/tank
29
29
29
29
replicates
4
4
4
4
Mean % mortality
0±0
1 ±2
3 ±7
38 ±6
Following the NF stage 66 cull, 10 individuals were left in each tank to continue exposure until
test termination. Within this timeframe, significant mortality was experienced in the high test
concentration (Jonckeere-Terpstra, Table 5.2.6-2).
Table 5.2.6-2
Mean percent juvenile mortality post-cull. Highlighted cell is
significantly different than control.
Nominal BP2 conc. (mg/L)
0
1.5
3
6
n/tank
10
10
10
10
replicates
4
4
4
4
Mean % mortality
0±0
0±0
5 ±6
25 ± 13
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37
Larval and Juvenile Growth
Summary of BP-2 growth endpoints.
All endpoints with lowest concentration statistically
different than control. Concentrations listed as
nominal regardless of mean-measured concentrations.
Lab E
Nominal conc. 6, 3, 1.5 mg/L
Larval sample
Weight 6 mg/L
Length 6 mg/L
Juvenile Males
Weight 6 mg/L
Length 6 mg/L
LSI 6 mg/L
Juvenile Females
Weight 6 mg/L
Length 6 mg/L
LSI 1.5 mg/L*
* Non-monotonic (i.e. the only treatment significantly different than control)
There was a significant increase in larval growth in the 6 mg/L treatment as compared to the
control. There was also a significant delay in time to reach NF stage 62 in the 6 mg/L treatment
(Appendix 8.4.4, Table 8.4.4-3). This increase in tadpole size is consistent with previous reports
following exposure to thyroid axis disruptors which produce a significant developmental delay
(Degitz et al., 2005; Tietge el al., 2005). Another potential contributor to increased growth in
the 6 mg/L treatment could be the significant mortality in that treatment allowing the survivors
more access to food. Growth in male and female juveniles was significantly reduced in the 6
mg/L treatment compared to the control, presumably due to overt toxicity (Appendix 8.4.4,
Table 8.4.4-4). LSI appeared to be affected by BP-2 exposure; however, statistical analyses were
inconsistent between genders due to non-monotonicity of the data in both cases. There was a
significant decrease in male LSI in the 6 mg/L treatment and a significant increase in female LSI
in the 1.5 mg/L treatment (Appendix 8.4.4, Table 8.4.4-4).
Liver and Kidney pathology
Summary of BP-2 liver and kidney pathology.
All endpoints with lowest concentration statistically
different than control. Concentrations listed as
nominal regardless of mean-measured concentrations.
Lab E
Nominal conc. 6, 3, 1.5 mg/L
Juvenile Males
Liver pathology 3 mg/L
Kidney pathology 3 mg/L
Juvenile Females
Liver pathology 3 mg/L
Kidney pathology 1.5 mg/L
Treatment-related findings in the liver were limited to phenotypic females and genetic males
which showed mixed sex gonads (i.e., 3.0 and 6.0 mg/L treatments). These included
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38
concentration-dependent increases in the prevalence and severity of basophilia, decreased
hepatocellular vacuolation, and intravascular proteinaceous fluid (Appendix 8.4.4, Table 8.4.4-6).
As compared to control frogs, frogs of the 3 and 6 mg/L treatment groups additionally had higher
prevalence of increased pigment, which also tended to be concentration-dependent. Furthermore,
the few findings of individual hepatocyte necrosis and foci of hepatocellular alterations were
restricted to frogs of the 3 and 6 mg/L treatment groups and 6 mg/L treatment group,
respectively. Overall, these liver changes are likely related to estrogenic activity of BP-2 and
significant up-regulation of vitellogenin synthesis {i.e., vitellogenesis).
BP-2 exposure caused profound effects in both genotypic male and female kidneys. These
effects were very similar between genders and widespread in the 3 and 6 mg/L treatments
(Appendix 8.4.4, Table 8.4.4-8). However, these two treatments had 100% female phenotypes.
Interestingly, many of the pathologies had higher severity scores in the 3 mg/L treatment and
were slightly less severe in the 6 mg/L treatment.
Thyroid Endpoints
Summary of BP-2 thyroid endpoints.
All endpoints with lowest concentration statistically
different than control. Concentrations listed as
nominal regardless of mean-measured concentrations.
Lab E
Nominal conc. 6, 3, 1.5 mg/L
Larval sample
TimetoNF62 6 mg/L
Thyroid pathology 1.5 mg/L
Time to NF stage 62
There was a significant delay in time to NF stage 62 in the 6 mg/L treatment (Appendix 8.4.4,
Table 8.4.4-3).
Larval thyroid pathology
There were significant treatment-related effects on thyroid tissue including increased prevalence
and severity of follicular cell hypertrophy and hyperplasia and gland hypertrophy (Appendix
8.4.4, Table 8.4.4-5). The 6 mg/L treatment exhibited glandular hypertrophy and extreme
follicular cell hypertrophy and hyperplasia in 100% of the individuals. As noted above, there
was also a significant delay in the rate of metamorphosis.
Larval blood thyroxine
In the BP-2 study, heparinized plasma samples as opposed to serum were analyzed for T4 levels
using both the optimized ELISA described in LAGDA Guideline Appendix 7 and the
HPLC/ICP-MS method. There were no statistically significant effects on circulating T4 in either
sample set, and T4 levels from the two methods did not agree. The cause of the lack of
agreement between the two methods is unknown. Therefore, the data is not presented. The
effects of using plasma as opposed to serum for T4 analyses are currently under investigation.
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Reproductive Endpoints
Summary of BP-2 reproductive endpoints.
All endpoints with lowest concentration statistically
different than control. Concentrations listed as
nominal regardless of mean-measured concentrations.
Lab E
Nominal
conc. 6, 3, 1.5 mg/L
Juvenile Males
Plasma VTG 1.5 mg/L
Gonad pathology 1.5 mg/L
Duct pathology 3 mg/L
Juvenile Females
Plasma VTG 1.5 mg/L
Gonad pathology 1.5 mg/L
Duct pathology 1.5 mg/L
Juvenile plasma vitellogenin
There was significant induction of vitellogenin in all treatments compared to the control and the
response was very consistent between genders (Appendix 8.4.4, Table 8.4.4-4). The overall
response was a > 5000-fold induction in the 6 mg/L treatment in both genetic males and females
compared to controls. This illustrates the estrogenic properties of BP-2 and emphasizes the
potential magnitude of response of this endpoint.
Gonad histopathology
Genotypic males experienced feminization of the gonad resulting in both intersex gonads (e.g.,
presence of testicular oocytes and partial ovarian cavity) and sex-reversed gonads in the 1.5
mg/L treatment (Figure 5.2.4-1). In the 3 and 6 mg/L treatments, 100% of the genotypic males
had been completely sex reversed exhibiting only ovarian tissue (i.e., phenotypic females). In
genotypic females, there was a significant treatment-related delay in gonad development based
on differences in developmental staging of ovaries in control versus treated samples (Appendix
8.4.4, Table 8.4.4-7). Similarly, there was a treatment-related delay in ovary development in
sex-reversed genotypic males. Thinning of the germinal epithelium is specific to testis tissue,
and significant increases in prevalence and severity of this pathology were observed in the 1.5
mg/L treatment. Mononuclear cellular infiltrate was observed in genotypic males and females
across all treatments and the control, but prevalence and severity were significantly attenuated in
the 3 and 6 mg/L treatments for genotypic females. Prevalence and severity of proteinaceous
fluid infiltrate were significantly increased in both genders in the 3 and 6 mg/L treatments.
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Figure 5.2.4-1 A) Testis, ducts, and kidney from a control male, provided for reference. Scale
bar = 206um. B) Testis of genetic male treated with 1.5 mg/L BP-2. The gonad consists
primarily of testicular tissue, but also contains several oocytes and a partial ovarian cavity
(arrow). Scale bar = 50um.
Reproductive duct histopathology
Oviduct development was rather variable within and between genders. In genotypic females,
there was a significant delay in oviduct development in all treatments (Appendix 8.4.4, Table
8.4.4-9). However, in both genders, there were some individuals in the 1.5 and 3 mg/L
treatments that are two developmental stages more advanced than the others. Both genders
experienced advancement of Wolffian duct development in the 3 and 6 mg/L treatments.
6 DISCUSSION OF THE INTER-LABORATORY STUDIES
6.1 Comparison of control performance
Larval growth and development
A summary of control larval weight, length, and time to NF stage 62 across all the inter-
laboratory studies is shown in Table 6.1-1. The measurements are ranked by weight in
descending order from a maximum control larval NF stage 62 weight of 2.07 + 0.33 g to a
minimum of 0.46 + 0.14 g. The overall mean weight was 1.0 g with a standard deviation of 0.5 g.
Two studies, B-OP and C-OP, fell outside of one standard deviation from the overall mean
weight. Within studies conducted by B, mean larval weight doubled from the PZ study to the OP
study and resulted in notable incidences of scoliosis in the OP study. Unpublished feeding
studies performed at A have demonstrated that excess feeding through larval development leads
to higher incidences and increased severity of scoliosis, which is generally considered adverse
for toxicological studies. Upon follow-up investigation, it was reported that feeding rates were
increased in the B-OP study by 20-30% because they opted to start the test with 25% more
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organisms per tank and subsequently increased feeding rates proportionately. Increasing the
number of organisms per tank for test initiation, in order to account for mortality, is acceptable
according to the LAGDA guideline. However, increasing feeding rates proportionately proved
to be excessive. The lowest rates of larval growth among the labs occurred in C-PZ and C-OP.
These studies also exhibited the highest level of variance in all three growth and development
metrics. This lab utilized its own established feeding rates which were lower than the
recommended feeding rates in the LAGDA guideline. These data and observations emphasize
the need for strict adherence to the guideline's recommended feeding rates to achieve
comparable and optimal organism growth across studies. It is suggested that the criterion for
control larval growth be 1.0 ± 0.2 grams at NF stage 62, which should be reached in less than 45
days.
Table 6.1-1
Inter-laboratory control larvae growth and development performance comparison.
Entries are listed in descending order according to weight (mean ± SD).
Study
Weight (g)
cv
Length (mm)
CV
Time to NF62 (d)
CV
A-PZ
-
-
-
B-OP
2.07 ±0.33
16
23.1 ± 1.3
6
42 ±2
5
E-BP2
1.18 ± 0.18
15
22.2± 1.1
5
38 ± 5
13
B-PZ
1.08 ± 0.17
16
20.8 ± 1.0
5
41 ± 3
7
A-OP
0.96 ± 0.15
16
19.2 ± 1.0
5
42 ±5
12
A-TB
0.82 ± 0.14
17
18.7 ± 1.1
6
40 ±4
10
D-PZ
0.79 ±0.13
17
18.8 ± 1.0
5
43 ± 2
5
C-PZ
0.62 ±0.14
23
17.1 ± 1.4
8
44 ± 6
14
C-OP
0.46 ±0.14
30
15.2 ± 1.6
11
41 ± 10
24
Overall means
1.0 ±0.49
49
19.4 ±2.6
13
41 ±2
4
Juvenile growth and development
As with the larval comparison, Table 6.1-2 contains juvenile male and female growth metrics
(length, weight, LSI) and VTG for each gender ranked by weight in descending order. Across all
studies, the male mean weight was 11.2 ± 3.9 g, and female mean weight was 11.7 ± 4.3 g. Two
studies had control juvenile weights more than one standard deviation from the overall mean in
both juvenile males and females but in different directions: D-PZ had higher mean weights, and
C-PZ had lower mean weights. Lab D used the recommended diet formulation but fed at a
higher rate than what was recommended in the LAGDA guideline. This was not necessarily
adverse; however, there was a significant treatment-related decrease in weight in this study
whereas studies from the other labs did not show this effect from prochloraz. Lab C used their
own diet formulation but fed at rates similar to that recommended in the LAGDA guideline.
This diet formulation was clearly inadequate for supporting growth rates consistent with the
other studies and led to confounding effects on LSI in their prochloraz study (explained in
Section 5.2.1). Body size does not become sexually dimorphic until later in development, so
significant differences in growth are not expected between genders at test termination. Therefore,
criteria for juvenile growth can be set the same for both sexes and is suggested to be 11.5 ± 3 g.
It is worth noting the VTG levels in control juveniles. These values are an artifact of detection
limits within each ELISA and are 100 to 1000-fold less than levels in mature females (Olmstead
et al., 2009b) and 10 to 1000-fold less than levels induced by OP and BP-2 exposures. The
utility of this endpoint is discussed further in Section 6.3.
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Table 6.1-2
Inter-laboratory control juvenile growth performance and vitellogenin titer comparison. Entries are
listed in descending order according to weight (mean ± SD).
Study
Weight (g)
CV
Length (mm)
CV
LSI
CV
VTG (mg/mL)
CV
Male
D-PZ
18.6 ±4.1
22
53.0 ±3.9
7
0.058 ± 0.005
9
0.01 ±0.02
158
A-PZ
13.1 ±2.0
15
47.2 ±2.8
6
0.059 ±0.010
17
0.03 ±0.02
59
A-TB
12.3 ± 3.3
27
46.2 ±4.1
9
0.060 ±0.010
17
0.07 ±0.07
108
B-PZ
11.5 ± 3.6
31
43.0 ±4.5
11
0.057 ± 0.009
16
0.00 ±0.00
176
E-BP2
11.3 ± 2.7
24
45.2 ±3.3
7
0.046 ±0.005
11
0.02 ±0.03
150
A-OP
10.9 ± 2.7
25
43.1 ± 3.3
8
0.058 ± 0.008
14
0.00 ±0.00
150
B-OP
10.1 ±2.6
26
41.6 ± 3.5
8
0.058 ± 0.007
12
0.01 ±0.01
107
C-OP
8.6 ±2.6
30
41.1 ± 3.4
8
0.072 ±0.008
11
0.01 ± 0.001
30
C-PZ
4.0 ±0.8
20
32.8 ± 2.2
7
0.035 ± 0.007
20
0.01 ± 0.002
44
Overall J1 means
11.2 ±3.9
35
43.7 ±5.5
12
0.056 ±0.010
18
0.02 ±0.02
122
Female
D-PZ
20.0 ±4.6
23
55.0 ±5.2
10
0.059 ±0.006
10
0.02 ±0.07
357
B-PZ
13.8 ± 5.0
36
46.4 ± 5.4
12
0.064 ± 0.014
22
0.01 ±0.02
251
A-PZ
12.9 ± 2.6
20
47.2 ±2.6
6
0.061 ± 0.007
12
0.03 ±0.03
76
A-TB
12.9 ± 3.7
29
47.5 ± 5.1
11
0.059 ±0.006
10
0.06 ±0.07
116
A-OP
12.0 ± 2.7
23
44.7 ±4.0
9
0.059 ±0.012
20
0.00 ±0.00
112
B-OP
10.9 ± 3.0
28
42.5 ±4.0
9
0.059 ±0.014
24
0.01 ±0.01
105
E-BP2
10.8 ± 2.1
19
44.7± 3.2
7
0.045 ± 0.006
13
0.04 ±0.11
323
C-OP
8.3 ± 1.7
21
40.8 ± 3.1
8
0.075 ± 0.007
9
0.04 ± 0.01
32
C-PZ
4.1 ±0.8
20
32.9 ±2.2
7
0.032 ±0.006
19
0.03 ±0.01
41
Overall 2 means
11.7 ±4.3
37
44.6 ±5.9
13
0.057 ±0.012
21
0.03 ±0.02
76
6.2 Effectiveness of the LAGDA
In total, the results for four chemicals are presented in this report. Two of these chemicals, 4-
/m-octylphenol and prochloraz, were tested in three and four laboratories respectively. The
other two test chemicals, benzophenone-2 and 17-/? trenbolone, were tested in a single laboratory
which prevents their use in inter-laboratory comparisons. However, these studies serve to
demonstrate the responsiveness of the LAGDA to additional modes of action. Table 6.2-1
summarizes the important findings for all of the studies which were conducted. All four
chemicals tested produced endocrine-related effects in these studies.
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Table 6.2-1.
Summary of significant findings in LAGD A studies on 4 chemicals representing different modes of action.
Chemical
Growth effects and
overt toxicity
Thyroid effects
Reproduction-related
effects
Prochloraz
(4 labs)
Liver and kidney
pathologies (A, C, D,
B)
Thyroid gland pathologies
consistent with hypothyroid
condition (A, D, and C)
VTG induction
Gonad/ reproductive duct
pathologies (all labs)
4-fcrt-octylphcnol
(3 labs)
Thyroid gland pathologies
consistent with hypothyroid
condition (A)
Delayed development (A)
VTG induction
Mild gonad/reproductive duct
pathologies (all labs)
11-\\ Trenbolone
(A only)
Liver and kidney
pathologies
Thyroid gland pathologies
Oviduct pathologies
Benzophenone-2
(E only)
Mortality
Thyroid gland pathologies
consistent with hypothyroid
condition
Delayed metamorphosis
VTG induction
Profound gonad/reproductive
duct pathologies
Prochloraz
At the highest test concentration, prochloraz produced liver and kidney pathologies consistent
with a toxic response across the four studies, although there was little evidence of an effect on
growth. LSI was also impacted by the highest prochloraz concentration across the studies. It
was also apparently decreased at two, three, or four concentrations in most studies, although it
appeared increased in C-PZ, but this was likely an artifact due to control juvenile growth in that
study being significantly less than in all other studies.
Effects on the thyroid axis (thyroid gland change) were also observed across the studies although
the metamorphic rate was not altered. This suggests a successful compensatory response by the
thyroid axis. Prochloraz has not been previously shown to have impacts on the thyroid axis and
a review of the literature found no evidence of prior investigations.
The degenerative testicular changes consistently observed across the four prochloraz studies is in
agreement with the anti-androgenic effects of prochloraz reported in species as divergent as
fathead minnows (Ankley et al., 2005) and rats (Blystone el al., 2007). In particular, germinal
epithelium thinning/loss with high doses of prochloraz has been described in rats (Noriega et al.,
2005). In male frogs, sporadic development of stage 3 oviducts and the retention of stage 2
oviducts with increasing prochloraz exposure were observed across the four studies and indicate
interference with normal oviduct regression. Oviduct regression in males is known to be an
androgen regulated process in amphibians (Kelley, 1996), and this finding is similar to the
feminization of the secondary sexual characteristics of male rats during perinatal prochloraz
exposure (Vingaard etal., 2005).
The LAGDA was successful in identifying prochloraz as anti-androgenic and demonstrated the
consequence of this mechanism of action on male gonad development.
4-tert-octylphenol
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44
In general, OP produced few signs of overt toxicity. No consistent effects on growth, liver and
kidney pathology or LSI were observed.
OP exposure resulted in VTG induction and female oviduct pathologies across all four studies.
These OP-induced changes are consistent with weak estrogenic activity. Vitellogenin has also
been shown to be elevated in males and females of multiple fish species exposed to OP (Seki et
al., 2002; Ortiz-Zarragoitia and Cajaraville, 2005; Rasmussen et al., 2005; Seki et al., 2002), in
X laevis primary cultured hepatocytes exposed to OP (Mitsui et al., 2007), and following in vivo
OP exposure in X. tropicalis (Porter et a I., 2011).
In these inter-laboratory studies, no consistent findings were observed in the developing male
and female gonads. Previous studies that have employed exposure during gonad development
have shown disruption in gonad development. These effects include decreased sperm count and
the presence of testis-ova in medaka (Seki et al., 2002), decreased gonadosomatic index and
increased testis-ova in eelpout (Rasmussen et al., 2005), and decreased spermatozoa and
increased testis anomalies in sheepshead minnows (Karels et al., 2003). In contrast, previous
studies with the X. tropicalis have shown no impact on male or female gonad development
(Porter et al., 2011). When taken together, it appears that these amphibian test species are less
sensitive to the endocrine disrupting effects of OP than are fish species. It must be pointed out
that it was not possible to test higher concentrations given the fact that concentrations above 60
|ig/L produced significant levels of embryo mortality mX. tropicalis (unpublished data, Lab A).
It is difficult to make extensive comments regarding across-laboratory performance given that
minimal effects were observed, other than to note that OP produced a consistent weak estrogenic
response. This was indicated by VTG induction and advancement of oviduct development
across the four studies, a response that it is consistent with OP activity reported in the published
literature.
77-/? Trenbolone
There were no significant treatment-related effects on larval or juvenile growth, although a
concentration-dependent increase in hepatocyte hypertrophy was observed. This finding
indicates that the test concentrations did not produce an appreciable level of overt toxicity.
The effects produced by trenbolone in this study were rather mild. Female gonads were
unaffected by exposure whereas there was increased spermatogonia and germ cell vacuolation in
the testis at 100 ng/L. The most significant finding was that 17-/? trenbolone induced the
regression of the female oviduct. The oviduct functions to transport mature oocytes from the
ovary to the external environment for fertilization by the male. Loss of or functional alterations
in the oviduct will have significant impacts on reproductive success and could lead to population
level impacts. There was also a mild increase in follicular cell hyperplasia at > 25 ng/L although
no significant alterations in developmental rate were observed.
In the absence of additional LAGDA studies with 17-/? trenbolone, comparisons to the published
literature served as a basis for evaluating the performance of the LAGDA in responding to
androgenic chemicals. Studies in zebrafish have demonstrated that aqueous exposure to
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trenbolone results in complete sex reversal of females and production of all male cohorts (Orn et
al., 2006; Holbech et al., 2006; Morthorst et al., 2010). Ovo-testes characterized by ovaries
which develop tubules containing spermatocytes were induced in mosquitofish exposed to
trenbolone (Sone et al., 2005). Females exposed to trenbolone have been reported to acquire
male secondary sex characteristics such as tubercles in adult fathead minnows (Ankley et al.,
2003) and papillary processes in Japanese medaka (Seki et al., 2006). Trenbolone exposure has
also been reported to reduce circulating estradiol and testosterone levels in fathead minnows
(Ankley et al., 2003). Decreased circulating vitellogenin in females has been demonstrated in a
number of different fish species exposed to this toxicant (Ankley et al., 2003; Sone et al., 2005;
Seki et al., 2006). Test concentrations of 17-/? trenbolone were set based on reported findings for
X tropicalis (Olmstead et al., 2012). In that study, the authors reported a much more dramatic
impact of 17-/? trenbolone on androgen responsive tissues. Concentrations >100 ng/L resulted in
a spectrum of androgenic responses which included hypertrophy of the larynx resulting in late
larval mortality, precocious development of nuptial pads, a mixed-sex phenotype in the ovaries
of females, and hypertrophy of the Wolffian duct. This comparison suggests thatX laevis may
be less sensitive thanX tropicalis to this synthetic androgen. Although the LAGDA showed a
lower sensitivity when compared to the literature, the LAGDA successfully identified the
endocrine disrupting potential and mode of action of trenbolone. Further, the LAGDA
demonstrated findings (female oviduct regression) which are useful in the context of risk
assessment.
Benzophenone-2
BP-2 treatment resulted in significant increases in larval and juvenile mortality at the highest test
concentration.
There were significant treatment-related effects on thyroid tissue including increased prevalence
and severity of follicular cell hypertrophy and hyperplasia and gland hypertrophy. The 6 mg/L
treatment exhibited remarkable pathologies in 100% of the subjects which were associated with a
significant delay in the rate of metamorphosis. BP-2 has been shown to be a thyroid peroxidase
inhibitor (Schmutzler et al., 2007), and exposure has been shown to result in disruption of the
thyroid axis in mammalian (Jarry et al., 2004) and aquatic species (Thienpont et al., 2011).
BP-2 has been found to be estrogenic in vitro (Molina-Molina et al., 2008) and in vivo (Kunz and
Fent, 2009) and is capable of impacting reproduction in fish (Weisbord et al., 2007). The results
of this study are consistent with these published reports. Plasma vitellogenin levels were
considerably elevated in all treatments (Appendix 8.4.4, Table 8.4.4-4). In addition to this
finding, liver and renal pathologies which were observed are consistent with estrogen exposure
(Pawlowski et al., 2004; Folmar etal., 2001) and are presumably a consequence of excessive
VTG accumulation in these organs. Genotypic males experienced feminization of the gonad in a
concentration-dependent fashion; both intersex gonads and sex-reversed gonads were observed
in the 1.5 mg/L treatment whereas in the 3 and 6 mg/L treatment, 100% of the genotypic males
had been completely sex reversed exhibiting only ovary tissue. In genotypic females, there was
a significant treatment-related delay in gonad development. Similarly, there was a treatment-
related delay in ovary development in sex-reversed genotypic males.
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6.3 Lessons learned from the inter-laboratory studies
Inconsistencies in Growth
Inconsistencies in organism growth were observed (see Section 6.1) which may have impacted
the results of the test chemicals. Diet formulation and feeding rates have been shown to be
correlated with organismal growth and developmental rates in the LAGDA. In one instance,
excessive control growth was observed when all the studies were considered as a group. In this
case, treatment-related effects on growth were observed. In two other studies (C-PZ and C-OP),
control growth was reduced relative to the broader group, and this was a confounding factor in
interpreting chemical related effects on LSI. This laboratory chose to use its own feed
formulation in lieu of that recommended in the guidance. From this inter-laboratory comparison,
it can be concluded that this formulation is inadequate to support appropriate growth and
development. The recommended diet and feeding rates in the LAGDA provide for optimal
growth, and the Agency strongly encourages adherence to the provided guidance.
Inconsistencies in Pathology Findings
Another area of inconsistency was in the pathology findings. Over the course of these studies,
the contract laboratories hired to prepare and read slides changed resulting in different
pathologists conducting the assessments. In the prochloraz studies, Labs A and D gonad, oviduct
and kidney samples were read by a different pathologist than the Labs B and C studies.
Interestingly, the Lab A and D studies shared consistencies as did the Lab B and C studies. It is
not possible to definitively demonstrate this as a cause for the observed inconsistencies.
However, it does point to the need for adherence to the pathology guidance document. It is
necessary to point out that the gonad histopathology guidance document was not finalized when
the prochloraz studies were prepared and read. In the case of OP, the three studies were prepared
and read by one laboratory, and reported findings (although minimal) were much more consistent.
Similar anomalies were observed with liver pathology for the prochloraz studies. Lab A, B and
D studies were read by the same contracted pathologist whereas Lab C study was read by a
different contracted pathologist. It is interesting to point out that similar treatment-related liver
pathologies were reported for Labs A, B and D whereas a different spectrum of treatment-related
pathologies were reported for Lab C. It is worth noting that the animals in the C study showed
feeding-related reduced juvenile growth rates as compared to the other studies. It is possible that
disease issues may have factored into the apparent inconsistencies in liver findings. Regardless,
developing a pathology guidance document for both the liver and kidney is critical. The samples
from these studies will provide the necessary examples for development of such a guidance
document.
T4 Serum Measures
The B, C and D laboratories used commercially available ELISA kits specific for human or
canine T4. These laboratories either did not attempt to optimize or failed in any such effort. As
such, the laboratories were unsuccessful in making these measurements and these data were not
included in this analysis. Lab A measured T4 levels in serum using an HPLC/ICP-MS method
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which has been previously optimized for use with X laevis (Tietge et al., 2010; Tietge et al.,
2012). Additionally, Lab A has developed an extraction method which allows for the use of
these commercially available ELISA kits (See LAGDA Guideline Appendix 7) with X laevis
serum. In the Lab A OP study, both methods were used and compared.
Unfortunately, it is not possible to make inter-laboratory comparisons of the effects of the test
chemicals on T4 since studies that used appropriate methods for measuring T4 were single
laboratory studies. However, the collection of control data indicates that both methods are
reproducible. Further, these methods were effective in accurately identifying alterations in T4.
In the future, laboratories conducting the assay must demonstrate a competence in making this
measurement. These studies should serve as a foundation for establishing performance criteria
for this endpoint.
Vitellogenin (VTG)
Although the VTG ELISA registers a measurable signal in control males and females, this
should be interpreted with caution. These values are more likely background noise within the
assay. PZ, OP, and BP-2 exposures resulted in a concentration-dependent significant increase in
plasma VTG. However, the BP-2 data set provides the best representation of the magnitude of
induction that can occur with estrogenic chemical exposure in X laevis. Further, with the level
of induction exhibited in the BP-2 study, CVs approached more acceptable values. Taken
together, this emphasizes that VTG data from all of the other studies are likely background levels,
and VTG induction as a reproductive endpoint in this assay should be interpreted in the context
of overall potential magnitude of response.
6.4 Conclusions
The LAGDA protocol describes a chronic toxicity test with an amphibian species that considers
growth and development from fertilization through the early juvenile period. Individual and
inter-laboratory evaluations of the LADGA were conducted to evaluate the practical
transferability of the assay protocol and quantitative reproducibility of the results.
Although the results of the inter-laboratory validation were mixed, the LAGDA was an effective
test model with endocrine-related effects characterized for all four chemicals evaluated. Overall,
the authors conclude that the Tier 2 LAGDA is capable of sufficiently characterizing potential
disruption of the endocrine system by putative endocrine disrupting chemicals. However, careful
attention to recommendations in the Test Guideline is necessary to ensure consistent and reliable
findings across multiple testing laboratories.
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Villeneuve DL, Ankley GT, Makynen EA, Blake LS, Greene KJ, Higley EB, Newsted JL, Giesy
JP, Hecker M. 2007. Comparison of fathead minnow ovary explant and H295R cell-based
steroidogenesis assays for identifying endocrine-active chemicals. Ecotoxicology and
Environmental Safety 68(l):20-32.
Vinggaard AM, Christiansen S, Laier P, Poulsen ME, Breinholt V, Jarfelt K, Jacobsen H,
Dalgaard M, Nellemann C, Hass U. 2005. Perinatal exposure to the fungicide prochloraz
feminizes the male rat offspring. Toxicological Sciences 85(2):886-897.
Wake MH, Dickie R. 1998. Oviduct structure and function and reproductive modes in
amphibians. Journal of Experimental Zoology 282(4-5):477-506.
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Weisbrod CJ, Kunz PY, Zenker AK, Fent K. 2007. Effects of the UV filter benzophenone-2 on
reproduction in fish. Toxicology and Applied Pharmacology 225(3):255-266.
Ying G-G, Williams B, Kookana R. 2002. Environmental fate of alkylphenols and alkylphenol
ethoxylates—a review. Environment International 28(3):215-226.
Yoshimoto S, Okada E, Umemoto H, Tamura K, Uno Y, Nishida-Umehara C, Matsuda Y,
Takamatsu N, Shiba T, Ito M. 2008. A W-linked DM-domain gene, DM-W, participates in
primary ovary development in Xenopus laevis. Proceedings of the National Academy of Sciences
105(7):2469-2474.
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8 APPENDICES
8.1 LAGDA Guideline
GUIDELINE FOR THE TESTING OF CHEMICALS
The Larval Amphibian Growth and Development Assay
INTRODUCTION
The need to develop and validate an assay capable of identifying and characterizing the adverse
consequences of exposure to toxic substances, in amphibians, originates from concerns that
environmental levels of chemicals may cause adverse effects in both humans and wildlife. The
Food Quality Protection Act of 1996 requires EPA to develop and implement a program using
valid tests for determining the potential endocrine effects from pesticides. The EPA established
an advisory group, the Endocrine Disruptor Screening and Testing Advisory Committee
(EDSTAC), to obtain advice on developing this program. In accordance with EDSTAC's
recommendations, EPA proposed a two-tiered approach to the program: Tier 1 would identify
the potential of a substance to interact with the endocrine system whereas Tier 2 would confirm
the interaction and characterize the effects. One of the Tier 2 tests recommended by EDSTAC is
an amphibian full life cycle test to evaluate the adverse consequences of putative endocrine
disrupting chemicals, especially those active within the thyroid and reproductive systems, on the
development, growth and reproduction of amphibians.
PRINCIPLE OF l lll TEST
The Larval Amphibian Growth and Development Assay (LAGDA) is intended to identify and
characterize the adverse consequences of exposure to substances which interfere with the normal
development and growth of amphibians through larval development and metamorphosis. It is an
important assay to address potential inciting contributors to amphibian population declines by
evaluating the effects from exposure to contaminants, both through endocrine and non-endocrine
mechanisms, during the larval stage that may adversely affect populations. It should be noted
that to date, no validated assay exists that serves this function for amphibians.
The general experimental design entails exposing NF stage 8 Xenopus laevis embryos to four
different concentrations of a test chemical and a control until 10 weeks after the median time to
NF stage 62 in the control with one interim sub-sample at NF stage 62 (See Nieuwkoop and
Faber 1994 for staging details). There are four replicates in each test concentration with eight
replicates for the control. Endpoints evaluated during the course of the exposure include those
indicative of generalized toxicity: mortality, abnormal behavior, and growth determinations
(length and weight), as well as endpoints designed to characterize specific endocrine toxicity
modes of action targeting estrogen-, androgen-, or thyroid-mediated physiological processes.
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DESCRIPTION OF THE METHODS
Environmental Conditions
The inflow of water is provided to tanks housing tadpoles and post-metamorphic juvenile frogs
at a rate of >10 tank additions per day (4.0 - 10.0 L tanks). Water temperature is maintained at
21±1°C with a photoperiod of 12 hours of light and 12 hours of dark. Dissolved oxygen (%),
tank temperature, and pH are measured twice weekly. Conductivity (|iS), alkalinity (mg/L of
CaCCb), and hardness (mg/L of CaCCte) are measured once a month. For post-metamorphic
juvenile frogs, humidity levels should be held above 30%. The occurrences of lesions on the
head just posterior to the nostrils are indications of insufficient humidity levels.
Exposure tanks should be siphoned on a daily basis to remove uneaten food and waste products.
Care should be used to minimize stress and trauma to the animals, especially during movement,
cleaning of aquaria, and manipulation. Stressful conditions/activities should be avoided such as
loud and/or incessant noise, tapping on aquaria, vibrations in the aquaria, excessive activity in
the laboratory, and rapid changes in environmental media (light availability, temperature, pH,
DO, water flow rates, etc).
Feeding
Feed and feeding rates change with life stages of Xenopus, which is unique to this test species
and is a very important aspect of the LAGDA protocol. Excessive feeding during the larval
phase typically results in increased incidences and severity of scoliosis and should be avoided.
Conversely, inadequate feeding during the larval phase results in highly variable developmental
rates among controls potentially compromising statistical power or confounding test results.
Therefore, adherence to the provided feeding regime is strongly advised (Appendix 2).
Larvae
The larval diet consists of Silver Cup Trout Starter (Nelson and Sons, Murray, UT, USA),
Spirulina algae discs (Wardley, Secaucus, NJ, USA) and TetraFin® flakes (Tetra Sales,
Blacksburg, VA, USA) blended together in culture water. This mixture is fed three times daily
on weekdays and once daily on weekends. Tadpoles are also fed 24-hour old live brine shrimp
(Bio-Marine® Brand, Bio-Marine, Hawthorne, CA, USA) twice daily on weekdays and once
daily on the weekends starting on day 8 post-fertilization. Sera Micron® (Sera GmbH,
Heinsberg, Germany) can be used in place of the diet previously described but feeding rates need
to be adjusted to meet control performance criteria.
Juvenile
Once metamorphosis is complete, the feeding regime consists of 3/32" premium sinking frog
food by Xenopus Express. For juveniles, the pellets are briefly run in a coffee grinder or blender
in order to reduce their size. Appendix 2 provides information regarding the type and amount of
feed used during the juvenile life stage. The animals should be fed once per day.
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Control organisms must meet all of the following performance criteria in order to ensure
reproducibility and transferability of the LAGDA:
1) The mean time to NF stage 62 is < 45 days.
2) The mean weight atNF stage 62 is 1.0±0.2 grams.
3) The mean juvenile weight at test termination is 11,5±3 grams.
Chemical Testability
The LAGDA is based upon an aqueous exposure protocol whereby the test chemical is
introduced into the test chambers via a flow-through system. Flow-through methods however,
introduce constraints on the types of chemicals that can be tested, as determined by the
physicochemical properties of the compound. Therefore, prior to using this protocol, baseline
information about the chemical should be obtained that is relevant to determining its testability.
Both the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and
Mixtures and the USEPA Guidance Document on Special Considerations for Conducting
Aquatic Laboratory Studies should be consulted (OECD 2000; OCSPP 850.1000).
Characteristics which indicate that the chemical may be difficult to test in aquatic systems
include: high octanol-water partitioning coefficients (log Kow), high volatility, susceptibility to
hydrolysis, and susceptibility to photolysis under ambient laboratory lighting conditions. Other
factors may also be relevant to determining testability and should be determined on a case by
case basis. If a successful test is not possible for the chemical using a flow-through test system,
then it is recommended that this chemical not be tested using this protocol.
Exposure System
A flow-through diluter system is required for this protocol. The system components should have
water-contact components of glass, stainless steel, and/or Teflon®. However, suitable plastics
can be utilized if they do not compromise the study. Exposure tanks should be glass or stainless
steel aquaria, equipped with standpipes that result in an approximate tank volume between 4.0
and 10.0 L and minimum water depth of 10 to 15 cm. The system should be capable of
supporting all exposure concentrations, a control, and a vehicle control, if necessary, with four
replicates per treatment (eight in the control). The flow rate to each tank should be constant in
consideration of both the maintenance of biological conditions and chemical exposure. It is
recommended that flow rates accommodate at least 10 tank turnovers per day to avoid chemical
concentration declines due to metabolism by both the test organisms and aquatic microorganisms.
The treatment tanks should be randomly assigned to a position in the exposure system to reduce
potential positional effects, including slight variations in temperature, light intensity, etc.
Fluorescent lighting should be used to provide a photoperiod of 12 hr light and 12 hr dark at an
intensity that ranges from 600 to 2,000 lux (lumens/m ) at the water surface. Water temperature
should be maintained at 21° ± 1°C, pH maintained between 6.5 to 8.5, and the dissolved oxygen
(DO) concentration maintained at >3.5 mg/L (>40% of the air saturation) in each test tank. At a
minimum, water temperature, pH and dissolved oxygen should be measured twice a week.
Temperature should be measured continuously in at least one test vessel. Humidity within the
system should be kept above 30%. Appendix 1 outlines the experimental conditions under
which the protocol should be executed. For further information on setting up flow-through
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exposure systems, please refer to the ASTM Standard Guide for Conducting Acute Toxicity
Tests on Test Materials with Fishes, Macroinvertebrates, and Amphibians (ASTM 2002) and
general aquatic toxicology texts.
Water Quality
Any water that is locally available (e.g. spring water or charcoal-filtered tap water) and permits
normal growth and development of X. laevis can be used. Because local water quality can differ
substantially from one area to another, analysis of water quality should be undertaken,
particularly, if historical data on the utility of the water for raising amphibian larvae is not
available. Special attention should be given that the water is free of copper, chlorine and
chloramines, all of which are toxic to amphibians. It is further recommended to analyze the
water to determine background levels of estrogenic and androgenic chemicals, fluoride,
perchlorate and chlorate (by-product of drinking water disinfection) as all of these may confound
the study outcome. Analyses should be performed before testing begins and the testing water
should normally be free from these chemicals.
Iodide Concentration in Test Water
In order for the thyroid gland to synthesize thyroid hormones to support normal metamorphosis,
sufficient iodide needs to be available to the larvae through a combination of aqueous and dietary
sources. Currently, there are no empirically derived guidelines for minimum iodide
concentrations. However, iodide availability may affect the responsiveness of the thyroid system
to thyroid active agents and is known to modulate the basal activity of the thyroid gland, an
aspect that deserves attention when interpreting the results from thyroid histopathology.
Therefore, measured aqueous iodide concentrations from the test water should be reported.
Based on previous work, the protocol has been demonstrated to work well when test water iodide
(I") concentrations ranged between 0.5 and 10 (J,g/L. Ideally, the minimum iodide concentration
in the test water should be 0.5 (J,g/L. If the test water is reconstituted from deionized water,
iodine must be added at a minimum concentration of 0.5 (J,g/L. Supplementation of the test water
with iodine or other salts must be noted in the report.
Analytical Chemistry
Prior to conducting a study, the stability of the test compound should be evaluated using existing
information on its solubility, degradability, and volatility. Test solutions from each replicate
tank at each concentration should be sampled for analytical chemistry analyses at test initiation
(day 0), and weekly throughout the exposure period. It is also recommended that each test
concentration be analyzed during system preparation, prior to test initiation, to verify system
performance and that stock solutions be analyzed when they are changed, especially if the
volume of the stock solution does not provide adequate amounts of chemical to span the duration
of routine sampling periods. In the case of chemicals which cannot be detected at some or all of
the concentrations used in a test, stock solutions should be measured and system flow rates
recorded in order to calculate actual concentrations.
Chemical Delivery
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The method used to introduce the test chemical to the system can vary depending on its
physicochemical properties. Water soluble compounds can be dissolved in aliquots of test water
at a concentration which allows delivery at the target test concentration in a flow-through system.
Chemicals which are liquid at room temperature and moderately soluble in water can be
introduced using liquid:liquid saturator methods. Chemicals which are solid at room temperature
and are moderately soluble in water can be introduced using glass wool column saturators (Kahl
et al. 1999). The preference is to use a carrier-free test system; however, different test chemicals
will possess varied physicochemical properties that will likely require different approaches for
preparation of chemical exposure water. All efforts should be made to avoid solvents or carriers
because: (1) certain solvents themselves may result in toxicity and/or undesirable or unexpected
responses, (2) testing chemicals above their water solubility (as can frequently occur through the
use of solvents) can result in inaccurate determinations of effective concentrations, and (3) the
use of solvents in longer-term tests can result in a significant degree of "biofilming" associated
with microbial activity. For difficult to test substances, a solvent may be employed as a last
resort, and the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances
and Mixtures should be consulted (OECD 2002) to determine the best method. The choice of
solvent will be determined by the chemical properties of the substance. Solvents which have
been found to be effective for aquatic toxicity testing include acetone, ethanol, methanol,
dimethyl formamide and triethylene glycol. In case a solvent carrier is used, solvent
concentrations should be below the chronic No Observed Effect Concentration (NOEC); the
OECD Guidance Document recommends a maximum of 100 [j,L/L and a recent review
recommends that solvent concentrations as low as 20 [xL/L of dilution water be used (Hutchinson
et al. 2006). If solvent carriers are used, appropriate solvent controls (clean water) must be
evaluated in addition to non-solvent controls. If it is not possible to administer a chemical via
the water, either because of physicochemical characteristics (low solubility) or limited chemical
availability, introducing it via the diet may be considered. Preliminary work has been conducted
on dietary exposures; however, this route of exposure is not commonly used. The choice of
method should be documented and analytically verified.
Establishing the High Test Concentration
For the purposes of this test, results from Tier I amphibian studies should be used to what extent
possible in determining the high test concentration so as to avoid concentrations that are overtly
toxic. Prior to running the LAGDA a range finding experiment should be conducted. The
exposure should be initiated within 24 hours of fertilization and continue for 7-10 days. The test
concentrations should be set such that the intervals between test concentrations are no greater
than 1 log. The results of the range finding experiment should serve to set the highest test
concentration in the LAGDA. Once the highest test concentration is established a 50% dilution
series is to be used to establish the 3 lower test concentrations.
Test Concentration Range
There is a required minimum of four test concentrations and a clean water control (and vehicle
control if necessary). The minimum test concentration differential between the highest and
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lowest should be about one order of magnitude. The maximum concentration separation should
be no greater than 0.5.
PROCEDURE
Assay Overview
This assay is intended for use in determining the chronic effects of chemicals on amphibian
larval growth and development. The assay is initiated with newly spawned larvae and continues
into juvenile development. Animals are examined daily for mortality and any signs of abnormal
behavior. At NF stage 62, a sub-sample of animals is collected and various endpoints are
examined (see Table 1). After all animals have reached NF stage 66, a cull is carried out to
reduce the number of animals per tank, and the remaining animals continue exposure until 10
weeks after the median time to NF stage 62 in the control. At test termination additional
measurements are collected (see Table 1).
Table 1. Timing of apical endpoint measurements over the course of
the LAGDA.
Apical Endpoints
Daily
Interim
Sampling
Test
Termination
Mortality
X
Clinical signs of disease and/or general toxicities
X
Time to NF stage 62
X
Thyroid histology
X
Serum thyroxine (T4)
X
Morphometries (growth)
X
X
Liver-somatic index (LSI)
X
Vitellogenin
X
Genetic/phenotypic sex ratios
X
Gonad histology
X
Reproductive duct histology
X
Kidney histology
X
Liver histology
X
Test Initiation
It is preferred that animals for test initiation have previously been shown to produce offspring
that can be genetically sexed (see Appendix 3). After spawning of adults, embryos are collected,
cysteine treated to remove the jelly coat and screened for viability according to ASTM standards
(ASTM 2004). Cysteine treatment allows the embryos to be handled during screening without
sticking to surfaces. Screening takes place under a dissecting microscope using an appropriately
sized eye dropper to remove non-viable embryos. It is preferred that a spawn resulting in greater
than 70% viability be used for the test. Embryos from a single spawn are randomly distributed
into exposure treatment tanks containing an appropriate volume of dilution water until each tank
contains 20 embryos. Embryos should be carefully handled during this transfer in order to
minimize handling stress and to avoid any injury. At 96 hours post spawning, the tadpoles should
have moved up the water column and begun clinging to the sides of the tank.
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Mortality
All test tanks should be checked daily for dead animals and the numbers recorded for each tank.
The date, concentration and tank number for any observation of mortality is recorded. Dead
animals should be removed from the test tank as soon as observed. The developmental stage of
dead animals should be categorized as either pre-NF stage 58 (pre-forelimb emergence), NF
stage 58-NF stage 62, NF stage 63-NF stage 66 (between NF 62 and complete tail absorption), or
post-NF stage 66 (post-larval). Mortality rates exceeding 20% may indicate inappropriate test
conditions or overtly toxic effects of the test chemical. During the first few days of development
after the spawning event and during metamorphic climax, the animals tend to be most sensitive
to non-chemical induced mortality events.
Additional Observations
The date, treatment, and tank number for any observation of abnormal behavior, grossly visible
malformations (e.g. scoliosis), or lesions should be recorded. Normal behavior for larval animals
is characterized by suspension in the water column with tail elevated above the head, regular
rhythmic tail fin beating, periodic surfacing, operculating, and being responsive to stimuli.
Abnormal behaviors would include, for example, floating on the surface, lying on the bottom of
the tank, inverted or irregular swimming, lack of surfacing activity, and being nonresponsive to
stimuli. For post-metamorphic animals, in addition to the above abnormal behaviors, gross
differences in food consumption between treatments should be recorded. Gross malformations
and lesions could include morphological abnormalities (e.g., limb deformities), hemorrhagic
lesions, abdominal edema, and bacterial or fungal infections, to name a few. These
determinations are qualitative and should be considered akin to clinical signs of disease/stress
and made in comparison to control animals. If the rate of occurrence is greater in exposed tanks
than in the controls, then these should be considered as evidence for overt toxicity.
Larval Sub-Sample
Those tadpoles that have reached NF stage 62 should be removed from the tanks and either
sampled or moved to the next part of the exposure in a new tank, or physically separated from
the remaining tadpoles in the same tank with a divider. Tadpoles are checked daily, and the time
at which an individual tadpole reaches NF stage 62 is recorded. The defining characteristic for
use in this assessment is the shape of the head. Once the head has become reduced in size such
that it is the same width as the trunk of the tadpole, then that individual would be counted as
having attained NF stage 62. One tadpole from every four removed from a given tank is
randomly selected for the larval sub-sampling. Those tadpoles not sampled are moved to new
tanks or physically separated from the remaining tadpoles for continued exposure. The random
procedure used must give each organism on test equal probability of being selected. This can be
achieved by using any randomizing method but requires that each tadpole be netted.
For the larval sub-sampling, the following endpoints are obtained:
1. Time to NF stage 62 (number of days between fertilization and NF stage 62)
2. External abnormalities
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3. Morphometries (growth - weight and length)
4. Thyroid histology
5. Serum T4
Outline of Procedure for Larval Sampling:
1. Euthanize tadpole by immersion in Tricaine Methanesulfonate (MS-222)
appropriately buffered to achieve pH 7.0.
2. Rinse and blot dry.
3. Weigh the tadpole (mg) and measure snout to vent length (mm).
4. Note any gross morphologic abnormalities and/or clinical signs of toxicity.
5. Collect blood from the heart using a non-heparinized capillary tube for serum
collection (-25 |iL).
6. Remove and discard the lower torso below the forelimbs.
7. Fix the upper torso for 48 hours in Davidson's solution with agitation and then rinse
with tap water and store tissues in 10% NBF.
Euthanasia
NF stage 62 tadpoles should be euthanized by immersion in a 0.01% MS-222 solution (500 mL).
This solution should be buffered with sodium bicarbonate to a pH of approximately 7.0.
(Unbuffered MS-222 solution is acidic and irritating to frog skin resulting is poor absorption and
unnecessary additional stress to the organisms.) The characteristics of the water used to make
this solution may impact the pH and therefore the amount of sodium bicarbonate needed to
neutralize this solution. In general, however, a recipe of one part MS-222 to two parts of sodium
bicarbonate should be used as a starting point.
Using a mesh dip net, a tadpole is removed from the experimental chamber and transported to
the necropsy area in a transport container. The tadpole is placed into the euthanasia solution. It
is ready for necropsy when it is unresponsive to external stimuli such as pinching the hind limb
with a pair of forceps.
Growth: Weight and Length
Measurements of wet weight and snout to vent length for each tadpole (see Figure 1) should be
made immediately after it becomes non-responsive. These measurements are included in the test
protocol to assess possible effects of test substances on the growth rate of organisms and are
useful in detecting generalized toxicity. Tadpoles should be blotted dry before weighing to
remove excess adherent water. After measurements of body size are made, any gross
morphological abnormalities and/or clinical signs of toxicity should be noted, recorded, and any
digital documentation made. These include scoliosis, petechiae, hemorrhage, etc.
a. Larval sub-sampling (NF stage 62)
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b. NF stage 66 cull and juvenile sampling
Blood Collection for T4 A nalyses
Blood is collected from the heart using a non-heparinized capillary tube for serum collection
(-25 |iL). The tube is sealed (e.g., with Crit-o-seals) and placed on ice until clots form (1-4 hrs.),
at which time the tubes should be centrifuged to remove the clots from the serum. The capillary
tube is scored and broken at the serum-clot interface, the volume of serum collected for each
individual is recorded, and the serum is expelled into a microcentrifuge tube. Serum samples
from individuals are stored at -80°C for subsequent quantification of T4. Given the limited
volume of blood that can be collected from an individual NF stage 62 tadpole, analyses of
circulating T4 has to be performed on pooled samples consisting of serum collected from all
individuals in a tank (i.e., replicate). A pooled sample is formed by combining equal volumes of
serum from every individual in a tank. The volume of serum contributed by each individual to
the pool is equal to the least volume of serum collected among all individuals in a tank.
Professional judgment should be used to exclude samples with low yields of collected serum due
to technical difficulties.
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Tissue Collection and Fixation for General Histology
For the sub-sampling, thyroid glands are assessed for histology. The lower torso below the
forelimbs is removed and discarded. The trimmed carcass is placed into individually labelled
histological cassettes and fixed in Davidson's fixative. The volume of fixative in the container
should be at least 10 times the approximated volume of the tissues. Appropriate agitation or
circulation of the fixative should be achieved to adequately fix the tissues of interest. All tissues
remain in Davidson's fixative for at least 48 hours, but no longer than 96 hours at which time
they are rinsed in deionized water and stored in 10% NBF.
Davidson's Fixative:
a) Glacial acetic acid 115 ml
b) Formaldehyde (35-40%) 220 ml
c) 95% Ethyl Alcohol 330 ml
d) Distilled water 335 ml
10% Neutral Buffered Formalin:
a) 100 ml formalin, full strength (37-40%) formaldehyde)
b) 6.5 g sodium phosphate dibasic (anhydrous)
c) 4.0 g sodium phosphate monobasic
d) 900 ml distilled water
Refer to Appendix 5 for procedural information on processing tissues for histological
examinations and Appendix 6 (Appendix 8.3 of ISR) for information regarding the interpretation
of diagnostic criteria.
End of Larval Exposure
Given the initial number of tadpoles, it is expected that there will likely be a small percentage of
individuals that do not develop normally and do not complete metamorphosis in a reasonable
amount of time. The larval portion of the exposure should not exceed 70 days. Any tadpoles
remaining at the end of this period should be euthanized by immersion in 0.02% buffered MS-
222 (pH 7.0). They should be weighed, staged (Nieuwkoop and Faber, 1994), and any
developmental abnormalities noted.
Cull after NF stage 66
Ideally, only 10 individuals per tank should continue until termination of the exposure.
Therefore, after all animals have reached NF stage 66 or after 70 days (whichever occurs first), a
cull should be conducted. Animals that will not continue the exposure should be selected at
random. These animals are euthanized by immersion in a 0.02% MS-222 solution (500 mL)
buffered with sodium bicarbonate to a pH of approximately 7.0. Measurements of wet weight
and snout to vent length (see Figure 1) for each froglet are made, and a gross necropsy is
conducted. The phenotypic sex (based on gonad morphology) is noted as female, male, or
indeterminate.
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64
Juvenile Sampling
At the end of the exposure period, the remaining animals are euthanized, and the following
endpoints are evaluated and recorded:
1. Morphometries (growth - weight and length)
2. Phenotypic/genotypic sex ratios
3. Plasma vitellogenin
4. Gonad histopathology
5. Reproductive duct histopathology
6. Liver histopathology
7. Liver weight (Liver-somatic Index)
8. Kidney histopathology
Methods:
1. Euthanize frogs by injection of buffered 10% MS-222 in PBS (0.01 ml per g frog).
2. Measure whole body weight (g) and snout-vent length (mm).
3. Record any developmental abnormalities and obvious signs of disease (e.g. scoliosis,
hemorrhage).
4. Place the frog in dorsal recumbency.
5. Expose the heart by removing the skin and associated cartilage.
6. Carefully expose the heart by opening the pericardial sac without cutting any major
blood vessels.
7. Snip a major blood vessel coming from the heart and collect blood using a heparinized
capillary tube for plasma collection (-50 |iL).
8. Open the abdominal cavity. Dissect out, weigh, and fix the liver.
9. Carefully remove the digestive organs (e.g. stomach, intestines) from the lower
abdomen.
10. Assess gross morphological abnormalities in gonads.
11. Remove a foot for a DNA sample and snap freeze in liquid nitrogen or dry ice.
12. Remove hind limbs and fix remaining carcass.
Euthanasia
Post-metamorphic frogs are euthanized by an intraperitoneal injection of 10% MS-222 in an
appropriately buffered solution (e.g., phosphate buffered solution). Dosage for frogs is 0.01 mL
per gram of frog. This can be approximated to 0.1 mL for a typical male-sized individual and
0.2 mL for a typical female-sized individual. Using this procedure, frogs may be sampled after
becoming unresponsive, usually around 2 min after injection. Samples should be collected from
frogs shortly after euthanasia. Therefore, unless multiple prosectors are available, numerous
frogs should not be euthanized simultaneously.
Growth: Weight and Length
Measurement of weight and length are identical to those outlined for the larval sub-sampling.
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65
Blood Collection for Vitellogenin Analyses
The euthanized juveniles are placed in dorsal recumbency. The heart is exposed by removing the
skin and cartilage that covers it. The pericardial sac is opened and the heart exposed. In order
for adequate blood collection to be made, it is imperative that care be taken not to cut or tear any
blood vessels during this process. The exposed heart should still be beating at this point. A
major blood vessel at the top of the heart is snipped, and blood is collected with a heparinized
capillary tube. The tube is sealed (e.g., with Crit-o-seal®) and placed on ice until the samples can
be centrifuged. At the end of the sampling, tubes should be centrifuged to remove the blood
cells from the plasma. The capillary tube is scored and broken at the plasma-cell interface, and
the plasma is expelled into a microcentrifuge tube that has been coated with aprotinin. Plasma
samples are stored at -80°C for subsequent quantification of vitellogenin levels with an ELISA.
Vitellogenin is an established estrogen responsive biomarker in a number of fish and frogs
species. If a valid commercially available ELISA kit exists for X laevis vitellogenin it may be
used. Alternatively, an ELISA can be developed as described in Appendix 4.
Tissue Collection and Fixation for General Histology
Gonads and livers are collected for histological analysis during the final sampling. The
abdominal cavity is opened, and the liver is dissected out, weighed and fixed. Next, the digestive
organs (e.g. stomach, intestines) are carefully removed from the lower abdomen to reveal the
gonads. Any gross morphological abnormalities in the gonads should be noted. Finally, the hind
limbs should be removed, and the carcass should be fixed with the gonads left in situ. Collected
livers and carcasses are placed into labelled histological cassettes and fixed in Davidson's
fixative. The volume of fixative in the container should be at least 10 times the approximated
volume of the tissues. All tissues remain in Davidson's fixative for at least 48 hours, but no
longer than 96 hours at which time they are rinsed in de-ionized water and stored in 10% NBF.
The same fixatives used in the larval sub-sampling as described previously are used with adult
samples. Refer to Appendix 5 for details on histological procedures.
Genetic Sexing
DNA samples from frogs can be obtained from any tissue. One hind limb removed during
dissection is easy to collect and store in a microcentrifuge tube. Tissue can be stored at -20°C
until DNA isolation. There are several commercially available kits for the isolation of DNA
from tissue that can be used. Testing for the presence or absence of a marker is done by PCR as
described in Appendix 3. Development of the markers used for genetic sexing is described in
Yoshimoto etal. 2008 (X laevis).
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66
DATA COLLECTION AND REPORTING
All data should be collected using electronic or manual systems which conform to good
laboratory practices (GLP). Study data should include:
1. Test substance: Characterization of the test substance: physical-chemical properties;
information on stability and biodegradability.
2. Chemical observations and data: method and frequency of preparation of dilutions.
Test chemical information includes actual and nominal concentrations of the test
chemical, and in some cases, non-parent chemical, as appropriate. Test chemical
measurements may be required for stock solutions as well as for test solutions.
3. Solvent (if other than water): justification of the choice of solvent, and
characterization of solvent (nature, concentration used).
4. Test conditions:
a) Operational records: these consist of observations pertaining to the functioning
of the test system and the supporting environment and infrastructure. Typical
records include: ambient temperature, test temperature, photoperiod, status of
critical components of the exposure system (e.g. pumps, cycle counters,
pressures), flow rates, water levels, stock bottle changes, and feeding records.
General water quality parameters include: pH, dissolved oxygen, conductivity,
total iodine, alkalinity, and hardness.
b) Deviations from the test method: this information should include any
information or narrative descriptions of deviations from the test method.
5. Results:
a) Biological observations and data: these include daily observations of mortality,
food consumption, abnormal swimming behavior, lethargy, loss of equilibrium,
malformations, lesions, etc. Observations and data collected at predetermined
intervals include: growth (weight and length), clinical signs of disease or general
toxicities, time to metamorphosis, T4 and vitellogenin measurements, and genetic
sex.
b) Histological data including narrative descriptions, as well as graded severity
and incidence scores of specific observations.
6. Statistical analyses: Statistical analytical techniques and justification of techniques
used; results of the statistical analysis preferably in tabular form.
7. Ad hoc observations: these observations should include narrative descriptions of the
study that do not fit into the previously described categories.
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67
PERFORMANCE CRITERIA AND TEST ACCEPTABILITY/VALIDITY
Performance Criteria
Generally, gross deviations from the test method will result in unacceptable data for
interpretation or reporting. Therefore, the following criteria in Table 2 have been developed as
guidance for determining the quality of the test performed and the general performance of the
control organisms.
Table 2. Performance criteria for the LAGDA.
Criterion
Test concentrations
Mortality in controls
Development in controls
Dissolved Oxygen
pH
Water temperature
Test concentrations without overt toxicity
Replicate performance
Special conditions for use of a solvent
Acceptable limits
Maintained at < 20% CV(variability of
measured test concentration) over the entire
test
< 20% mortality in any one replicate in the
controls.
The mean time to NF stage 62 is < 45 days,
and the mean weight at NF stage 62 is > 1.0
± 0.2 g. The mean weight at test
termination is 11.5 ± 3 g.
> 40% air saturation
pH should be maintained between 6.5-8.5.
The inter-replicate/inter-treatment
differentials should not exceed 0.5.
Target temperature ± 1°C - the inter-
replicate/inter-treatment differentials should
not exceed 1.0 °C
>2
< 2 replicates across the test can be
compromised
If a carrier solvent is used, both a solvent
control and clean water control must be
used.
Statistically significant differences between
solvent control and water control groups are
treated specially. See below for more
information.
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68
Test Validity
The following requirements must be met to deem a test acceptable/valid:
1. For any given treatment (including controls), mortality cannot exceed 20%, otherwise
the replicate is considered compromised.
2. At least two treatment levels, with all four uncompromised replicates, must be
available for analysis.
3. At least two treatment levels without overt toxicity must be available for analysis.
STATISTICAL ANALYSES
Statistical analyses of the data should preferably follow procedures described in the document
Current Approaches in the Statistical Analysis of Ecotoxicity Data: A Guidance to Application
(OECD 2006). Data for continuous endpoints 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 significant 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
independently of any ANOVA F-test and the Mann-Whitney test is applied independently of any
overall Kruskall-Wallis test. In addition, regardless of the results of the previous tests, a step-
down Jonckheere-Terpstra trend (JT) test should also be applied to the data as long as a
monotonic response was observed.
Mortality data should be analyzed for time periods encompassing larval development and the full
test. Tadpoles that do not complete metamorphosis in the given time frame, those tadpoles that
are in the larval sub-sample cohort, those juvenile frogs that are culled, and any animal that dies
due to experimenter error should be treated as censured data. Mortality data as expressed as the
percentage that died should be arcsine square root transformed prior to analysis.
Time to metamorphosis data should be treated as time to event data, with any mortalities treated
as censured data. Median time to completion of metamorphosis should be determined by
Kaplan-Meier product-limit estimators and used in the statistical analysis.
Body weight and snout-vent lengths should be analyzed using the means of a given tank. Males
and females are not sexually-dimorphic at the completion of metamorphosis and data for the
sexes are combined when analyzing larval sub-sampling data. Male and female juvenile body
size data should be analyzed separately.
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69
Vitellogenin plasma concentrations should be log transformed before statistical analysis and
separated by sex.
Liver weights should be normalized as proportions of whole body weights (LSI), arcsine square
root transformed and analyzed separately for each sex.
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 >5
mortalities in any replicate that cannot be explained by 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.
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. The most relevant endpoints for consideration in this analysis are
growth determinants (weight and length), as these can be affected through generalized toxicities.
If statistically significant differences are detected in these endpoints between the clean water
control and solvent control groups, determine the study endpoints for the response measures
using the clean water control. If there is no statistically significant difference between the clean
water control and solvent control for all measured response variables, determine the study
endpoints for the response measures using the pooled dilution-water and solvent controls.
REFERENCES
ASTM (2002). Standard Guide for Conducting Acute Toxicity Tests on Test Materials with
Fishes, Macroinvertebrates, and Amphibians. (A. S. f. T. a. Materials, ed., Philadelphia, PA.
ASTM (2004). Standard Guide for Conducting the Frog Embryo Teratogenesis Assay - Xenopus
(FETAX).
Hutchinson, T. H., Shillabeer, N., Winter, M. J., and Pickford, D. B. (2006). Acute and chronic
effects of carrier solvents in aquatic organisms: a critical review. Aquat Toxicol 76, 69-92.
Kahl, M. D., Russom, C. L., DeFoe, D. L., and Hammermeister, D. E. (1999). Saturation units
for use in aquatic bioassays. Chemosphere 39, 539-551.
Nieuwkoop, P. D. and Faber, J., (1994). Normal Table of Xenopus laevis (Daudin). Garland
Publishing, Inc, New York, NY.
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70
OECD (2000). Guidance Document on Aquatic Toxicity Testing of Difficult Substances and
Mixtures.
OECD (2002). Guidance Document on Aquatic Toxicity Testing of Difficult Substances and
Mixtures. In OECD Guidelines for the Testing of Chemicals, Volume 1, Number 5, Paris, France.
OECD (2006). Current Approaches in the Statistical Analysis of Ecotoxicity Data: A Guidance
to Application Environmental Health and Safety Publications, Paris.
USEPA. 1996 Endocrine Disruptor Screening Protram Test Guidelines OPPTS 850.1000:
Special Considerations for Conducting Aquatic Laboratory Studies. Office of Chemical Safety
and Pollution Prevention [OCSPP] formerly Prevention, Pesticides and Toxic Substances
[OPPTS] (7101) EPA 712-C-96-113. April 1996.
Yoshimoto, S., Okada, E., Umemoto, H., Tamura, K., Uno, Y., Nishida-Umehara, C., Matsuda,
Y., Takamatsu, N., Shiba, T., Ito, M. (2008). A W-linked DM-domain gene, DM-W,
participates in primary ovary development in Xenopus laevis. Proc Natl Acad Sci USA
105(7):2469-2474.
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2665 Appendix 1: Experimental Conditions for the LAGDA
2666
Test animal
Xenopus laevis
Initial larval stage
Nieuwkoop and Faber (NF) stage 8
Exposure period
Until 10 weeks after the median time to NF
stage 62 in the control
Test concentrations
Minimum of 4 different concentrations
Dilution water / laboratory control
Any water that permits normal growth and
development of X. laevis (e.g., spring water or
charcoal-filtered tap water)
Replication
4 replicate test vessels / test concentration
8 replicate test vessels / control
Exposure regime
Flow-through
Test system flow-rate
Constant, in consideration of both the
maintenance of biological conditions and
chemical exposure (e.g., flow rate sufficient to
provide 10 tank volume additions/day)
Test solution / test vessel
4 - 10L(10 - 15 cm minimum water) / glass or
stainless steel
Initial larval density
20 larvae / test vessel
Larval density after NF stage 66 cull
10 juveniles / test vessel (decided randomly)
Feeding
See Appendix 2
Lighting
Photoperiod
12 hr light: 12 hr dark
Intensity
600 to 2000 lux (lumens/m2) at the water
surface
Water temperature
2i° ± rc
pH
6.5 to 8.5
Dissolved oxygen (DO) concentration
>3.5 mg/L (> 40% of the air saturation)
Analytical chemistry schedule
initiation (day 0), and weekly thereafter for each
replicate tank at each concentration
2667
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2670
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72
Appendix 2: Diet Preparation
Larval diet
This diet, consisting of Trout Starter, algae/TetraFin®, and brine shrimp, allows control
organisms to meet both of the following criteria:
1) mean time to NF stage 62 is < 45 days, and
2) mean weight at NF stage 62 is > 1.0±0.2 g
Food Preparation
A. 1:1 (v/v) Trout Starter: algae/TetraFin®:
1. Trout Starter: blend 80 mL of Trout Starter and 300 mL of suitable filtered on high 20
seconds
2. Algae/TetraFin® mixture: blend 12 g spirulina algae disks and 500 mL filtered LSW on
high for 40 seconds, blend 12 g Tetrafin® with 500 mL filtered LSW and then combine
these to make up one L of 12 g/L spirulina algae and 12 g/L Tetrafin®
3. Combine equal volumes of the blended Trout Starter and the algae/TetraFin® mixture
B. Brine shrimp: Fifteen mL brine shrimp eggs are hatched in 1 L of salt water (prepared by
adding 20 mL of NaCl to 1 L deionized water). After aerating 24 hours at room temperature
under constant light, the brine shrimp are harvested. Briefly, the brine shrimp are allowed to
settle for 30 min by stopping aeration. Cysts that float to the top of the canister are poured
off and discarded, and the shrimp are poured through the appropriate filters and brought up to
30 mL with LSW.
Feeding Protocol
Table 3 provides a reference regarding the type and amount of feed used during the larval stages
of the exposure. The animals should be fed three times per day Monday through Friday and once
per day on the weekends*.
Table 3. Feeding regime forX laevis larvae in flow-through conditions (Trout Starter,
Algae/TetraFin Diets, and Brine Shrimp Quantities for 26 tadpoles).
Time
Post-F ertilization
(Day 0=Date of
Injection)
Weekday
Feeding Volume
(3X per day)
Weekend
Feeding Volume
(IX per day)
Brine Shrimp**
2X/day
(IX per day
weekend)
4 or 5 days***-14
days
0.33 ml
1.2 ml
0.5 ml (d 8)**
(0.5 ml)*
2 weeks
0.67 ml
2.4 ml
1 ml (d 16)**
(1 ml)*
3 weeks
1.3 ml
4.0 ml
1 ml (1 ml)*
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2712
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73
4 weeks
1.5 ml
4.0 ml
1 ml (1 ml)*
5 weeks
1.6 ml
4.4 ml
1 ml (1 ml)*
6 weeks
1.6 ml
4.6 ml
1 ml (1 ml)*
7 weeks
1.7 ml
4.6 ml
1 ml (1 ml)*
8-12 weeks
1.7 ml
4.6 ml
1 ml (1 ml)*
* All numbers in parentheses are feeding rates for the weekend (only fed once/day). Brine shrimp are fed once per
day on the weekend, and the volume remains either 0.5 ml (8-15 days) or 1 ml (16 days).
** Brine Shrimp:
Add 0.5 ml of live brine shrimp to the diet twice per day (once per day on the weekend) after 8 days.
Add 1.0 ml of live brine shrimp to the diet twice per day (once per day on the weekend) after 16 days.
***Start feeding when animals are free swimming.
Juvenile diet
Once metamorphosis is complete, the feeding regime changes to 3/32" premium sinking frog
food by Xenopus Express.
Food preparation
Pellets are briefly run in a coffee grinder or blender in order to reduce the size of the pellets.
Feeding protocol
Table 4 provides a reference regarding the type and amount of feed used during juvenile and
adult life stages. The animals should be fed once per day. It should be noted that as animals
metamorph, they continue receiving a portion of the brine shrimp until >95% animals complete
metamorphosis.
Table 4. Feeding regime forX laevis post-metamorphic juveniles in flow-through
conditions.
Weeks post-median metamorphosis
date*
Crushed pellet
volume (mL) per
froglet
Whole pellet volume
(mL) per froglet
As animals complete metamorphosis
0.05
0
0-1
0.05
0.05
2-3
0
0.2
4-5
0
0.3
6-9
0
0.4
*The first day of week 0 is the median metamorphosis date.
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74
Appendix 3: Genetic Sexing
X. laevis primers
DM-W marker
Forward: 5'-CCACACCCAGCTCATGTAAAG-3'
Reverse: 5' -GGGC AGAGT C AC AT AT ACTG-3'
Positive Control
Forward: 5'-AACAGGAGCCCAATTCTGAG-3'
Reverse: 5'-AACTGCTTGACCTCTAATGC-3'
DNA purification
Purify DNA from muscle or skin tissue using Qiagen DNeasy Blood and Tissue Kit (cat # 69506)
or similar product according to kit instructions. DNA can be eluted from the spin columns using
less buffer to yield more concentrated samples if deemed necessary for PCR.
PCR
TM
Sample protocol using JumpStart Tag from Sigma is outlined below:
Master Mix
lx (uL)
TFinall
NFW
11
-
10X Buffer
2.0
-
MgCl2 (25mM)
2.0
2.5 mM
dNTP's (lOmM ea)
0.4
200 |iM
Marker for primer (8 |iM)
0.8
0.3 |iM
Marker rev primer (8 |iM)
0.8
0.3 |iM
Control for primer (8 |iM)
0.8
0.3 |iM
Control rev primer (8 |iM)
0.8
0.3 |iM
JumpStart™ Taq
0.4
0.05 units/|il
DNA template
1.0
-200 pg/|il
Note: When preparing Master Mixes, prepare extra to account for any loss that may occur while
pipetting (example: 25x should be used for only 24 reactions).
Reaction: Master Mix 19.0 |iL
Template 1.0
Total 20.0 |iL
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2768
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2770
2111
2772
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2800
2801
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75
Thermocycler Profile: 1.
94°C
1 min
2.
94°C
30 sec
3.
60°C
30 sec
4.
72°C
1 min
5.
Goto
step 2. 35 c
6.
72°C
1 min
7.
4°C
PCR products can be ran immediately in a gel or stored at 4°C.
Agarose Gel Electrophoresis (3%)
5 OX TAE
24.2 g Tris
5.71 mL glacial acetic acid
3 .72 g Na2(EDTA) 2H20
Add water to 100 mL
IX TAE
392 mL H20
8 mL 5 OX TAE
3:1 Agarose
3 parts NuSieve agarose GTG
1 part Fisher agarose low EEO
Method
1. Prepare a 3% gel by adding 1.2 g agarose mix to 43 mL IX TAE. Swirl to disassociate
large clumps.
2. Microwave agarose mixture until completely dissolved (avoid boiling over). Let cool
slightly.
3. Add 1.0|iL ethidium bromide (10 mg/mL). Swirl flask.
4. Pour gel into mold with comb. Cool completely.
5. Add gel to apparatus. Cover gel with IX TAE.
6. Add 1 |iL of 6x loading dye to each 10|il PCR product.
7. Pipette samples into wells.
8. Run at 160 constant volts for -20 minutes.
-------
2812
2813
2814
2815
2816
2817
2818
2819
2820
2821
2822
2823
2824
2825
2826
2827
2828
2829
2830
2831
2832
2833
2834
2835
2836
2837
2838
2839
2840
2841
2842
2843
2844
2845
2846
2847
2848
2849
2850
2851
2852
2853
2854
2855
2856
2857
76
LAGDA Appendix 4: Vitellogenin ELISA
Measurement of Vitellogenin (Vtg) in Xenopus laevis by ELISA
The procedure is based on an ELISA originally developed for fathead minnow Vtg (Parks et al.,
1999, Comp. Biochem. Physiol C 123:113-125). See Olmstead et al. (2009) Gen. Comp.
Endocrinol. 160:117-123 for a description of the assay as modified for Vtg in X tropicalis. The
method uses an antibody made against X tropicalis Vtg, but we know it also works for X. laevis
Vtg.
Materials and Reagents:
Preadsorbed 1° Antibody (Ab) serum: mix 1 part anti-Xtropicalis Vtg 1° Ab serum with 2 parts
control male plasma and leave at RT for ~ 75 minutes, put on ice for 30 min, centrifuge > 20K x
G for 1 h at 4 °C, remove supernatant, aliquot, store at -20 °C.
2° Antibody: Goat Anti-Rabbit IgG-HRP conjugate (e.g., Bio-Rad 172-1019)
Vtg Standard: purifiedX laevis Vtg at 3.3 mg/ml.
TMB (3,3',5,5' Tetramethyl-benzidine) (e.g., KPL 50-76-00; or Sigma T0440)
Normal Goat Serum (NGS) (e.g., Chemicon S26-100ml)
96 well EIA polystyrene microtiter plates (e.g., ICN:76-381-04, Costar:53590, Fisher:07-200-35)
37 °C hybridization oven (or fast equilibrating air incubator) for plates, water bath for tubes
Other common laboratory equipment, chemicals, and supplies.
Recipes:
Coating Buffer (50mMCarbonate Buffer, pH 9.6):
1.26g NaHC03
0.68gNa2CO3
428 ml water
1 OX PBS (0.1 M phosphate, 1.5MNaCl):
0.83gNaH2PO4H2O
20.1 g Na2HP04-7 H20
71 gNaCl
810 ml water
Wash Buffer (PBST):
100 ml 10XPBS
900 ml water
Adjust pH to 7.3 with 1 MHCL
Then add 0.5 ml Tween-20
Assay Buffer:
3.75 ml Normal Goat Serum (NGS)
-------
2858
2859
2860
2861
2862
2863
2864
2865
2866
2867
2868
2869
2870
2871
2872
2873
2874
2875
2876
2877
2878
2879
2880
2881
2882
2883
2884
2885
2886
2887
2888
2889
2890
2891
2892
2893
2894
2895
2896
2897
2898
2899
2900
2901
2902
77
146.25 ml Wash Buffer
Sample collection:
Blood is collected with heparinized microhematocrit tube and placed on ice. After centrifugation
for 3 min, the tube is scored, broken open, and the plasma expelled into 0.6 ml microcentrifuge
tubes which contain 0.13 units of lyophilized aprotinin. (These tubes are prepared in advance by
adding the appropriate amount of aprotinin, freezing, and lyophilizing in a speed-vac at low heat
until dry.) Place plasma at -80 °C until analyzed.
Procedure for One Plate:
Coating the Plate:
Mix 20 [jl of purified Vtg with 22 ml of carbonate buffer (final 3 |ig/ml). Add 200 j_il to each
well of a 96-well plate using. Cover the plate with adhesive sealing film and allow to incubate
overnight at 4 °C (or 37 °C for 2 hours).
Blocking the Plate:
Blocking solution is prepared by adding 2 ml of Normal Goat Serum (NGS) to 38 ml of
carbonate buffer. Remove coating solution and shake dry. Add 350 j_il of the blocking solution
to each well. Cover with adhesive sealing film and incubate at 37 °C for 2 hours (or at 4 °C
overnight).
Preparation of Standards:
5.8 [jl of purified Vtg standard is mixed with 1.5 ml of assay buffer in a 12 x 75 mm borosilicate
disposable glass test tube. This yields 12,760 ng/ml. Then a serial dilution is performed by
adding 750 j_il of the previous dilution to 750 j_il of assay buffer to yield final concentrations of
12,760, 6380, 3190, 1595, 798, 399, 199, 100, and 50 ng/ml.
Preparation of Samples:
Start with a 1:300 (e.g., combine 1 (_il plasma with 299 j_il of assay buffer) or 1:30 dilution of
plasma into assay buffer. If a large amount of Vtg is expected, additional or greater dilutions
may be needed. Try to keep B/B0 within the range of standards. For samples without
appreciable Vtg, e.g., control males and immature females, use the 1:30 dilution. Samples
diluted less than this may show unwanted matrix effects.
Additionally, it is recommended to run a positive control sample on each plate. This comes from
a pool of plasma containing highly induced levels of Vtg. The pool is initially diluted in NGS,
divided in aliquots and stored at -80 C. For each plate, an aliquot is thawed, diluted further in
assay buffer and run similar to a test sample.
Incubation with 1° Antibody:
The 1° antibody is prepared by making a 1:2,000 dilution of preadsorbed 1° antibody serum in
assay buffer (e.g., 8 jal to 16 ml of assay buffer). Combine 300 j_il of 1° antibody solution with
300 [jl of sample/standard in a glass tube. The B0 tube is prepared similarly with 300 j_il of assay
buffer and 300 |il of antibody. Also, a NSB tube should be prepared using 600 jal of assay buffer
-------
2903
2904
2905
2906
2907
2908
2909
2910
2911
2912
2913
2914
2915
2916
2917
2918
2919
2920
2921
2922
2923
2924
2925
2926
2927
2928
2929
2930
2931
2932
2933
2934
2935
2936
2937
2938
2939
2940
2941
2942
2943
2944
2945
2946
2947
2948
78
only, i.e., no Ab. Cover the tubes with parafilm and vortex gently to mix. Incubate in a 37 °C
water bath for 1 hour.
Washing the Plate:
Just before the 1° antibody incubation is complete, wash the plate. This is done by shaking out
the contents and patting dry on absorbent paper. Then fill wells with 350 j_il of wash solution,
dump out, and pat dry. A multi-channel repeater pipette or plate washer is useful here. The wash
step is repeated two more times for a total of three washes.
Loading the Plate:
After the plate has been washed, remove the tubes from the water bath and vortex lightly. Add
200 [jl from each sample, standard, B0, and NSB tube to duplicate wells of the plate. Cover plate
with adhesive sealing film and allow to incubate for 1 hour at 37 °C.
Incubation with the 2° Antibody:
At the end of the incubation from the previous step, the plate should be washed three times again,
like above. The diluted 2° antibody is prepared by mixing 2.5 j_il of 2° antibody with 50 ml of
assay buffer. Add 200 jal of diluted 2° antibody to each well, seal like above, and incubate for 1
hour at 37 °C.
Addition of Substrate:
After the incubation with the 2° antibody is complete, wash the plate three times as described
earlier. Then add 100 jal of TMB substrate to each well. Allow the reaction to proceed for 10
minutes, preferably out of bright light. Stop the reaction by adding 100 j_il of 1 M phosphoric
acid. This will change the color from blue to an intense yellow. Measure the absorbance at 450
nm using a plate reader.
Calculate B/B0:
Subtract the average NSB value from all measurements. The B/B0 for each sample and standard
is calculated by dividing the absorbance value by the average absorbance of the B0 sample.
Obtain the Standard Curve and Determine Unknown Amounts:
Generate a standard curve with the aid of some computer graphing software (e.g., Slidewrite or
Sigma Plot®) that will extrapolate quantity from B/B0 of sample based on B/B0 of standards.
Typically, the amount is plotted on a log scale and the curve has a sigmoid shape. However, it
may appear linear when using a narrow range of standards. Correct sample amounts for dilution
factor and report as mg Vtg/ml of plasma.
Determination of Minimum Detection Limits (MDL):
Often, particularly in normal males, it will not be clear how to report results from low values. In
these cases, the 95% "Confidence limits" should be used to determine if the value should be
reported as zero or as some other number. If the sample result is within the confidence interval
of the zero standard (B0), the result should be reported as zero. The minimum detection level will
be the lowest standard which is consistently different from the zero standard; that is, the two
-------
79
2949 confidence intervals don't overlap. For any sample result which is within the confidence limit of
2950 the minimum detection level, or above, the calculated value will be reported. If a sample falls
2951 between the zero standard and the minimum detection level intervals, one half of the minimum
2952 detection level should be reported for the value of that sample.
2953
2954
2955
2956
-------
2957
2958
2959
2960
2961
2962
2963
2964
2965
2966
2967
2968
2969
2970
2971
2972
2973
2974
2975
2976
2977
2978
2979
2980
2981
2982
2983
2984
2985
2986
2987
2988
2989
2990
2991
Appendix 5: Histology Protocol
Larval Subsampling (Stage 62)
Dissection
- Froglets euthanized by submersion in MS-222
- Wet weight is measured
- Blood is collected directly from aorta
- Legs are removed at knee joint
Fixation
- Davidson's 48 hours
a) Glacial acetic acid 115 ml
b) Formaldehyde (35-40%) 220 ml
c) 95% Ethyl Alcohol 330 ml
d) Distilled water 335 ml
- Rinse with distilled water
10%) neutral buffered formalin
Trimming
Gonad:
Transverse cuts posterior to the arms and anterior to the pelvis
Thyroid:
Transverse cut through middle of arms leaving some shoulder joint
Transverse cut anterior to eyes to remove snout
Processing
Decalcification
- By fixation in Davidson's or Bouin's
Dehydration
Thyroid and gonad (1 hour stations)
Station
Reagent
70% EtOH
80% EtOH
95% EtOH
95% EtOH
100%
EtOH
100%
EtOH
Time
1 hour
1 hour
1 hour
1 hour
1 hour
1 hour
retort
temp
-ambient-
-ambient-
-ambient-
-ambient-
-ambient-
-ambient-
p/v
on
on
on
on
on
on
Station
8
10
11
12
13
Reagent
Clear-rite
Clear-rite
Clear-rite
Paraffin
Paraffin
Paraffin
Time
1 hour
1 hour
1 hour
1 hour
1 hour
1 hour
retort
temp
-ambient-
-ambient-
-ambient-
60°
60°
60°
p/v
on
on
on
on
on
on
Embedment
Gonad:
-------
2992
2993
2994
2995
2996
2997
2998
2999
3000
3001
3002
3003
3004
3005
3006
3007
3008
3009
3010
3011
3012
3013
3014
3015
3016
3017
3018
3019
3020
3021
3022
3023
3024
3025
3026
3027
3028
3029
81
- Embed anterior side down.
I \
E3
Thyroid:
- Embed posterior side down, with the nose pointing up. The nose can be trimmed off anterior
to the eyes if needed to fit into embedding mold.
Sectioning Protocol
Gonad: (4 |im sections)
- Face block
Step section and collect one good section to be mounted on a glass slide at the following
levels:
~
0|jm
~
900|jm
~
1900pm
~
100pm
~
1100pm
~
2100pm
~
300|jm
~
1300(jm
~
2300pm
~
500|jm
~
1500pm
~
2500pm
~
700|jm
~
1700pm
~
2700pm
Thyroid: (4 |im sections)
- Face block
Take 1 section, mount on slide, and inspect in microscope for both thyroid glands.
No glands?
Take 150-200 micron step, examine a section for thyroid glands, and repeat until both glands
appear on sections.
Section has both glands?
Take 1 section, cut 50 microns, take 1 section, and mount together on slide.
Seal block
Staining
- H&E and coverslip with glass using a permanent mounting medium
Juvenile Sampling & Processing
Dissection
- Frogs euthanized by MS-222 injection
- Wet weight, length measured
- Blood is collected from heart
- Liver is removed, weighed and fixed
One foot is removed for DNA analysis
- Remaining carcass is placed in an appropriately sized tissue cassette and fixed
-------
3030
3031
3032
3033
3034
3035
3036
3037
3038
3039
3040
3041
3042
3043
3044
3045
3046
3047
3048
3049
3050
3051
3052
3053
3054
3055
3056
3057
3058
3059
3060
3061
3062
3063
3064
3065
3066
3067
3068
3069
3070
3071
3072
3073
3074
3075
82
Male Female
Fixation
- Davidson's 48 hours
Glacial acetic acid
Formaldehyde (35-40%)...
95% Ethyl Alcohol
Distilled water
- Rinse with distilled water
10% neutral buffered formalin
Trimming
Testis/Kidney/ducts
Transverse cuts on anterior and posterior margins of testis.
Ovary/Kidney/ducts
Transverse cuts on anterior and posterior margins of the ovary.
Thyroid
Transverse cut through middle of arms leaving some shoulder joint
Transverse cut anterior to eyes to remove snout
w
115 ml
220 ml
,330 ml
.335 ml
-------
3076
3077
3078
3079
3080
3081
3082
3083
3084
3085
3086
3087
3088
3089
3090
3091
3092
3093
3094
3095
3096
3097
3098
3099
3100
3101
83
Processing
Decalcification
Bone surrounding thyroid and kidney samples should be decalcified for 24-48 hours
depending on strength of decalcifier. Decalcification not necessary for ovaries.
Dehydration
**Ovaries (V.2 hour stations) Thyroid and kidney (1 hour stations)
Station
1
2
3
4
5
6
7
Reagent
70% EtOH
80% EtOH
95% EtOH
95% EtOH
100%
EtOH
100%
EtOH
100%
EtOH
Time
**
**
**
**
**
**
**
retort
temp
-ambient-
-ambient-
-ambient-
-ambient-
-ambient-
-ambient-
-ambient-
p/v
on
on
on
on
on
on
on
Station
8
9
10
11
12
13
14
Reagent
Clear-rite
Clear-rite
Clear-rite
Paraffin
Paraffin
Paraffin
Paraffin
Time
**
**
**
**
**
**
**
retort
temp
-ambient-
-ambient-
-ambient-
60
60
60
60
p/v
on
on
on
on
on
on
on
Embedment
Testis/kidney & Oviduct/kidney: Embed anterior side down
Ovaries: Embed normally; a large mold will be needed
Thyroid: Embed posterior side down, nose up in large mold
Sectioning
Testis/kidney & Oviduct/kidney: (4|im sections)
- Face block
Step section and collect one good section to be mounted on a glass slide at the following
levels:
~
~
~
~
~
0pm
100pm
300pm
500pm
700|jm
~
~
~
~
~
900pm
1100pm
1300pm
1500pm
1700pm
~
~
~
~
~
1900pm
2100pm
2300pm
2500pm
2700pm
Ovaries: (7|im sections)
- Face block
On the side of the paraffin block, mark %, V2,3A way through entire ovary.
-------
3102
3103
3104
3105
3106
3107
3108
3109
3110
3111
3112
3113
3114
3115
3116
3117
3118
3119
3120
3121
3122
3123
3124
3125
3126
84
Take a ribbon of 2-3 sections at each mark, mount on slide.
Thyroid: (4|im sections)
- Face block
Take 1 section, mount on slide, and inspect in microscope for both thyroid glands.
No glands?
Take 150-200 micron step, examine a section for thyroid glands, and repeat until both glands
appear on sections.
Section has both glands?
Take 1 section, cut 50 microns, take 1 section, and mount together on slide.
Seal block
Staining
- H&E and coverslip with glass using a permanent mounting medium
-------
3127
3128
3129
3130
3131
3132
3133
3134
3135
3136
3137
3138
3139
3140
3141
3142
3143
3144
3145
3146
3147
3148
3149
3150
3151
3152
3153
3154
3155
3156
3157
3158
3159
3160
3161
3162
3163
3164
3165
3166
3167
3168
3169
3170
3171
3172
3173
85
Appendix 6: Histopathology Guidance for Reading LAGDA Studies
See ISR Appendix 8.3
Appendix 7: T4ELISA
Adaptation of a Commercially Available ELISA Kit to Measure Total
Thyroxine (T4) in the Serum/Plasma oiXenopus laevis.
Materials
T4 ELISA kit: (e.g.MP Biomedicals 07BC1007)
Buffer: 0.01 M Tris-Cl, pH 7.3, 1% methanol
95% ethanol
Vacuum centrifuge concentrator (e.g. Savant Speed Vac SVC 100)
Other common laboratory equipment
Background
The ELISA kits are based on a competition between T4 in a sample and a constant amount of
enzyme-labeled T4 to bind an immobilized antibody recognizing T4. There do not appear to be
any commercially available ELISA kits for the measurement of circulating T4 in Xenopus laevis.
There are kits available for human and several other species, but these kits appear to be highly
dependent on the use of standards made with homologous serum. The kits are formulated to be
compatible with the presence of proteins in the blood which bind T4. These proteins include
thyroxine binding globulin, transthyretin (prealbumin), and albumin. Initial attempts to measure
T4 in X laevis using a commercially available kit produced results which were highly variable
and greatly different from the values obtained with non-immuno-metric methods (i.e. ICP-MS).
It was thought that this variability was to due to inappropriate interaction between the binding
proteins present in the sample, the amount of T4 in the sample, and the labeled T4 included in the
kit. It was thought that these problems would be avoided if the T4 binding ability of the sample
proteins was removed. This also necessitated using a standard which was free of binding
proteins. Reviewing literature of early methods for T4 measurement indicated that a
deproteination step with acid or ethanol was often used. Consistent and sensible results were
obtained with the ELISA kits when this adaptation was used.
Procedure
Follow the kit instructions, with these three exceptions:
1 Standards
A superstock solution of T4 at 154 |ig/ml was made in 0.05 M Na2CC>3. This was diluted
to 20 |ig/dl (200 ng/ml) in buffer. From this, additional dilutions in buffer were made to
give standards of 0.25, 0.5, 1.0, 2.0, 4.0 and 10.0 |ig/dl.
-------
3174
3175
3176
3177
3178
3179
3180
3181
3182
3183
3184
3185
3186
3187
3188
3189
3190
3191
3192
3193
3194
3195
3196
3197
3198
3199
3200
3201
3202
3203
3204
3205
3206
3207
3208
3209
3210
3211
3212
3213
3214
3215
3216
3217
3218
3219
86
2 Extraction/deproteination
Blanks (buffer), standards, and samples should be processed at room temperature as
follows. Add 60 |il of buffer, standards, or serum/plasma to a 0.6 ml micorcentrifuge
tube. If lesser amounts of sample are used, make up the volume to 60 |il with buffer.
Add 180 |il of ethanol and mix vigorously by vortexing. Repeat vortexing two more
times within a 10 minute period. Centrifuge for 10 minutes at 20,000 times g. Remove
supernatant to another tube and repeat extraction on pellet with another 180 |il of ethanol.
Centrifuge like above for 5 minutes. Combine supernatants and mix. Bring samples to
dryness in a vacuum centrifuge concentrator. Thoroughly resuspend residue in 60 |il of
buffer. Centrifuge for 10 minutes at 20,000 times g. Remove 55 ju.1 to another 0.6 ml
tube.
3 Combine samples/standards with T4-HRP conjugate before loading on the EIA
Plate
Add 220 |il of diluted T4-conjugate to the 55 |il from above and mix. Add 125 |il to
duplicate wells of the EIA plate. Continue with kit instructions.
Comments
Although this adaptation has only been tried with the kit from MP Biomedicals, it is expected
that kits from other suppliers that use a similar format should work as well.
-------
3220
3221
3222
3223
3224
3225
3226
3227
3228
3229
3230
3231
3232
3233
3234
3235
3236
3237
3238
3239
3240
3241
3242
3243
3244
3245
3246
3247
3248
3249
3250
3251
3252
3253
3254
3255
3256
3257
3258
3259
3260
3261
3262
3263
3264
3265
3266
87
8.2 Histological Development of the Gonad in Juvenile Xenopus laevis
Chad A. Blanksma1, Allen W. Olmstead2, Sigmund J. Degitz2, Jonathan Haselman2, Rodney D. Johnson2
'ORISE Research Participation Program at U.S. Environmental Protection Agency, ORD, NHEERL, Mid-Continental
Ecology Division, Duluth, MN
2U.S. Environmental Protection Agency, Office of Research and Development, National Health and Environmental
Effects Research Laboratory, Mid-Continental Ecology Division, Duluth, MN
Introduction
As directed by the Food Quality Protection Act, the US Environmental Protection
Agency is developing a screening program for endocrine disrupting compounds. The Larval
Amphibian Growth and Development Assay (LAGDA) is a tier II test intended to identify and
characterize the adverse consequences of toxicants that interfere with normal growth and
development of larval and juvenile frogs with particular emphasis on disruption of estrogen,
androgen, and thyroid mediated processes. A particularly important aspect of this assay is the
detection of alterations in gonad differentiation and development.
Assessments of gonad malformations in anuran species have traditionally relied on gonad
histology of tadpoles at the moment of metamorphosis completion (NF stage 66). Preliminary
experiments using Xenopus exposed to three model endocrine disruptors possessing estrogenic,
androgenic, or anti-aromatase activities indicated that gonad histology at this developmental
stage was inadequate for the goals of the assay. Specifically the gonads were too immature, with
the detection of mixed sex phenotypes being subjective and not informative with respect to a
toxicant's mechanism of action. In addition, exposure to endocrine disruptors such as
ethynylestradiol and fadrozole, which induce testicular oocytes in older animals, did not in frogs
sampled at metamorphosis.
We propose that assessing the gonads at a later stage of development would allow for
improved detection of aberrant gonad differentiation and development, yielding more
informative results for risk assessment purposes. In order to identify a more appropriate level of
gonad development for use in the LAGDA, we characterized normal gonad development in the
test species, X laevis, at two week intervals from completion of metamorphosis until 12 weeks
post-metamorphosis (wpm).
Materials and Methods
X laevis were cultured in-house at EPA-MED using filtered Lake Superior water at 21°C
with 12:12 hour light:dark photoperiod. No fewer than 4 males and 4 females were subsampled
at completion of metamorphosis (NF stage 66) and at 2 week time periods up to 12 weeks post
metamorphosis (wpm).
Frogs were euthanized by injection of MS-222 in accordance with animal care and use
guidelines. Specimens were trimmed by removing the gastrointestinal tract and liver to expose
the gonad, kidney, and reproductive ducts, followed by transverse cuts to the anterior margin of
the gonad and the caudal margin of the kidneys. The trimmed trunks containing gonads were
processed for histological analysis. Samples were embedded with the anterior surface down for
transverse sectioning so gonads could be assessed for morphological changes along their
longitudinal axis. Sections were cut at a thickness of 5 microns and stained with H&E.
Incremental sectioning produced 4-6 slides per sample, arranged anterior to posterior.
A representative image of the anterior region of each sample's gonad was taken using a
40x objective and each sample was assessed for developmental landmarks.
-------
88
Regions evaluated for analysis
Female Male Bar=5 mm
3267
Regions evaluated
Female Male Bar=5 mm
3268
3269
3270
3271
3272
3273
3274
3275
3276
3277
3278
3279
-------
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Stage V/Vi Ov.ir*
Tf*tekJllc Prophase ] - Leptotene,
r%go*ene. pachytene, d«pfcCysts with secondary spermatogone
& pnmiry vprtm#tc«ytrs
^•Diameter erf semrvferous coeds
increases due to ax^evjismg rsMmber &
germ cells iMthn cysts
—"^efe re54rs - cenflral d«?s comecl
semiMrous cords *tt3i r^fefent ducts
Suge W/X tessli
=>Semr*/erous tytnAn term with lumens
connected to rele testis and efferent
djcts
;
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3287
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3307
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90
Gonadal Development Timeline
Testis
NF 66 (~50d) 2 wpm (64d) 4wpm(78d) 6 wpm (92d) 8wpm(106d) 10wpm(120d) 12wpm(134d)
•Primary 'Early cyst •Secondary 'Primary
spermatogonium formation spermatogonium spermatocytes
•Undifferentiated -Primordial -Early rete testis 'Rete testis
somatic cells seminiferous cords formation
•Elongating
spermatids
•Seminiferous
tubules with
lumen
•All stages of germ
cell development
•Well developed
tubules and ducts
•New cyst
formation
Ovary
NF66(~50d) 2 wpm (64d) 4wpm(78d) 6 wpm (92d) 8wpm(106d) 10wpm(120d) 12wpm(134d)
•Primary 'Cysts with first 'First appearance "Diplotene oocyte •Diplotene oocyte 'Diplotene oocyte 'Diplotene oocyte
oogonium primary oocytes diplotene oocyte growth in size and growth growth continued growth continued
•Cyst formation 'Open ovarian number 'Germ cell nests (previtellogenic)
•Opening ovarian cavity 'Ovarian cavitily fewer and located
cavity lumen thins in periphery
Conclusions
The evaluation of gonad development is an important endpoint for protocols that test
chemicals for possible endocrine disruption effects. At metamorphosis, NF stage 66, the gonads
are only partially developed and sex-specific germ cells cannot be identified histologically.
Extending the test protocols past metamorphosis allows more comprehensive histological
evaluation of possible effects on gonad development. This study provides information which can
be used to optimize amphibian EDC test protocols. Extending the test protocol to at least 6
weeks post-metamorphosis provides substantially more insight into possible EDC effects on
gonad development. When sampling at this time or later, the gonads express:
• Nearly complete maturation of oocytes and spermatocytes
• Developed seminiferous tubules and associated ducts in males
• Developing oviduct system in females
• Developed supporting cells and tissues such as granulosa cells, Sertoli and Leydig cells.
References
Nieuwkoop PD, J Faber. 1967 Normal table of Xenopus laevis (Daudin): A Systematical and
Chronoligical Survey of the Development from the Fertilized Egg Till the End of
Metamorphosis. 2nd ed. Amsterdam: North Holland
Reproduction of Amphibians. Biological Systems in Vertebrates. 2009. Edited by Maria
Ogielska. Enfield (New Hampshire): Science Publishers
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3321
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91
Dumont, James N. Oogenesis in Xenopus laevis (Daudin). I. Stages of oocyte development in
laboratory maintained animals. Journal of Morphology Volume 136, Issue 2, pp. 153-179.
February 1972
8.3 Histopathology Guidance for Reading LAGDA Studies
Histopathology Guidance for Reading LAGDA Studies
Chad A. Blanksma1, Rodney D. Johnson2
1ORISE Research Participation Program at U.S. Environmental Protection Agency, ORD, NHEERL, Mid-Continental Ecology
Division, Duluth, MN
2U.S. Environmental Protection Agency, Office of Research and Development, National Health and Environmental Effects
Research Laboratory, Mid-Continental Ecology Division, Duluth, MN
The slides shall be evaluated by a pathologist for treatment-induced effects. The pathologist
shall perform the evaluation according to the Best Practices Guideline: Toxicologic
Histopathology (Crissman et al. 2004). It is recommended that evaluation begin with examining
representative slides from specimens selected from control and the two highest treatments to
determine likely treatment-related effects. Then, images of representative treatment-related
effects and associated severity scores shall be prepared to be used as references for scoring the
remainder of the specimens. Finally, after the initial evaluation, the study pathologist shall
perform a masked re-evaluation of all the specimens, but for only the previously observed
treatment-related lesions.
Each section shall be evaluated for gonad pathology, whereas kidney, reproductive duct, and
liver pathologies shall be based on the assessment of one representative section. The prevalence
and extent of the effects shall be assessed in the various treatments as compared to controls.
Effects data containing diagnoses and associated severity grades shall be reported in an Excel
spreadsheet similar to the example provided below. Diagnoses of treatment effects shall be
based on the columns in the example pathology spreadsheet. However, for pathologies not listed
in the spreadsheet, the pathologist shall provide new diagnostic criteria to the spreadsheet.
Pathology not related to treatment shall also be noted as a comment in the spreadsheet. Create
digital images of representative diagnostic features in an uncompressed file format. Images of
representative control tissues shall be included for comparison. Each image shall be at an
appropriate magnification to illustrate the features using the best judgment of the pathologist. An
embedded calibrated scale bar shall be included in all pictures. The spreadsheet shall indicate
imaged sections.
A report shall be prepared which includes a short summary of the protocols used in slide
preparation and a description of treatment-related effects based on concentration responses and
comparisons with control frogs. The narrative report shall include tables, figures, figure legends,
and citations, as necessary to describe treatment effects. Citations, tables, figures, figure legends,
shall be placed at the end of the narrative report. Other supporting histopathology data, images,
and histology technical reports shall be provided as spreadsheets, images without legends or
annotations, and documents. The titles and file names of reports and documentation shall
include: laboratory; species name; developmental stage; test chemical; and tissue examined. The
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3397
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3401
3402
3403
3404
92
file names of digital images shall identify the test chemical, treatment, and specimen ID. A
tabular cross-reference of image files and report figure numbers and titles shall be provided.
General approach to reading studies
From OECD guidance document for the Diagnosis of Endocrine-Related Histopathology of Fish
Gonads. (2009)
Studies are to be read by individuals experienced in reading toxicologic pathology studies, and
who are familiar with normal frog gonad histology, with gonadal physiology, and with general
responses of the gonads to toxicologic insult. Pathologists may be board certified (e.g., American
College of Veterinary Pathologists, The European Centre of Toxicologic Pathology, or other
certifying organizations); however, certification is not a requirement as long as the pathologist
has obtained sufficient experience with, and knowledge of, frog histology and toxicologic
pathology. Technicians should not be used to conduct readings due to the subtle nature of some
changes and the need for subjective judgments based on past experience.
It is recognized that there is a limited pool of pathologists with the necessary training and
experience that are available to read the gonadal histopathology for the Larval Amphibian
Growth and Development Assay. If an individual has toxicological pathology experience and is
familiar with gonadal histology in amphibian species, he/she may be trained to read the frog
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). This is because endocrine effects on histomorphology tend to be
incremental, and subtle differences between exposed and unexposed animals may not be
recognizable unless tissue sections from high dose animals can be knowingly compared to those
from controls. Thus, the aim of the initial evaluation is to ensure that diagnoses are not missed
(i.e., to avoid false-negative results). However, it is expected that any potential compound-
related findings will be re-evaluated by the pathologist in a blinded manner prior to reporting
such findings, when appropriate. Certain diagnostic criteria, such as relative increases or
decreases in cell populations, cannot be read in a blinded manner due to the diagnostic
dependence on control gonads. As a rule, treatment groups should be evaluated in the following
order: control, high-dose, intermediate-dose, and low-dose.
It is suggested that the pathologists be provided with all available information related to the
study prior to conducting their readings. Information regarding gross morphologic abnormalities,
mortality rates, and general test population performance and health are useful for pathologists to
provide comprehensive reports and to aid in the interpretation of findings. For a more
comprehensive discussion of standard reading approaches for toxicologic pathology studies,
please refer to the Society of Toxicologic Pathology Best Practices for reading toxicologic
histopathology studies (Crissman et al., 2004).
Severity Grading
In toxicologic pathology, it is recognized that compounds may exert subtle effects on tissues that
are not adequately represented by simple binary (positive or negative) responses. Severity
grading involves a semi-quantitative estimation of the degree to which a particular
histomorphological change is present in a tissue section (Shackelford et al., 2002). The purpose
of severity grading is to provide an efficient, semi-objective mechanism for comparing changes
(including potential compound-related effects) among animals, treatment groups, and studies.
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3405
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3407
3408
3409
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3423
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3453
93
Severity grading will employ the following system:
Not remarkable
Grade 1 (minimal)
Grade 2 (mild)
Grade 3 (moderate)
Grade 4 (severe)
A grading system needs to be flexible enough to encompass a variety of different tissue changes.
In theory, there are three broad categories of changes based on the intuitive manner in which
people tend to quantify observations in tissue sections:
(1) Discrete, these are changes that can be readily counted. Examples include atretic follicles,
oocytes in the testis, and clusters of apoptotic cells.
(2) Spatial, these are changes that can be quantified by area measurements. This includes lesions
that are typically classified as focal, multifocal, coalescing, or diffuse. Specific examples include
granulomatous inflammation and tissue necrosis.
(3) Global, these are generalized changes that would usually require more sophisticated measurement
techniques for quantification. Examples include increased hepatocyte basophilia,
Sertoli cell/interstitial cell hypertrophy, or quantitative alterations in cell populations.
General severity grading scale
• Not Remarkable. This grade is used if there are no findings associated with a particular diagnostic
criterion.
• Grade 1: Minimal. Ranging from inconspicuous to barely noticeable but so minor, small, or
infrequent as to warrant no more than the least assignable grade. For discrete changes, grade
1 is used when there are fewer than 2 occurrences per microscopic field, or 1 - 2 occurrences
per section. For multifocal or diffusely-distributed alterations, this grade is used for
processes where <20 % of the tissue in the section is involved.
• Grade 2: Mild. A noticeable feature of the tissue. For discrete changes, grade 2 is used when
there are 3 - 5 occurrences per microscopic field or per tissue section. For multifocal or diffusely-
distributed alterations, this grade is used for processes where 20 - 50 % of the tissue in
the section are involved.
• Grade 3: Moderate. A dominant feature of the tissue. For discrete changes, grade 3 is used
when there are 6 - 8 occurrences per microscopic field or per tissue section. For multifocal or
diffusely-distributed alterations, this grade is used for processes where 50 - 80 % of the tissue
in the section are involved.
• Grade 4: Severe. An overwhelming feature of the tissue. For discrete changes, grade 4 is
used when there are more than 9 occurrences per microscopic field or per tissue section. For
multifocal or diffusely-distributed alterations, this grade is used for processes where > 80 %
of the tissue in the section are involved.
At least some of the histomorphological changes that have been associated with EDCs in fish are
considered to be exacerbations of "normal", physiologic findings (e.g., oocyte atresia
[Nagahama, 1983; Tyler and Sumpter, 1996]). At the discretion of the pathologist, the severity
of a given change should be scored according to one of the following two methods:
(1) score compound-exposed animals relative to the severity of the same change in control
animals, or
(2) score all animals relative to "normal" as determined by the pathologist's experience.
For each important (i.e., treatment-associated) finding, the method that was used should be stated
in the Materials and Methods section of the pathology narrative report. By convention, severity
grading should not be influenced by the estimated physiologic importance of the change, since
this would add a further layer of subjectivity to the findings that complicates inter-laboratory
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94
results comparisons. For example, the presence of two oocytes in the testis should not be graded
as "severe", even if the pathologist considers this finding to be highly significant in terms of
endocrine modulation.
Data Recording.
An Excel worksheet form has been created that includes worksheets for primary, secondary, and
additional diagnoses to facilitate histopathology data collection. In this worksheet, each data
entry cell represents an individual fish. Additional sheets are available for comments and
additional findings. For each fish, the pathologist records a severity score associated with the
diagnosis. Diagnostic criteria with non-remarkable findings shall be denoted using (-). If there is
no reasonably appropriate diagnostic term for a particular finding, the pathologist can create a
term that can be recorded in the "Additional diagnoses" worksheet. If insufficient tissue is
available for diagnosis, this should be recorded as IT (insufficient tissue). If a target tissue is
missing, this should be recorded as MT (missing tissue).
Histopathology Report Format.
Each histopathology narrative report should contain the following five sections:
Introduction
Materials and Methods
Results
Discussion
Summary/Conclusions
(References)
The Introduction section briefly outlines the experimental design. The Materials and Methods
section describes any items or procedures that are essentially different from Section 1: Post-
mortem and Histotechnical Procedures. As applicable, specific severity grading criteria (see
Severity Grading) should also be listed in this section. The Results section should report findings
that are: (1) compound-related; (2) potentially compound-related; (3) novel or unusual. Detailed
histomorphological descriptions need only be included for findings that differ substantially from
diagnoses presented in Section 4 (Glossary and diagnostic criteria). It is intended that the Results
section should be as objective as possible {i.e., opinions and theories should be reserved for the
Discussion section). The Discussion section, which contains subjective information, should
address relevant findings that were reported in the Results section. Opinions and theories can be
included in this section, preferably backed by references from peer-reviewed sources, but
unsupported speculation should be avoided. The Summary/Conclusions section should
encapsulate the most important information from the Results and Discussion sections.
Developmental Staging of Xenopus gonads and Reproductive ducts.
Xenopus Testis Staging Criteria (Figure 1.)
Stage 1: Undifferentiated gonad.
Stage 2: Medullary region populated with individual primary spermatogonia and basophillic
undifferentiated somatic cells.
Stage 3: Somatic and germ cells organize into early seminiferous tubules populated with primary
spermatogonia and cysts of secondary spermatogonia.
Stage 4: Germ cell cysts contain developmental stages up to primary spermatocytes with
formation of rete testis; may have occasional spermatocysts that contain round or elongated
spermatids.
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95
3500
3501
3502
3503 Figure X* Example of staging system applied to Xenopus laevis testis. Bar = 50 urn
3504 Xenopus Ovary Staging Criteria (Figure 2.)
3505 Stage 1: Undifferentiated gonad. Gonad consists of an undifferentiated structure, germ cells, and
3506 somatic cells.
3507 Stage 2: Gonad identifiable as an ovary based on the presence of a discontinuously open lumen
3508 lined with epithelial cells. The germ cells within the cortex (the region outside the epithelial
3509 layer) consist of primary oogonia, cysts of primary mitotic oogonia, secondary oogonia, and very
3510 early meiotic oocytes.
3511 Stage 3: First appearance of diplotene oocytes in cortex. Structure and shape of the ovary
3512 similar to stage 1, but the appearance of diplotene oocytes is the first morphological difference
3513 between male and female germ cells. The most prevalent germ cell types at this stage are cysts
3514 of secondary oogonia and cysts of leptotene-pachytene primary meiocytes.
3515 Stage 4: Pre-vitellogenic (Dumont Stage I) diplotene oocytes are the most prevalent germ cell
3516 type observed by area and absolute cell counts. The central lumen is proportionately smaller
3517 while the whole ovary grows greatly in size and volume due to the growth of the oocytes. Cysts
3518 in earlier stages of oogenesis become fewer in number and are located along the periphery of
3519 ovary.
3520 Stage 5: Ovary consists almost entirely of vitellogenic oocytes (Dumont Stage IV). Pre-
3521 vitellogenic diplotene oocytes can be found along the periphery of the ovary and germ patches of
3522 primary and secondary oogonia are sparse and difficult to locate along the germinal epithelium.
Organization and development of seminiferous
Stage 5: All stages of spermatogenesis evident.
tubules and rete testis apparent.
Stage 2
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96
3523
3524
3525
3526
3527
3528
3529
3530
3531
3532
3533
3534
Figure 2. Staging system applied to Xenopus laevis ovaries. Bar = 50 jum (Stage 2 and 3); Bar = 100
jLini (Stage 4); Bar = 1 mm (Stage 5).
Xenopus Wolffian Duct Staging Criteria (Figure 3.)
Stage 1: The Wolffian ducts are paired ducts each located along the lateral edges of the kidneys,
usually adjacent to the suspensory connective tissue of the organ. At this stage, the epithelium of
the Wolffian duct consists of a single layer of cuboidal cells.
Stage 2: Pseudostratified epithelial cells line the duct asymmetrically. The height of the
pseudostratified epithelium is less than 3 times the height of the other epithelial cells.
Stage 3: The asymmetric epithelial lining of the duct is more pronounced with the
pseudostratified cells greater than 3 times the height of the other epithelial cells.
Stage 4: The duct is lined symmetrically with tall columnar epithelial cells.
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3537
3538
3539
3540
3541
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3547
3548
3549
97
Stage 1
20 pm
20 pm
Figure 3. Wolffian duct staging. W = Wolffian duct; K = kidney tubule; Bar = 20 uni.
Oviduct Staging (Figure 4.)
Stage 1: A discrete, solid, fibrous tag with proliferative fibroblasts in the location of normal
oviduct formation.
Stage 2: Formation of a rudimentary oviduct with a central lumen surrounded by a single layer
of cuboidal to columnar epithelium. The ductular lumen is frequently filled with cell debris.
Stage 3: Oviduct is comprised of an undulating, pseudostratified, ciliated columnar epithelium, a
discrete lumen, and patchy areas of glandular formation. Ductular lumen may contain cell debris.
Stage 4: Size of oviduct enlarged with prominent gland formation.
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98
3550 9Figure 4. Oviduct Staging. W = Wolffian duct; G = glandular region; Bar = 100 jim.
3551 Diagnostic Criteria for Specific Histopathological findings in EDC tests.
3552
Diagnostic Criteria for Specific Histopathological Findings
1
Undifferentiated gonad
2
Individual primary spermatogonia and undifferentiated somatic cells populate the medullary region
Testis Stage
3
Seminiferous tubules with primary spermatogonia and cysts of secondary spermatogonia
4
Primary spermatocytes with rete testis formation; may have occasional spermatocysts that contain
round or elongated spermatids
5
All stages of spermatogenesis evident
Tubule Lumen
Development
Score
1
Less than 5094 of seminiferous tubules have visible lumens
2
50% or greater seminiferous tubules have visible lumens
1
Undifferentiated gonad
Gonad
2
Gonad identifiable as an ovary based on the presence of a discontinuously open lumen lined with
epithelial cells; germ cells within the cortex consist of primary oogonia, cysts of primary mitotic
oogonia, secondary oogonia, and very early meiotic oocytes
3
First appearance of diplotene oocytes in cortex; the most prevalent germ cell types at this stage are
cysts of secondary oogonia and cysts of leptotene-pachytene primary meiocytes
Ovary Stage
4
Pre-vitellogenic (Dumont Stage I) diplotene oocytes are the most prevalent germ cell type observed
by area and absolute cell counts; the central lumen is proportionately smaller while the whole ovary
grows greatly in size and volume due to the growth of the oocytes; cysts in earlier stages of oogenesis
become fewer in number and are located along the periphery of ovary
5
Ovary consists almost entirely of vitellogenic oocytes (Dumont Stage IV); pre-vitellogenic diplotene
oocytes can be found along the periphery of the ovary and germ patches of primary and secondary
oogonia are difficult to locate
Gonad
Degeneration
1
A single degenerating germ cell to less than three clusters of degenerating germ cells per gonad
2
Three or more clusters of degenerating germ cells, but less than 25% of gonad affected
3
Gonad consists of greater than 25? o but less than 50% of degenerating germ cells
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99
4
Gonad consists of 50% or more of degenerating germ cells
Mononuclear
Cell Infiltrates
1
Small, focal infiltrates in one gonad
2
Small focal infiltrates in both gonads, or large area of infiltrates in one gonad
3
Large areas of infiltrates in both gonads
4
Gonads contain greater than 50% of infiltrates by area
Kidney
Mineralization
1
Less than three small foci of mineralization per gonad
2
Three or more small foci of mineralization per gonad, or less than three large foci
3
Three or more large foci of mineralization per gonad
4
Mineralized deposits in 50% or more tubules
Tubule Dilation
1
Minimal to mild dilation of less than three tubule clusters
2
Mild to moderate dilation of three or more tubule clusters
3
Moderate dilation affecting 50% or more tubules
4
Massive dilation of one or more tubules
Oviduct
Oviduct Stage
1
Duct is either a fibrous connective tissue tag, or is essentially non-existent
2
Duct has an epithelial cell lining, and is comparable in size to the Wolffian duct
3
Duct has an epithelial cell lining, and is substantially larger (1.5x or greater) than the size of the
Wolffian duct
4
Duct is substantially larger (1.5x or greater) than the size of the Wolffian duct Epithelial cells have
formed large, basophilic glandular structures
Wolffian
Duct
Wolffian Duct
Stage
1
Epithelial lining of Wolffian duct is completely or focally less than two cell layers thick
2
Epithelial lining of Wolffian duct is two cell layers thick or greater, but clear apical portion of cell (if
present) is less than half the cell height
3
Epithelial lining of Wlffian duct is two cell layers thick or greater, and clear apical portion of the
columnar cells is at least half the cell height but less than 2/3 of the cell height
4
The clear apical portion of the columnar cells represents 2/3 or greater of the total cell height
3553
3554 References:
3555
3556 Crissman, J.W., Goodman, D.G., Hildebrandt, P.K., Maronpot, R.R., Prater, D.A., Riley, J.H., Seaman,
3557 W.J., Thake, D.C. (2004) Best practices guideline: Toxicologic pathology. Toxicol. Pathol. 32,
3558 126-131.
3559 Johnson, R., Wolf, J., and Braunbeck T. (2009). "OECD Guidance Document for the Diagnosis
3560 of Endocrine-Related Histopathology of Fish Gonads" Organization for Economic and
3561 Cooperative Development, Paris, France.
3562 Nieuwkoop PD, J Faber. 1967 Normal table of Xenopus laevis (Daudin): A Systematical and
3563 Chronoligical Survey of the Development from the Fertilized Egg Till the End of Metamorphosis.
3564 2nd ed. Amsterdam: North Holland
3565 Reproduction of Amphibians. Biological Systems in Vertebrates. 2009. Edited by Maria
3566 Ogielska. Enfield (New Hampshire): Science Publishers
3567 Dumont, James N. Oogenesis in Xenopus laevis (Daudin). I. Stages of oocyte development in
3568 laboratory maintained animals. Journal of Morphology Volume 136, Issue 2, pp. 153-179.
3569 February 1972
3570
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3589
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3598
3599
3600
3601
3602
3603
100
8.4 Inter-laboratory validation study results
8.4.1 Prochloraz results
Measured test concentrations
All four labs met the criteria of maintaining CVs of <20% over the entire test for all
concentrations with the exception of the 6.67 and 20|ig/L nominal concentrations at C which had
CVs of 0.38 and 0.23 and were on average 84% and 25% above nominal respectively (Table
8.4.1-1). Measured concentrations did not show any crossover with other treatment levels in all
labs except C which had several overlaps in treatment concentrations through week five of
exposure. CV values between replicates could not be calculated on a weekly basis for B and C
due to measurements of only one replicate tank per week. Treatment levels will be referred to as
nominal concentrations regardless of mean-measured concentrations reported by each of the labs.
Table 8.4.1-1
Inte(laboratory comparison of measured prochloraz concentrations throughout the duration of the LAGDA (mean ± SD). NR: not
reported. Highlighted cells have CVs >20%,
Nominal PZ concentration
A
D
B
C
0 |jg/L
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3604
3605
3606
3607
3608
3609
3610
3611
3612
3613
3614
3615
3616
3617
3618
3619
3620
3621
3622
3623
3624
3625
3626
101
body weight and snout-vent length in the 180|ig/L treatment (ANOVA with Dunnett). There
were no concentration-related effects on larval growth in the C study (Table 8.4.1-3).
Table 8.4.1-3
Inte(laboratory comparison of larval growth and development (NF62) following prochloraz exposure (mean
± SD). Highlighted cells are significantly different than control (a = 0.05).
Lab D
B
C
Nominal PZ concentration
Weight (g)
0 |jg/L
0.791 ± 0.130
1.08 ±0.17
0.616 ±0.139
6.7 Mg/L
0.742 ± 0.244
1.21 ±0.22
0.627 ±0.104
20 Mg/L
0.722 ± 0.204
1.08 ±0.22
0.640 ±0.124
60 |jg/L
0.723 ±0.153
1.13 ± 0.14
0.707 ±0.154
180 |jg/L
0.708 ±0.168
0.92 ±0.18
0.658 ±0.174
Length (mm)
o |jg/L
18.8 ± 1.0
20.8 ± 1.0
17.1 ±1.4
6.7 Mg/L
18.5 ± 1.8
21.3 ± 1.2
17.5 ± 1.0
20 Mg/L
18.4 ± 1.6
20.7 ± 1.2
16.9 ± 1.3
60 |jg/L
18.3 ± 1.0
21.0 ±0.9
18.1 ±1.3
180 |jg/L
18.1 ±1.2
19.6 ± 1.2
16.8 ± 1.3
Time to NF62 (d)
o |jg/L
43 ±2
41 ± 3
44 ±6
6.7 Mg/L
42 ± 2
39 ±2
44 ±4
20 Mg/L
41 ± 2
40 ±3
43 ±5
60 |jg/L
43 ±2
41 ± 3
45 ±5
180 ng/L
42 ± 2
40 ±2
45 ± 10
Larval blood thyroxine
A measured T4 levels in serum using an HPLC/ICP-MS method whereas the other labs used an
ELISA method to measure T4 in plasma. The ELISA method was not optimized so data could
not be included. There was a significant increase in T4 concentrations in the 60 and 180|ig/L
treatment at A (ANOVA with Dunnett, Table 8.4.1-4).
Table 8.4.1-4
Interlaboratory comparison of larval blood thyroxine
concentrations following prochloraz exposure (mean ±
SD). Highlighted cells are significantly different than
control (a = 0.05).
Nominal PZ concentration
T4 (ng/mL)
Lab A (NF66)
0 |jg/L
5.4 ± 1.7
6.7 Mg/L
6.5 ± 1.9
20 Mg/L
7.7 ± 1.9
60 |jg/L
8.2 ± 2.7
180 ng/L
11.8 ±3.3
Juvenile growth
Juvenile female weight and length was significantly impeded in the 180|ig/L treatment at D
whereas only length in the 20, 60 and 180 |ig/L treatments showed a significant decrease at B
(Jonckeere-Terpstra). Juvenile male weight and length showed a significant decrease in the 60
and 180 |ig/L treatments also at D. The other labs showed no significant concentration-related
effect on juvenile growth (Table 8.4.1-5).
Juvenile liver-somatic index (LSI)
-------
3627
3628
3629
3630
3631
3632
3633
3634
3635
3636
102
A, D and B all showed significant decreases in both male and female LSI (Table 8.4.1-5). C
showed a significant female LSI increase in the 180|ig/L treatment.
Juvenile plasma vitellogenin
A, D and C showed a significant increase in both juvenile male and female plasma vitellogenin
titers (Table 8.4.1-5). B showed no significant concentration-related trend but also reported titer
values that were much lower on average than the other three labs.
-------
Table 8.4.1-5
Interlaboratory comparison of juvenile growth and plasma vitellogenin titers following prochloraz exposure (mean ± SD). Highlighted cells are significantly different than control (a = 0,05),
Male Female
A
D
B
c
A
D
B
C
Nominal PZ concentration
Weight (g)
Weight (g)
0 |jg/L
13.1 ±2.0
18.6 ±4.1
11.5 ±3.6
4.0 ±0.8
12.9 ±2.6
20.0 ±4.6
13.8 ±5.0
4.1 ±0.8
6.7 Mg/L
12.5 ±2.6
19.4 ±3.8
13.2 ±5.4
3.7 ±0.5
13.8 ±3.4
18.2 ±3.0
12.3 ±5.0
3.7 ±0.6
20 Mg/L
11.8 ±2.8
17.6 ±3.8
12.9 ±5.0
4.0 ± 1.1
14.1 ±2.3
20.3 ±4.9
12.2 ±4.8
4.2 ±1.1
60 |jg/L
12.6 ±2.3
16.1 ±3.9
12.5 ±3.3
3.7 ±0.8
13.8 ±2.6
18.1 ±4.4
12.0 ±5.4
3.4 ±0.7
180 |jg/L
12.5 ±2.7
10.8 ±5.2
11.4 ±4.3
4.0 ± 1.2
13.3 ±2.8
14.3 ±7.1
11.4 ±4.5
4.7 ± 1.6
Length (mm)
Length (mm)
o |jg/L
47.2 ±2.8
53.0 ±3.9
43.0 ±4.5
32.8 ±2.2
47.2 ±2.6
55.0 ±5.2
46.4 ±5.4
32.9 ±2.2
6.7 Mg/L
46.4 ±3.0
53.9 ±4.1
44.8 ±6.7
32.5 ± 1.3
47.9 ±4.0
51.4 ±8.6
43.8 ±6.8
32.2 ±1.9
20 Mg/L
45.1 ±4.2
52.0 ±3.8
44.7 ±5.4
31.9 ±2.5
47.8 ±2.0
55.2 ±4.7
43.3 ±5.4
33.5 ±2.9
60 |jg/L
46.9 ±2.5
50.7 ±4.0
44.1 ±3.8
32.5 ±2.4
47.7 ±2.9
52.9 ±4.5
43.0 ±6.9
32.1 ±1.8
180 |jg/L
46.4 ±3.8
43.6 ±8.5
42.7 ±6.2
32.2 ±3.0
48.1 ±3.7
48.3 ±9.8
42.2 ±6.8
33.9 ±3.9
LSI
LSI
o |jg/L
0.059 ±0.010
0.058 ± 0.005
0.057 ± 0.009
0.035 ± 0.007
0.061 ±0.007
0.059 ± 0.006
0.064 ±0.014
0.032 ± 0.006
6.7 Mg/L
0.056 ±0.012
0.053 ±0.012
0.064 ±0.015
0.031 ± 0.005
0.056 ±0.013
0.057 ± 0.006
0.059 ±0.014
0.032 ± 0.007
20 Mg/L
0.055 ±0.012
0.054 ± 0.007
0.053 ±0.014
0.034 ± 0.006
0.060 ± 0.007
0.055 ± 0.004
0.055 ±0.010
0.032 ± 0.006
60 |jg/L
0.052 ± 0.007
0.049 ± 0.005
0.052 ± 0.007
0.032 ± 0.007
0.051 ± 0.006
0.051 ± 0.006
0.056 ±0.010
0.031 ± 0.005
180 |jg/L
0.050 ± 0.004
0.040 ± 0.009
0.048 ± 0.009
0.037 ± 0.005
0.052 ± 0.005
0.044 ± 0.007
0.048 ±0.011
0.037 ± 0.006
VTG (mg/mL)
VTG (mg/mL)
o |jg/L
0.03 ± 0.02
0.01 ±0.02
0.00 ± 0.00
0.00 ± 0.00
0.03 ± 0.03
0.02 ± 0.07
0.01 ±0.02
0.03 ±0.01
6.7 Mg/L
0.05 ± 0.06
0.02 ± 0.04
0.01 ±0.02
0.01 ± 0.002
0.07 ±0.10
0.01 ±0.01
0.03 ± 0.05
0.03 ± 0.02
20 Mg/L
0.05 ± 0.04
0.01 ±0.02
0.00 ± 0.00
0.01 ±0.001
0.07 ±0.10
0.01 ±0.01
0.00 ± 0.00
0.04 ±0.01
60 |jg/L
0.12 ± 0.13
0.02 ± 0.03
0.00 ± 0.00
0.01 ± 0.004
0.12 ± 0.13
0.04 ± 0.07
0.00 ±0.01
0.09 ± 0.05
180 ng/L
0.10±0.10
0.02 ± 0.02
0.00 ±0.01
0.01 ± 0.002
0.25 ±0.32
0.04 ± 0.07
0.01 ±0.01
0.10 ±0.04
-------
104
Larval thyroid histopathology
Common treatment-related observations in larval thyroid glands across all four studies were follicular cell hypertrophy and hyperplasia. Although
these pathologies were present in larvae at B, there were no clear concentration-dependent trends in their study. However, the other three labs
showed significant increases in follicular cell hypertrophy and two of those labs also showed significant increases in follicular cell hyperplasia.
Gland hypertrophy and increased or decreased follicular lumen area were observed minimally in the 180 |ig/L treatments inconsistently throughout
the labs, but each were found to be significantly different than control in the 180 |ig/L treatment in at least one of the labs (Table 8.4.1-6).
Table 8.4.1-6
Prevalance (%) and severity of larval thyroid gland histopathological observations showing significant treatment-related effects in at least one of the four studies (NR: not remarkable, highlighted cells are significantly
different than control based on RSCABS analysis; dash: not observed in any treatment),
Lab A (NF66) D (NF62) B (NF62) C (NF62)
Prochloraz concentration ([jg/L)
0
6.7
20
60
180
0
6.7
20
60
180
0
6.7
20
60
180
0
6.7
20
60
180
n
40
23
25
25
24
56
28
28
28
28
40
20
20
20
20
39
17
16
18
18
replicates
8
5
5
5
5
8
4
4
4
4
8
4
4
4
4
8
4
4
4
4
Thyroid Gland Hypertrophy
NR
100
100
100
100
93
100
100
100
100
94
Mild
0
0
0
0
7
0
0
0
0
6
Moderate ...
0
0
0
0
0
0
0
0
0
0
Severe ...
0
0
0
0
0
0
0
0
0
0
Follicular Cell Hypertrophy
NR
38
65
28
28
8
93
86
89
71
46
85
70
95
75
70
31
18
6
11
11
Mild
58
35
68
60
75
7
14
11
29
54
13
30
5
20
30
56
35
56
28
39
Moderate
5
0
4
12
17
0
0
0
0
0
3
0
0
5
0
10
47
38
61
44
Severe
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
3
0
0
0
6
Follicular Cell Hyperplasia
NR
78
87
80
76
67
95
86
79
68
46
95
80
95
90
90
85
59
50
39
61
Mild
23
13
20
24
33
5
14
21
32
54
5
20
5
10
10
13
41
44
56
33
Moderate
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
3
0
6
6
6
Severe
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Incresed Follicular Lumen Area
NR
100
100
100
100
93
Mild
0
0
0
0
7
Moderate ...
0
0
0
0
0
Severe ...
0
0
0
0
0
Decreased Follicular Lumen Area
NR
100
100
100
100
92 .....
100
94
100
100
100
Mild
0
0
0
0
8 .....
0
6
0
0
0
Moderate
0
0
0
0
0 .....
0
0
0
0
0
Severe
0
0
0
0
0 .....
0
0
0
0
0
-------
105
Liver histopathology
Consistencies in treatment-related findings were hepatocellular degeneration and hepatocellular atrophy in two of the four labs. A and B showed
significant hepatocellular degeneration in the 180 |ig/L treatment whereas hepatocellular atrophy showed slightly higher sensitivity to prochloraz
treatment at D and B with both labs showing significant effects in the 20, 60 and 180 |ig/L treatments. C exhibited a different suite of treatment-
related liver pathologies including basophilia, increased hepatocellular vacuolization and granulomatous inflammation, all of which were
significantly different than control at 60 and 180 |ig/L. Cellular hypertrophy was also observed in C samples and was significantly different than
control in the 20, 60 and 180 |ig/L treatments (Table 8.4.1-7). Prevalence, severity, sensitivity and pathologies were extremely similar between
males and females with slight variation in severity and sensitivity (Tables 8.4.1-7 and 8.4.1-8). The stark difference between findings at C compared
to the other three labs could be due to the fact that tissues were processed and evaluated by a different pathologist, or could be due to the extreme
lack of growth of Cs organisms compared to the other three labs.
Table 8.4.1-7
Prevalence (%) and severity of juvenile male liver histopathological observations showing significant treatment-related effects in at least one of the four studies (NR: not remarkable; highlighted cells are significantly
Lab A
D
B
Prochloraz concentration (yg/L) 0 6.7 20 60 180
0
6.7
20
60
180
0
6.7
20
60
180
n 16 10 10 10 10
23
12
12
12
12
24
12
12
11
11
replicates 8 5 5 5 5
8
4
4
4
4
8
4
4
4
4
Basophilia
NR
Minimal
Mild
Moderate
Severe
Increased Hepatocellular Vacuolization
NR
Minimal
Mild
Moderate
Severe
Granulomatous Inflammation
Cellular Hypertrophy
Hepatocellular Degeneration
NR
Minimal
Mild
Moderate
Severe
NR
Minimal
Mild
Moderate
Severe
NR
100
100
90
70
0
100
100
100
91
9
Minimal
0
0
10
30
10
0
0
0
9
73
Mild
0
0
0
0
70
0
0
0
0
18
Moderate
0
0
0
0
20
0
0
0
0
0
Severe
0
0
0
0
0
0
0
0
0
0
0
6.7
20
60
180
37
14
18
15
20
8
4
4
4
4
100
100
100
67
0
0
0
0
27
35
0
0
0
7
35
0
0
0
0
10
0
0
0
0
20
100
100
100
67
0
0
0
0
27
35
0
0
0
7
35
0
0
0
0
10
0
0
0
0
20
49
36
28
0
5
49
50
72
87
55
3
14
0
13
35
0
0
0
0
5
0
0
0
0
0
100
100
50
13
10
0
0
50
67
40
0
0
0
20
50
0
0
0
0
0
0
0
0
0
0
-------
106
Hepatocellular Atrophy
NR
100
100
75
25
0
100
100
83
27
18
Minimal -
0
0
8
17
0
0
0
8
27
18
Mild
0
0
17
42
50
0
0
8
36
36
Moderate -
0
0
0
17
42
0
0
0
9
27
Severe -
0
0
0
0
8
0
0
0
9
27
Table 8.4.1-8
Prevalence (%) and severity of juvenile female liver histopathological observations showing significant treatment-related effects in at least one of the four studies (NR: not remarkable; highlighted cells are significantly different
Lab
A
D
B
C
Prochloraz concentration (pq/L) 0
6.7
20
60
180
0
6.7
20
60
180
0
6.7
20
60
180
0
6.7
20
60
180
n 16
10
10
10
10
25
12
12
12
12
24
12
12
13
13
43
26
22
25
20
replicates 8
5
5
5
5
8
4
4
4
4
8
4
4
4
4
8
4
4
4
4
Basophilia
NR
Minimal
Mild
Moderate
Severe
Increased Hepatocellular Vacuolization
NR
Minimal
Mild
Moderate
Severe
Granulomatous Inflammation
Cellular Hypertrophy
Hepatocellular Degeneration
Hepatocellular Atrophy
NR
Minimal
Mild
Moderate
Severe
NR
Minimal
Mild
Moderate
Severe
96
100
100
100
100
0
0
0
0
0
4
0
0
0
0
0
0
0
0
0
0
0
0
0
0
NR
100
100
90
60
0
-
-
-
-
-
100
100
92
100
31
Minimal
0
0
10
40
60
-
-
-
-
-
0
0
8
0
62
Mild
0
0
0
0
10
-
-
-
-
-
0
0
0
0
8
Moderate
0
0
0
0
30
-
-
-
-
-
0
0
0
0
0
Severe
0
0
0
0
0
-
-
-
-
-
0
0
0
0
0
NR
_
_
_
_
_
100
100
83
42
0
100
100
92
77
15
Minimal
-
-
-
-
-
0
0
8
33
8
0
0
0
23
23
Mild
-
-
-
-
-
0
0
0
17
33
0
0
8
0
46
Moderate
-
-
-
-
-
0
0
8
0
33
0
0
0
0
8
Severe
-
-
-
-
-
0
0
0
8
25
0
0
0
0
8
100
100
100
48
0
0
0
0
28
25
0
0
0
24
45
0
0
0
0
30
0
0
0
0
0
100
100
100
48
0
0
0
0
28
25
0
0
0
24
50
0
0
0
0
25
0
0
0
0
0
44
42
27
12
5
49
58
64
84
70
7
0
9
4
25
0
0
0
0
0
0
0
0
0
0
100
100
77
12
15
0
0
23
88
35
0
0
0
0
50
0
0
0
0
0
0
0
0
0
0
-------
107
Male gonad histopathology
Histopathological findings in the testes of prochloraz-exposed male frogs were widespread and varied in consistency across laboratories. Of the ten
observed pathologies showing significant concentration-dependent trends in at least one of the four labs, increased spermatogonia, germinal epithelial
thinning, and gonadal degeneration (necrosis/apoptosis) were represented as significant findings in all labs except B. Interstitial cell
hyperplasia/hypertrophy, asynchronous development within cysts, and intratubular eosinophilic droplets were represented as significant and
consistent between A and D but varied in sensitivity and severity. Interstitial fibrosis was also substantially observed at A and D but only showed a
significant treatment-related effect in A samples at 180 |ig/L. Mononuclear cellular infiltrate was observed (minimal to mild) across all four labs, but
only showed significantly increased prevalence in A and B, although the labs differed in observed sensitivity. C showed significant, albeit slight,
advancement of tubule lumen development in the 180 |ig/L treatment whereas this observation was not noted in the A and D reports (different
histopathology contract). Finally, B had a significant minimal increase in spermatids in the 180 |ig/L treatment and was the only lab exhibiting this
pathology (Table 8.4.1-9).
Table 8.4.1-9
Prevalence (%) and severity of juvenile male gonad histopathological observations showing significant treatment-related effects in at least one of the four studies (NR: not remarkable; highlighted cells are significantly different
than control based on RSCABS analysis; dash: not observed in any treatment). Note: "Severe" rating only shown if observed in at least one of the four studies,
Lab
A
D
B
C
Prochloraz concentration ([jg/L)
0
6.7
20
60
180
0
6.7
20
60
180
0
6.7
20
60
180
0
6.7
20
60
180
n
36
25
24
24
25
39
25
22
21
18
42
22
23
13
15
37
14
18
15
20
replicates
8
5
5
5
5
8
4
4
4
4
8
4
4
4
4
8
4
4
4
4
Testicular interstitial cell hyperplasia/hypertrophy
NR
83
68
96
50
16
85
68
77
71
44
100
100
96
100
100
Minimal
17
32
4
46
52
15
32
23
29
56
0
0
0
0
0
-
-
-
-
-
Mild
0
0
0
4
32
0
0
0
0
0
0
0
4
0
0
-
-
-
-
-
Moderate
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
-
-
-
-
-
Increased spermatids
NR
100
100
96
100
87
Minimal
-
-
-
-
-
-
-
-
-
-
0
0
4
0
13
-
-
-
-
-
Mild
-
-
-
-
-
-
-
-
-
-
0
0
0
0
0
-
-
-
-
-
Moderate
-
-
-
-
-
-
-
-
-
-
0
0
0
0
0
-
-
-
-
-
Increased spermatogonia
NR
94
96
92
88
76
90
80
82
86
39
100
93
94
87
70
Minimal
6
4
4
8
12
10
20
18
10
50
-
-
-
-
-
0
7
6
13
30
Mild
0
0
0
0
8
0
0
0
5
11
-
-
-
-
-
0
0
0
0
0
Moderate
0
0
4
4
4
0
0
0
0
0
-
-
-
-
-
0
0
0
0
0
Tubule lumen development
NR
0
0
0
0
0
0
0
0
0
0
Minimal
-
-
-
-
-
-
-
-
-
-
95
100
96
85
93
54
64
44
33
15
Mild
-
-
-
-
-
-
-
-
-
-
5
0
4
15
7
46
36
56
67
85
Moderate
-
-
-
-
-
-
-
-
-
-
0
0
0
0
0
0
0
0
0
0
Germinal epithelial thinning
NR
92
84
92
54
12
85
56
41
29
17
95
100
100
100
100
100
93
100
67
35
Minimal
0
8
4
38
40
8
36
45
43
11
2
0
0
0
0
0
7
0
27
25
Mild
6
4
4
8
16
5
4
9
14
22
2
0
0
0
0
0
0
0
7
30
Moderate
3
4
0
0
28
3
4
0
14
44
0
0
0
0
0
0
0
0
0
10
Severe
0
0
0
0
4
0
0
5
0
6
0
0
0
0
0
0
0
0
0
0
Gonadal degeneration (necrosis / apoptosis)
NR
92
64
96
83
32
77
56
73
71
50
50
9
4
0
13
32
14
0
0
5
Minimal
8
32
4
17
64
23
44
27
29
50
38
73
65
77
60
65
86
94
93
75
Mild
0
4
0
0
4
0
0
0
0
0
12
18
30
23
27
3
0
6
7
20
-------
108
Moderate
Mononuclear cellular infiltrate
Asynchronous development within cysts
Interstitial fibrosis
Intratubular eosinophilic droplets
NR
92
92
79
88
56
69
44
73
33
72
Minimal
8
8
21
13
40
31
52
18
62
22
Mild
0
0
0
0
4
0
4
9
5
6
Moderate
0
0
0
0
0
0
0
0
0
0
NR
100
80
75
67
40
97
92
91
90
56
Minimal
0
16
17
25
44
3
8
9
10
39
Mild
0
4
8
8
12
0
0
0
0
6
Moderate
0
0
0
0
4
0
0
0
0
0
NR
75
52
71
75
12
69
56
45
62
61
Minimal
19
40
29
21
36
28
40
50
29
39
Mild
6
8
0
0
52
3
4
5
10
0
Moderate
0
0
0
4
0
0
0
0
0
0
NR
94
96
100
96
76
79
60
82
67
28
Minimal
3
4
0
4
24
15
20
14
10
50
Mild
3
0
0
0
0
5
16
5
24
17
Moderate
0
0
0
0
0
0
4
0
0
6
81
27
52
62
53
86
79
89
93
90
14
50
35
38
40
8
21
11
7
10
5
23
13
0
7
5
0
0
0
0
0
0
0
0
0
0
0
0
0
0
95
100
100
100
100
5
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Female gonad histopathology
Histopathological findings in the ovaries of prochloraz-exposed female frogs were somewhat inconsistent and mostly minimal. Mononuclear cellular
infiltrate was observed across all four laboratories (minimal to mild) but only showed significant concentration-related trends at A and C, though C
exhibited higher sensitivity with all four treatments being significantly different than control. Germ cell degeneration showed significant
concentration-related trends at B and C down to 20 and 6.7 |ig/L respectively and was not observed in either A or D studies. Granulomatous
inflammation, ovarian hypoplasia, and interstitial fibrosis were primarily observed at A and D (minimal) and only the 180 |ig/L treatments were
significantly different than control in one of the two labs for each diagnosis. Gonadal degeneration (necrosis/apoptosis) was only observed in the D
study (minimal) with the 60 and 180 |ig/L treatments being significantly different than control. Finally, ovary stages were evaluated in the B and C
studies and there appeared to be significant acceleration of ovary development, albeit slight, in Cs 60 and 180 |ig/L treatments compared to control
(Table 8.4.1-10).
Table 8.4.1-10
Prevalence (%) and severity of juvenile female gonad histopathological observations showing significant treatment-related effects in at least one of the four studies (NR: not remarkable; highlighted cells are significantly different
than control based on RSCABS analysis; dash: not observed in any treatment). Note: There were no diagnoses rated "severe" so data not shown,
Lab
ACEHR
Prochloraz concentration ([jg/L)
0
6.7
20
60
180
0
6.7
20
60
180
0
6.7
20
60
180
0
6.7
20
60
180
n
43
26
22
24
25
40
15
16
19
22
37
18
16
26
25
42
26
22
25
20
replicates
8
5
5
5
5
8
4
4
4
4
8
4
4
4
4
8
4
4
4
4
Gonad Stage
Germ cell degeneration
1 ....
0
0
0
0
0
0
0
0
0
0
2 - ... -
0
0
0
4
0
2
0
0
0
5
3 - - - - -
0
6
0
4
4
52
23
64
24
15
4 ....
100
94
100
92
96
45
77
36
76
80
5 - - - - -
0
0
0
0
0
0
0
0
0
0
NR
95
89
63
73
84
38
4
18
4
25
Minimal .....
5
11
38
27
16
62
81
50
88
50
-------
Mild
Moderate
Gonadal degeneration (necrosis / apoptosis)
NR
Minimal
Mild
Moderate
Mononuclear cellular infiltrate
NR
Minimal
Mild
Moderate
Granulomatous inflammation
NR
Minimal
Mild
Moderate
Ovarian hypoplasia
NR
Minimal
Mild
Moderate
Interstitial fibrosis
NR
Minimal
Mild
Moderate
-
-
-
-
-
80
-
-
-
-
-
18
-
-
-
-
-
0
-
-
-
-
-
3
72
77
77
50
44
50
26
15
18
50
48
40
2
8
5
0
8
10
0
0
0
0
0
0
100
100
91
100
92
95
0
0
9
0
4
5
0
0
0
0
0
0
0
0
0
0
4
0
95
92
95
96
72
95
5
8
5
4
28
5
0
0
0
0
0
0
0
0
0
0
0
0
93
96
86
92
80
70
7
0
14
8
12
28
0
4
0
0
8
0
0
0
0
0
0
3
-
-
-
-
0
0
0
0
-
-
-
-
0
0
0
0
67
88
47
45
_
_
_
_
33
13
53
55
-
-
-
-
0
0
0
0
-
-
-
-
0
0
0
0
-
-
-
-
20
50
11
55
41
22
19
27
53
44
79
41
46
67
81
62
27
6
11
5
14
11
0
12
0
0
0
0
0
0
0
0
93
100
100
91
7
0
0
9
0
0
0
0
0
0
0
0
100
100
95
95
0
0
5
5
0
0
0
0
0
0
0
0
47
69
42
32
47
31
53
59
7
0
5
9
0
0
0
0
109
0
0
15
32
8
25
0
0
0
0
0
0
40
43
15
18
12
5
44
57
50
59
64
90
16
0
35
23
24
5
0
0
0
0
0
0
-
100
96
100
100
100
-
0
4
0
0
0
-
0
0
0
0
0
-
0
0
0
0
0
Male reproductive duct histopathology
Male oviducts exhibited very consistent responses to prochloraz treatment across all four labs. Significant concentration-dependent delays in
involution were evident in the 60 and 180 |ig/L treatments for all labs and down to 20 |ig/L for A, B and C (Table 8.4.1-11). Male Wolffian duct
stages were similar between prochloraz treatments and controls for the B and C studies (data not shown).
Table 8.4.1-11
Histological staging (%) of juvenile male oviducts from four different labs performing the LAGDA with prochloraz (highlighted cells are significantly different than control based on RSCABS analysis).
Lab
A
D
B
C
Prochloraz concentration ([jg/L)
0
6.7
20
60
180
0
6.7
20
60
180
0
6.7
20
60
180
0
6.7
20
60
180
n
43
25
20
23
24
39
25
22
21
18
42
22
23
13
15
37
14
18
15
20
replicates
8
5
5
5
5
8
4
4
4
4
8
4
4
4
4
8
4
4
4
4
duct stage
1
84
88
50
31
4
31
24
18
10
11
78
68
52
15
7
89
79
50
20
10
2
16
8
42
58
92
69
76
82
90
89
22
32
48
85
93
11
21
50
80
90
3
0
4
8
12
4
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
-------
110
Female reproductive duct histopathology
There were no treatment-related effects on female oviduct development across all four studies. However, female Wolffian duct development was
slightly advanced in the 180 |ig/L treatment at B and minimal mononuclear inflammation of the Wolffian duct was significantly present in the 180
|ig/L treatment at A (Table 8.4.1-12).
Table 8.4.1-12
Histological staging and pathology (%) of juvenile female reproductive ducts from four different labs performing the LAGDA with prochloraz (highlighted cells are significantly different than control based on RSCABS
analysis).
Lab
A
D
B
C
Prochloraz concentration ([jg/L)
0
6.7
20
60
180
0
6.7
20
60
180
0
6.7
20
60
180
0
6.7
20
60
180
n
43
26
22
24
25
40
15
16
19
22
37
18
16
27
25
43
26
21
25
20
replicates
8
5
5
5
5
8
4
4
4
4
8
4
4
4
4
8
4
4
4
4
Oviduct stage
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
2
0
5
0
0
2
44
0
9
4
12
100
100
100
100
100
100
100
100
100
100
98
100
62
96
100
3
56
100
91
96
88
0
0
0
0
0
0
0
0
0
0
0
0
33
4
0
Wolffian duct stage
1
-
-
-
-
-
-
-
-
-
-
0
6
6
4
12
74
92
95
84
65
2
-
-
-
-
-
-
-
-
-
-
97
67
81
81
64
26
8
5
16
35
3
-
-
-
-
-
-
-
-
-
-
3
28
13
15
24
0
0
0
0
0
Wolffian duct mononuclear inflammation
NR
98
96
95
96
80
Minimal
2
4
5
4
20
Mild
0
0
0
0
0
Moderate
0
0
0
0
0
Severe
0
0
0
0
0
-------
Ill
Male kidney histopathology
The most consistent treatment-related finding in the juvenile male kidney was tubular amphophilic intracytoplasmic inclusions, which were
significantly present in the 180 |ig/L treatment at A, D and C. Tubule mineralization/casts were observed throughout the B and C studies but showed
a significant increase in prevalence; however severity and sensitivity varied between the two labs. Presumably, tubule dilation is a response to
mineralization and follows accordingly with significant increases in prevalence at B and C. The other diagnoses, tubule protein, interstitial
proteinaceous fluid and edema were primarily mild in severity and not likely related to prochloraz treatment (Table 8.4.1-13).
Table 8.4.1-13
Prevalence (%) and severity of juvenile male kidney histopathological observations showing significant treatment-related effects in at least one of the four studies (NR: not remarkable; highlighted cells are significantly different
than control based on RSCABS analysis; dash: not observed in any treatment).
A
Prochloraz concentration ([jg/L)
0
7
20
60
180
n
37
25
24
26
25
replicates
8
5
5
5
5
Tubules, protein
Interstitial fluid (proteinaceous)
Amphophilic intracytoplasmic bodies in tubule
epithelium
NR
92
88
96
96
92
Minimal
8
12
4
4
8
Mild
0
0
0
0
0
Moderate
0
0
0
0
0
Severe
0
0
0
0
0
Interstitial fluid (non-proteinaceous edema)
NR
Minimal
Mild
Moderate
Severe
Tubules, Dilation
NR
Minimal
Mild
Moderate
Severe
Tubules, mineralization / casts
0
7
20
60
180
39
25
22
21
18
8
4
4
4
4
67
60
64
81
67
33
40
32
19
33
0
0
5
0
0
0
0
0
0
0
0
0
0
0
0
NR
100
100
96
100
92
97
100
95
100
100
Minimal
0
0
4
0
8
3
0
5
0
0
Mild
0
0
0
0
0
0
0
0
0
0
Moderate
0
0
0
0
0
0
0
0
0
0
Severe
0
0
0
0
0
0
0
0
0
0
NR
100
100
100
100
44
100
100
100
95
33
Minimal
0
0
0
0
56
0
0
0
5
67
Mild
0
0
0
0
0
0
0
0
0
0
Moderate
0
0
0
0
0
0
0
0
0
0
Severe
0
0
0
0
0
0
0
0
0
0
B
C
0
6.7
20
60
180
0
6.7
20
60
180
43
22
23
13
15
37
14
18
15
20
8
4
4
4
4
8
4
4
4
4
93
100
100
100
100
_
_
_
_
_
7
0
0
0
0
-
-
-
-
-
0
0
0
0
0
-
-
-
-
-
0
0
0
0
0
-
-
-
-
-
0
0
0
0
0
-
-
-
-
-
100
95
96
100
100
92
100
100
100
90
0
5
4
0
0
8
0
0
0
10
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
100
100
100
100
40
-
-
-
-
-
0
0
0
0
15
-
-
-
-
-
0
0
0
0
25
-
-
-
-
-
0
0
0
0
20
-
-
-
-
-
0
0
0
0
0
47
64
65
62
73
97
86
100
100
100
40
32
35
38
13
3
7
0
0
0
14
5
0
0
13
0
7
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
12
9
9
0
13
95
93
67
80
45
53
45
35
15
27
5
7
28
20
45
35
45
57
85
60
0
0
6
0
10
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
-------
112
NR
0
0
0
0
7
30
7
11
27
10
Minimal .....
5
0
0
0
7
65
57
78
67
35
Mild .....
49
23
22
0
7
5
36
11
7
55
Moderate .....
47
77
78
100
80
0
0
0
0
0
Severe .....
0
0
0
0
0
0
0
0
0
0
Female kidney histopathology
Like juvenile male kidneys, tubular amphophilic intracytoplasmic inclusions were the most consistent finding across three of the four labs - severity
and sensitivity were almost identical between genders. Tubule mineralization and dilation were again observed throughout the B and C studies, but
only C exhibited significant increases in prevalence for both pathologies. Unlike male kidneys, renal interstitial fibrosis (minimal to mild) and
glomerular protein (mild) showed significant increases in prevalence in female kidneys at A and D respectively. Interstitial fluid, both proteinaceous
and non-proteinaceous edema, was primarily mild, inconsistent across laboratories and not likely related to prochloraz treatment (Table 8.4.1-14).
Table 8.4.1-14
Prevalence (%) and severity of juvenile female kidney histopathological observations showing significant treatment-related effects in at least one of the four studies (NR: not remarkable; highlighted cells are significantly different
than control based on RSCABS analysis; dash: not observed in any treatment).
Lab
A
D
B
C
Prochloraz concentration ([jg/L)
0
7
20
60
180
0
7
20
60
180
0
6.7
20
60
180
0
6.7
20
60
180
n
43
26
22
24
25
41
15
16
19
22
37
18
16
27
25
43
26
21
25
20
replicates
8
5
5
5
5
8
4
4
4
4
8
4
4
4
4
8
4
4
4
4
Glomerular protein
NR
100
100
95
92
84
93
93
100
95
91
Minimal
0
0
5
8
16
5
7
0
5
9
Mild
0
0
0
0
0
2
0
0
0
0
Moderate
0
0
0
0
0
0
0
0
0
0
Severe
0
0
0
0
0
0
0
0
0
0
Interstitial fluid (proteinaceous)
NR
100
100
95
92
96
100
100
94
100
92
88
96
100
100
95
Minimal
0
0
5
4
4
0
0
6
0
8
12
4
0
0
5
Mild
0
0
0
4
0
0
0
0
0
0
0
0
0
0
0
Moderate
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Severe
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Renal interstitial fibrosis
NR
88
85
77
96
88
68
40
56
47
45
100
94
100
96
92
Minimal
9
12
18
4
8
24
60
25
26
32
0
6
0
0
8
Mild
0
4
5
0
4
5
0
19
26
23
0
0
0
4
0
Moderate
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Severe
2
0
0
0
0
2
0
0
0
0
0
0
0
0
0
Amphophilic intracytoplasmic bodies in tubule
epithelium
NR
100
100
100
100
36
100
100
100
100
50
97
100
100
96
100
100
100
100
92
45
Minimal
0
0
0
0
64
0
0
0
0
50
0
0
0
4
0
0
0
0
4
15
Mild
0
0
0
0
0
0
0
0
0
0
3
0
0
0
0
0
0
0
4
20
Moderate
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
20
Severe
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Interstitial fluid (non-proteinaceous edema)
NR
32
61
75
59
56
93
100
100
100
95
Minimal .....
38
39
19
30
40
7
0
0
0
5
Mild .....
27
0
6
11
4
0
0
0
0
0
Moderate .....
3
0
0
0
0
0
0
0
0
0
Severe .....
0
0
0
0
0
0
0
0
0
0
Tubules, Dilation
-------
Tubules, mineralization / casts
NR
Minimal
Mild
Moderate
Severe
NR
Minimal
Mild
Moderate
Severe
113
0
17
13
30
8
84
81
81
72
40
27
11
31
19
20
16
19
19
28
35
73
72
56
48
72
0
0
0
0
25
0
0
0
4
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
6
0
7
0
37
15
24
8
10
0
6
0
7
4
53
77
48
56
40
19
22
31
26
12
9
8
29
36
50
81
67
69
59
84
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
-------
114
8.4.2 4-tert-Octylphenol results
Measured test concentrations
All test concentrations at A were maintained with CVs < 20%. B maintained test concentrations
with CVs < 20% except for their lowest concentration. C had CVs < 20% in two of the four
concentrations and had measurable amounts of test chemical in control water on three different
occasions (Table 8.4.2-1). Weekly CV values could not be calculated for B and C due to only
one replicate tank being measured per week. C's concentrations were on average higher than
nominal with the lowest concentration > 140% of nominal. B's concentrations were on average
lower than nominal with all mean concentrations being between 73 and 77% of nominal.
Table 8.4.2-1
Inte(laboratory comparison of measured octylphenol concentrations throughout the duration of the LAGDA
(mean ± SD), Highlighted cells indicate CVs >20%,
Nominal OP concentration
A
B
C
0 |jg/L
-------
115
Time to NF62 (d)
0 pg/L
42 ±5
42 ±2
41 ± 10
6.25 |jg/L
41 ±3
43 ±2
43 ± 13
12.5 |jg/L
44 ±5
44 ±3
39 ± 10
25 Mg/L
43 ±4
43 ±2
39 ±8
50 ng/L
44 ±5
43 ±2
39 ±9
Larval blood thyroxine
A used an HPLC/ICP-MS method for measuring serum T4 whereas the other two labs used an
ELISA method to measure T4 in plasma. The ELISA method was not optimized so data could
not be included. Serum T4 levels were significantly decreased in the 25 and 50 |ig/L treatments
at A (Jonckeere-Terpstra, Table 8.4.2-3).
Table 8.4.2-3
I nte(laboratory comparison of larval blood thyroxine
concentrations following octylphenol exposure
(mean ± SD). Highlighted cells are significantly
different than control (a = 0.05).
Nominal OP concentration
T4 (ng/mL)
A
0 |jg/L
39.4 ± 14.7
6.25 |jg/L
47.4 ± 12.8
12.5 |jg/L
28.3 ±8.5
25 Mg/L
22.3 ± 13.0
50 ng/L
14.4 ±8.7
Juvenile growth
There were no treatment-related effects on growth in either gender across all three labs.
However, it is worth noting that again, C organisms were smaller on average than the other labs
(Table 8.4.2-4).
Juvenile liver-somatic index (LSI)
Juvenile LSI was significantly decreased in octylphenol-exposed males and females from C
whereas no treatment-related effects on LSI were measured at A or B (Table 8.4.2-4).
Juvenile plasma vitellogenin
Vitellogenin titers were significantly increased in octylphenol-exposed males from A (12.5, 25,
50 |ig/L) and C (6.25, 12.5, 25, 50 |ig/L). Female vitellogenin levels were significantly
increased at B (50 |ig/L) and C (6.25, 12.5, 25, 50 |ig/L). In most cases, CV values are close to,
or exceed 100% and control means differ across labs by up to 74-fold (Table 8.4.2.4).
-------
116
Table 8.4.2-4
Interlaboratory comparison of juvenile growth and plasma vitellogenin titers following octylphenol exposure (mean ± SD). Highlighted cells are significantly different
than control (a = 0.05).
Male
Female
A
B
C
A
B
C
Nominal OP
concentration
Weight (g)
Weight (g)
0 |jg/L
10.9 ±2.7
10.1 ±2.6
8.6 ±2.6
12.0 ±2.7
10.9 ±3.0
8.3 ± 1.7
6.25 |jg/L
11.1 ±2.0
10.0 ±2.5
8.8 ±2.8
12.4 ±3.1
11.5 ± 3.1
9.1 ±2.2
12.5 |jg/L
11.5 ±3.0
10.4 ±2.4
8.0 ± 1.6
11.7 ± 2.9
10.3 ±2.6
9.6 ±2.7
25 pg/L
11.3 ±3.0
10.8 ±2.6
8.4 ±2.0
12.1 ±2.4
9.2 ± 4.0
7.5 ± 1.9
50 |jg/L
10.9 ± 1.5
10.1 ±3.2
7.8 ±2.5
11.6 ±3.0
11.6 ±4.0
7.9 ±2.0
Length (mm)
Length (mm)
o |jg/L
43.1 ±3.3
41.6 ±3.5
41.1 ±3.4
44.7 ±4.0
42.5 ±4.0
40.8 ±3.1
6.25 |jg/L
44.1 ±2.8
40.7 ±4.1
41.4 ±3.9
46.1 ±3.7
43.7 ±4.3
41.9 ±3.5
12.5 |jg/L
43.1 ±4.1
42.7 ±3.5
41.8 ±2.3
44.3 ±4.0
43.5 ±4.8
44.2 ±4.0
25 pg/L
43.5 ±3.6
43.8 ±3.7
42.2 ±3.2
44.5 ±3.2
41.4 ±5.9
40.8 ±3.6
50 |jg/L
43.3 ±2.2
41.6 ±4.5
41.2 ±3.6
43.9 ±4.3
44.5 ±5.1
41.0 ±3.4
LSI
LSI
o |jg/L
0.058 ± 0.008
0.058 ± 0.007
0.072 ± 0.008
0.059 ±0.012
0.059 ±0.014
0.075 ± 0.007
6.25 |jg/L
0.060 ±0.010
0.056 ± 0.006
0.066 ± 0.009
0.059 ± 0.008
0.059 ± 0.005
0.073 ± 0.009
12.5 |jg/L
0.058 ± 0.007
0.058 ± 0.008
0.070 ± 0.008
0.058 ± 0.004
0.062 ± 0.007
0.073 ± 0.007
25 pg/L
0.062 ± 0.010
0.062 ± 0.006
0.066 ± 0.009
0.061 ± 0.008
0.057 ±0.012
0.069 ± 0.009
50 |jg/L
0.066 ±0.011
0.058 ± 0.007
0.063 ±0.010
0.062 ±0.013
0.057 ± 0.004
0.067 ± 0.006
VTG (mg/mL)
VTG (mg/mL)
o |jg/L
0.000 ± 0.001
0.013 ±0.014
0.005 ± 0.001
0.000 ± 0.001
0.008 ± 0.008
0.036 ±0.011
6.25 |jg/L
0.000 ± 0.001
0.009 ± 0.009
0.007 ± 0.003
0.000 ± 0.001
0.006 ± 0.007
0.042 ± 0.013
12.5 |jg/L
0.001 ± 0.002
0.015 ±0.034
0.018 ±0.021
0.001 ±0.001
0.010 ±0.014
0.055 ± 0.009
25 pg/L
0.002 ± 0.004
0.007 ± 0.006
0.021 ± 0.016
0.001 ± 0.000
0.015 ±0.017
0.097 ± 0.026
50 ng/L
0.001 ± 0.002
0.014 ±0.015
0.039 ±0.019
0.001 ±0.001
0.019 ±0.028
0.143 ±0.023
Larval thyroid histopathology
Follicular cell hypertrophy and hyperplasia were observed throughout all three studies but only
showed significant treatment-related increases in severity at A. Mild decreases in colloid size
were substantially observed only at A and showed significant attenuation in the 25 and 50 |ig/L
treatments (Table 8.4.2-5).
Table 8.4.2-5
Prevalance (%) and severity of larval thyroid gland histopathological observations showing significant treatment-related effects in at least one of the three studies (NR:
not remarkable, highlighted cells are significantly different than control based on RSCABS analysis; dash: not observed in any treatment),
ABC
Nominal OP concentration (|jg/L)
0 6.25
12.5
25
50
0 6.25
12.5
25
50
0 6.25
12.5
25
50
n
39 24
25
25
24
40 20
20
20
20
40 20
20
20
20
replicates
8 5
5
5
5
8 4
4
4
4
8 4
4
4
4
Follicular Cell Hypertrophy
NR
3
0
0
0
4
3
5
0
5
10
73
70
75
65
75
Mild
46
21
36
16
4
90
90
90
85
80
28
30
25
35
25
Moderate
51
75
56
68
71
8
5
10
10
10
0
0
0
0
0
Severe
0
4
8
16
21
0
0
0
0
0
0
0
0
0
0
Follicular Cell Hyperplasia
NR
74
58
56
76
58
83
70
75
90
85
85
80
80
65
80
Mild
26
42
44
24
33
18
30
25
10
15
15
20
20
35
20
Moderate
0
0
0
0
8
0
0
0
0
0
0
0
0
0
0
Severe
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Decreased Colloid
NR
36
46
28
72
71
98
100
100
100
100
Mild
62
50
72
28
29
3
0
0
0
0
Moderate
3
4
0
0
0
0
0
0
0
0
Severe
0
0
0
0
0
0
0
0
0
0
-------
117
Liver histopcithology
No treatment-related effects were present in any of the octylphenol-exposed livers from all three
studies. Granulomatous inflammation was observed in all three studies, but prevalence and
severity was comparable among treated and control samples. Cystic degeneration was observed
in one of the three studies (B), but prevalence and severity was also comparable among treated
and control samples (data not shown).
Male gonad histopathology
Effects of octylphenol exposure on male gonads were minimal. Mononuclear cellular infiltrate
was observed throughout all three studies but was not treatment-related, although statistically,
severity was significantly less than controls at B. There were very few observations of
minimally increased spermatogonia at B and C, but the 180 |ig/L treatment at B was significantly
different than control. Statistically, there appears to be slight advancement of testicular
development in the 180 |ig/L treatment at B and shows consistency across all three labs,
although not statistically significant at A or C (Table 8.4.2-6).
Table 8.4.2-6
Prevalence (%) and severity of juvenile male gonad histopathological observations showing significant treatment-related effects in at least one of the three studies (NR: not
remarkable; highlighted cells are significantly different than control based on RSCABS analysis; dash: not observed in any treatment),
ABC
Nominal OP concentration ([jg/L)
0 6.25
12.5
25
50
0 6.25
12.5
25
50
0 6.25
12.5
25
50
n
32 20
16
16
9
45 19
23
22
25
40 14
20
15
19
replicates
8 5
4
5
3
8 4
4
4
4
8 4
4
4
4
Gonad Stage
1
0
0
0
0
0
2
0
0
0
0
0
0
0
0
0
2
0
0
0
0
0
4
0
0
0
12
0
0
0
0
0
3
0
0
0
0
0
4
5
4
0
0
0
0
0
0
0
4
34
30
31
44
11
69
68
70
55
52
13
0
30
7
5
5
66
70
69
56
89
20
26
26
41
36
88
100
70
93
95
Increased Spermatogonia
NR
100
100
100
100
92
100
100
100
93
100
Minimal -
0
0
0
0
8
0
0
0
7
0
Mild
0
0
0
0
0
0
0
0
0
0
Moderate -
0
0
0
0
0
0
0
0
0
0
Severe -
0
0
0
0
0
0
0
0
0
0
Mononuclear Cellular Infiltrate
NR
66
85
56
69
78
62
79
57
64
76
90
93
95
87
84
Minimal
31
15
38
31
22
29
21
43
32
24
10
7
5
13
16
Mild
3
0
6
0
0
9
0
0
0
0
0
0
0
0
0
Moderate
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Severe
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Female gonad histopathology
Unlike male gonads, female gonads experienced treatment-related increases in severity of
mononuclear cellular infiltrate and was significant (minimal to mild) in the 12.5, 25 and 50 |ig/L
treatments at A and C. Granulomatous inflammation was minimal at B and C in the low
treatments and control, but was absent in the 25 and 50 |ig/L treatments (Table 8.4.2-7).
Table 8.4.2-7
-------
118
Prevalence (%) and severity of juvenile female gonad histopathological observations showing significant treatment-related effects in at least one of the three studies (NR:
not remarkable; highlighted cells are significantly different than control based on RSCABS analysis; dash: not observed in any treatment),
A
B
C
Nominal OP concentration ([jg/L)
0
6.25
12.5
25
50
0
6.25
12.5
25
50
0
6.25
12.5
25
50
n
30
20
16
21
12
33
20
17
18
15
40
24
20
25
20
replicates
8
5
4
5
3
8
4
4
4
4
8
4
4
4
4
Gonad Stage
1
0
0
0
0
0
0
0
0
6
0
0
0
0
0
0
2
0
0
0
0
0
3
5
6
0
13
0
0
0
0
5
3
0
0
0
0
8
0
0
29
0
0
18
29
25
28
15
4
100
100
100
100
92
97
95
65
94
87
83
71
75
72
80
5
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Mononuclear Cellular Infiltrate
NR
63
40
25
38
42
53
30
41
39
33
55
42
50
24
30
Minimal
33
60
56
57
50
44
55
47
61
53
38
42
25
44
50
Mild
3
0
19
5
8
3
15
12
0
13
8
17
25
32
20
Moderate
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Severe
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Granulomatous inflammation
NR
-
-
-
-
-
97
95
94
100
100
93
96
95
100
100
Minimal
-
-
-
-
-
3
5
6
0
0
5
4
5
0
0
Mild
-
-
-
-
-
0
0
0
0
0
3
0
0
0
0
Moderate
-
-
-
-
-
0
0
0
0
0
0
0
0
0
0
Severe
-
-
-
-
-
0
0
0
0
0
0
0
0
0
0
Male reproductive duct histopathology
There were significant delays in male oviduct regression at A (6.25, 12.5, 25, 50 |ig/L) and B (25,
50 |ig/L) and evidence of advanced male oviduct maturation in the 50 |ig/L treatment at B.
Although there was a significant delay in Wolffian duct development in the 50 |ig/L treatment at
B, stage distributions were somewhat consistent across labs and similar between treatments and
controls (Table 8.4.2-8).
Table 8.4.2-8
Histological staging (%) of juvenile male reproductive ducts from three different labs performing the LAGDA with octylphenol (highlighted cells are significantly different than
control based on RSCABS analysis),
A
B
C
Nominal OPconcentration ([jg/L)
0
6.25
12.5
25
50
0
6.25
12.5
25
50
0
6.25
12.5
25
50
n
32
20
16
16
9
45
19
23
22
25
40
14
20
15
19
replicates
8
5
4
5
3
8
4
4
4
4
8
4
4
4
4
Oviduct Stage
1
88
60
44
31
67
40
26
30
9
12
98
94
100
93
100
2
12
40
56
69
33
58
74
65
86
28
3
6
0
7
0
3
0
0
0
0
0
2
0
4
5
52
0
0
0
0
0
4
0
0
0
0
0
0
0
0
0
8
0
0
0
0
0
Wolffian Stage
1
3
10
0
25
0
16
26
4
5
28
10
19
25
20
5
2
88
60
50
56
67
60
68
70
86
72
48
44
50
67
37
3
9
25
50
19
33
24
5
26
9
0
43
31
25
13
58
4
0
5
0
0
0
0
0
0
0
0
0
0
0
0
0
Female reproductive duct histopathology
Female oviduct development was significantly accelerated with octylphenol exposure across all
three labs. However, sensitivity differed between labs with A only showing a significant effect
in the 50 |ig/L treatment whereas significant effects were exhibited down to 12.5 and 6.25 |ig/L
at B and C respectively (Table 8.4.2-9).
-------
119
Table 8.4.2-9
Histological staging (%) of juvenile female reproductive ducts from three different labs performing the LAGDA with octylphenol (highlighted cells are significantly different
than control based on RSCABS analysis).
A
B
C
Nominal OP concentration ([jg/L)
0 6.25
12.5
25
50
0 6.25
12.5
25
50
0 6.25
12.5
25
50
n
30 20
16
21
12
33 20
17
18
15
40 24
20
25
20
replicates
8 5
4
5
3
8 4
4
4
4
8 4
4
4
4
Oviduct Stage
1
0
0
0
0
0
0
0
0
0
7
0
0
0
0
5
2
87
80
75
67
25
85
70
65
56
0
98
79
50
64
57
3
13
20
25
33
75
15
30
35
44
93
3
21
50
36
38
Wolffian Stage
1
13
25
25
29
42
27
20
24
22
33
43
46
50
60
48
2
77
60
63
48
50
58
60
59
72
40
40
38
40
28
33
3
10
15
13
24
8
15
20
18
6
27
18
17
10
12
19
Male kidney histopathology
Male kidney tubule dilation and mineralization were observed throughout all three studies.
Prevalence and severity was comparable between treatments and control in most cases. However,
these pathologies were significantly attenuated with octylphenol exposure at B. Minimal
interstitial proteinaceous fluid was observed at B but is essentially not remarkable (Table 8.4.2-
10).
Table 8.4.2-10
Prevalence (%) and severity of juvenile male kidney histopathological observations showing significant treatment-related effects in at least one of the three studies (NR: not
remarkable; highlighted cells are significantly different than control based on RSCABS analysis; dash: not observed in any treatment),
ABC
Nominal OPconcentration ([jg/L)
0 6.25
12.5
25
50
0 6.25
12.5
25
50
0 6.25
12.5
25
50
n
33 20
16
16
9
45 19
23
22
25
40 16
20
15
19
replicates
8 5
4
5
3
8 4
4
4
4
8 4
4
4
4
Interstitial Fluid (Proteinaceous)
NR
100
100
100
100
96
Minimal -
0
0
0
0
4
Mild
0
0
0
0
0
Moderate -
0
0
0
0
0
Severe -
0
0
0
0
0
Tubules, Dilation
NR
18
20
6
13
11
4
11
9
9
16
10
19
15
27
16
Minimal
58
55
63
56
22
36
32
52
45
48
33
44
40
20
53
Mild
24
25
31
25
67
60
58
39
45
36
58
38
45
53
32
Moderate
0
0
0
6
0
0
0
0
0
0
0
0
0
0
0
Severe
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Tubules, Mineralization / Casts
NR
0
0
0
0
0
0
0
0
0
0
0
0
5
0
0
Minimal
21
20
19
19
0
0
5
0
0
0
0
6
0
0
0
Mild
73
70
69
63
100
42
47
61
64
60
33
88
50
80
58
Moderate
6
10
13
19
0
58
47
39
36
40
68
6
45
20
42
Severe
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Female kidney histopathology
Female kidney tubule dilation and mineralization were observed throughout all three studies. C
exhibited a similar pattern to B male kidneys where these pathologies are attenuated with
octylphenol treatment. A showed a significant, but slight increase in severity of tubule dilation
in the 50 |ig/L treatment which is inconsistent with the findings in the other studies. B females
did not show significant trends in these pathologies, but showed significant minimal to mild
severity of edema, although prevalence was very low making the finding essentially not
remarkable (Table 8.4.2-11).
-------
120
Table 8.4.2-11
Prevalence (%) and severity of juvenile female kidney histopathological observations showing significant treatment-related effects in at least one of the three studies (NR:
not remarkable; highlighted cells are significantly different than control based on RSCABS analysis; dash: not observed in any treatment).
Interstitial Fluid
(Non-Proteinaceous Edema)
Tubules, Dilation
Tubules, Mineralization / Casts
o'
3
CQ
I I
0
6.25
12.5
25
50
0
6.25
12.5
25
50
0
6.25
12.5
25
50
n
30
20
16
21
12
33
20
17
18
15
40
24
20
25
21
replicates
8
5
4
5
3
8
4
4
4
4
8
4
4
4
4
NR
91
100
100
94
93
Minimal
-
-
-
-
-
9
0
0
6
0
-
-
-
-
-
Mild
-
-
-
-
-
0
0
0
0
7
-
-
-
-
-
Moderate
-
-
-
-
-
0
0
0
0
0
-
-
-
-
-
Severe
-
-
-
-
-
0
0
0
0
0
-
-
-
-
-
NR
23
5
6
10
0
15
0
29
28
13
0
8
0
12
14
Minimal
43
55
69
48
42
33
35
41
17
20
15
42
35
56
62
Mild
33
40
25
43
58
52
65
29
56
67
85
50
65
32
24
Moderate
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Severe
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
NR
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Minimal
23
15
31
10
8
3
0
0
6
0
0
4
0
4
0
Mild
57
75
50
67
75
45
35
47
33
60
25
63
45
60
38
Moderate
20
10
19
24
17
52
65
53
61
40
75
33
55
36
62
Severe
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
-------
121
8.4.3 17-p Trenbolone results
Measured test concentrations
Mean ± SD (n = 40) measured concentrations of the 12.5, 25, 50 and 100 ng/L 17P-trenbolone
treatments were 9.0 ± 2.8, 19.2 ± 5.8, 41.4 ± 9.7 and 79.9 ± 24.1 ng/L, respectively, over the
duration of the experiment. Among-tank variability was < 32% with the coefficient of variation
(averaged across the duration of the exposure) of 31%, 30%, 23% and 30%, respectively for the
12.5, 25, 50 and 100 ng/L treatments. No 17p-trenbolone was detected in the control water
treatments.
Survival
All control tanks met the guideline performance criteria of <20% mortality and there were no
treatment-related effects on survival (data not shown).
Larval growth
There were no significant treatment-related effects on larval growth and development (Table
8.4.3-1).
Larval blood thyroxine
Serum samples were analyzed for T4 levels using the HPLC/ICP-MS method described
previously. There were no treatment-related effects on larval serum T4 levels (Table 8.4.3-1).
Table 8.4.3-1
Larval growth and serum thyroxine concentrations following trenbolone exposure at A (mean ± SD).
NF62 larvae
Nominal TB concentration Weight (g) Length (mm) Time to NF62 (d) T4 (ng/mL)
0 |jg/L 0.818 ±0.140 18.7 ±1.1 40 ± 4 23.4 ± 9.0
12.5 ng/L 0.835 ±0.146 18.7 ±1.0 41 ± 4 25.0 ±10.5
25 ng/L 0.853 ±0.154 18.8 ± 1.2 40 ± 5 25.1 ±9.7
50 ng/L 0.913 ±0.193 19.2 ±1.3 41 ± 6 30.6 ± 9.1
100 ng/L 0.867 ±0.166 18.8 ± 1.2 39 ± 4 24.2 ± 9.8
Juvenile growth
There were no significant treatment-related effects from 17(3-trenbolone exposure on juvenile
growth for either gender (Table 8.4.3-2).
Juvenile liver-somatic index (LSI)
There were no significant treatment-related effects on LSI for either gender (Table 8.4.3-2).
Juvenile plasma vitellogenin
Trenbolone treatment did not affect vitellogenin titers in juvenile males or females (data not
shown).
-------
122
Table 8.4.3-2
Juvenile growth and plasma vitellogenin titers following trenbolone exposure at A (mean ± SD).
Highlighted cells are significantly different than control (a = 0.05).
Weight (g) Length (mm) LSJ
Nominal TB concentration Female
Opg/L 12.9 ±3.7 47.5 ± 5.1 0.059 ± 0.006
12.5 ng/L 13.3 ±2.6 47.3 ± 3.3 0.058 ±0.010
25 ng/L 12.5 ±3.2 46.4 ± 4.4 0.061 ± 0.006
50 ng/L 12.5 ±3.1 46.7 ± 4.8 0.058 ± 0.008
100 ng/L 13.7 ±3.6 49.0 ± 4.8 0.058 ± 0.009
Male
46.2 ±4.1 0.06 ±0.010
45.4 ± 3.2 0.058 ± 0.009
46.3 ± 4.0 0.057 ± 0.006
47.4 ± 4.0 0.057 ± 0.008
46.4 ± 3.6 0.058 ± 0.007
0pg/L
12.5 ng/L
25 ng/L
50 ng/L
100 na/L
12.3 ±3.3
11.4 ±2.4
11.9 ± 2.7
13.0 ±2.5
11.7 ± 2.7
Thyroid histopathology
The two categories of morphologic findings in thyroid gland tissue, both of which were common,
were follicular cell hypertrophy (mild to moderate) and follicular cell hyperplasia (mild to
moderate). The severity of follicular cell hypertrophy was slightly decreased in the 50 and 100
ng/L trenbolone treatment groups as compared to controls, but those differences are not
statistically significant. The prevalence and severity of follicular cell hyperplasia in trenbolone-
treated frogs were mild but significantly different than controls in the 25, 50 and 100 ng/L
treatments (Table 8.4.3-3).
Table 8.4.3-3
Prevalance (%) and severity of larval thyroid gland histopathological
observations in trenbolone-exposed larvae (NR: not remarkable, highlighted cells
are significantly different than control based on RSCABS analysis).
Trenbolone concentration (ng/L)
0 12.5
25
50
100
n
40 25
24
25
25
replicates
8 5
5
5
5
Follicular Cell Hypertrophy
NR
3
12
13
16
4
Minimal
68
64
58
68
80
Mild
30
24
29
16
16
Moderate
0
0
0
0
0
Follicular Cell Hyperplasia
NR
43
52
25
44
48
Minimal
45
44
75
56
52
Mild
13
4
0
0
0
Moderate
0
0
0
0
0
Liver histopathology
Treatment-related effects were present in the liver. The prevalence of minimal cellular
hypertrophy of hepatocytes was significantly different than controls in the 50 and 100 ng/L
treatments. The prevalence and severity of the only other finding in the liver, granulomatous
inflammation, was comparable among treated and control frogs (Table 8.4.3-4).
-------
123
Table 8.4.3-4
Prevalence (%) and severity of juvenile liver histopathological observations following trenbolone exposure (NR: not
remarkable; highlighted cells are significantly different than control based on
RSCABS analysis).
Male
Female
Trenbolone concentration (ng/L)
0
12.5
25
50
100
0
13
25
50
100
n
38
21
20
25
25
37
25
29
24
25
replicates
8
5
5
5
5
8
5
5
5
5
Granulomatous Inflammation
NR
53
67
55
52
52
46
40
69
67
48
Minimal
47
33
45
48
48
54
60
31
33
52
Mild
0
0
0
0
0
0
0
0
0
0
Moderate
0
0
0
0
0
0
0
0
0
0
Cellular Hypertrophy
NR
100
100
100
88
80
100
100
100
100
76
Minimal
0
0
0
12
20
0
0
0
0
24
Mild
0
0
0
0
0
0
0
0
0
0
Moderate
0
0
0
0
0
0
0
0
0
0
Male gonad histopathology
The only two significant treatment-related findings in the testes of males were increased
spermatogonia (minimal) and germ cell vacuolation (minimal to mild) in frogs of the 100 ng/L
treatment (Table 8.4.3-5). Germ cell vacuolation was characterized by clear cytoplasmic
ballooning in small clusters of pachytene phase spermatocytes. Other common findings in the
testes included germ cell degeneration (minimal) and mononuclear cell infiltrates (minimal to
mild). The prevalence and severity of these findings were essentially comparable between
trenbolone-treated and control frogs. Tubule lumen development was also similar among the
various treatment groups (data not shown).
Table 8.4.3-5
Prevalence (%) and severity of juvenile male gonad histopathological
observations showing significant treatment-related effects following trenbolone
exposure (NR: not remarkable; highlighted cells are significantly different than
Trenbolone concentration (ng/L)
0
12.5
25
50
100
n
39
21
20
25
25
replicates
8
5
5
5
5
Increased spermatogonia
NR
97
100
100
100
76
Minimal
3
0
0
0
24
Mild
0
0
0
0
0
Moderate
0
0
0
0
0
Severe
0
0
0
0
0
Germ cell vacuolation
NR
100
95
95
96
76
Minimal
0
5
5
4
20
Mild
0
0
0
0
4
Moderate
0
0
0
0
0
Severe
0
0
0
0
0
Female gonad histopathology
There were no apparent effects of trenbolone exposure on the ovaries of juvenile females (data
not shown).
Male kidney histopathology
-------
124
Two common findings in male kidneys were renal tubule mineralization/casts (minimal to
moderate) and renal tubule dilation (minimal to mild). However, tubule dilation was
significantly attenuated in the 50 and 100 ng/L treatments whereas mineralization/casts were not
treatment-related. There was a low occurrence of interstitial fibrosis (minimal to mild) in the
100 ng/L treatment, but was statistically different than control (Table 8.4.3-6).
Table 8.4.3-6
Prevalence (%) and severity of juvenile male kidney histopathological
observations showing significant treatment-related effects following trenbolone
exposure (NR: not remarkable; highlighted cells are significantly different than
Trenbolone concentration (ng/L)
0 12.5
25
50
100
n
39 21
20
25
25
replicates
8 5
5
5
5
Interstitial fibrosis
Tubules, Dilation
NR
100
100
100
96
92
Minimal
0
0
0
4
4
Mild
0
0
0
0
4
Moderate
0
0
0
0
0
Severe
0
0
0
0
0
NR
18
24
10
0
8
Minimal
18
52
50
24
56
Mild
64
24
40
76
36
Moderate
0
0
0
0
0
Severe
0
0
0
0
0
Female kidney histopathology
There were no apparent effects of trenbolone exposure on the kidneys of juvenile females (data
not shown).
Reproductive duct histopathology
There were treatment-related effects on oviduct development in both genders. Males
experienced accelerated regression of oviducts in the 100 ng/L treatment as compared to controls
whereas females experienced prodigious oviduct regression in the 50 and 100 ng/L treatments
(Table 8.4.3-7). There were no treatment-related effects on Wolffian duct development or
pathology in either gender.
Table 8.4.3-7
Histological staging of juvenile oviducts following trenbolone exposure (highlighted cells are significantly different than
control based on RSCABS analysis),
Male
Female
Trenbolone concentration (ng/L)
0
12.5
25
50
100
0
12.5
25
50
100
n
39
21
20
25
25
38
25
29
24
25
replicates
8
5
5
5
5
8
5
5
5
5
Oviduct Stage
1
54
29
40
64
100
0
0
0
8
52
2
46
71
60
36
0
100
100
100
92
48
3
0
0
0
0
0
0
0
0
0
0
-------
125
8.4.4 Benzophenone-2 results
Measured test concentrations
Mean ± SD (n = 36) measured concentrations of the 1.5, 3.0 and 6.0 mg/L benzophenone-2
treatments were 1.6 ± 0.1, 3.5 ± 0.2 and 6.1 ± 0.1 mg/L, respectively over the duration of the
experiment. CVs (averaged across the duration of the exposure) were 4%, 6% and 1%,
respectively for the 1.5, 3.0 and 6.0 mg/L treatments. No benzophenone-2 was detected in the
control water treatments.
Survival
The test was initiated with 30 embryos per tank. Following hatch on day 4, all tanks were
balanced based on the lowest number left in any particular tank, which was 29 larvae.
Subsequently, there was significant treatment-related mortality during the larval phase of the
assay (Jonckeere-Terpstra, Table 8.4.4-1).
Table 8.4.4-1
Mean percent mortality prior to completion of NF62 sample. Highlighted cell is
significantly different than control.
Nominal BP2 concentration (mg/L)
0
1.5
3
6
n/tank
29
29
29
29
replicates
4
4
4
4
Mean % mortality 0 ± 0 1 ± 2 3 ± 7 38 ± 6
Following the NF66 cull, 10 individuals were left in each tank to continue exposure until test
termination. Within this timeframe, significant mortality was experienced in the high test
concentration (Jonckeere-Terpstra, Table 8.4.4-2).
Table 8.4.4-2
Mean percent juvenile mortality post-cull. Highlighted cell is significantly
different than control.
Nominal BP2 concentration (mg/L)
0
1.5
3
6
n/tank
10
10
10
10
replicates
4
4
4
4
Mean % mortality
0±0
0±0
5 ± 6
25 ± 13
Larval growth and development
There was a significant increase in growth in the 6 mg/L treatment, but this could be a result of
the significant mortality experienced by this treatment level allowing more food access for
surviving larvae (Jonckeere-Terpstra, Table 8.4.4-3). There was also a significant delay in time
to NF stage 62 in the 6 mg/L treatment as compared to control (Jonckeere-Terpstra, Table 8.4.4-
3).
Larval blood thyroxine
-------
126
In the case of the BP-2 study, heparinized plasma samples were analyzed for T4 levels as
opposed to serum using both the optimized ELISA described in LAGDA guideline Appendix 7
and the HPLC/ICP-MS methods. There were no statistically significant effects on circulating T4
in either sample set and T4 levels from the two methods did not agree. It is unknown what
caused the lack of agreement between the two methods in this case. Therefore, the data is not
presented. The effects of using plasma as opposed to serum for T4 analyses are currently under
investigation.
Table 8.4.4-3
Larval growth and serum thyroxine concentrations following BP2 exposure at E (mean
± SD), Highlighted cells are significantly different than control (a = 0,05),
NF62 larvae
Nominal TB
Time to NF62
concentration
Weight (g)
Length (mm)
(d)
0 mg/L
1.18 ± 0.18
22.2 ±1.1
38 ±5
1.5 mg/L
1.05 ±0.23
21.5 ± 1.3
37 ±3
3 mg/L
1.18 ± 0.18
22.2 ±1.3
38 ±3
6 mg/L
1.92 ±0.59
25.2 ±2.1
49 ± 7
Juvenile growth
Growth was significantly reduced in the 6 mg/L treatment compared to controls for both males
and females (Jonckeere-Terpstra, Table 8.4.4-4).
Juvenile liver-somatic index (LSI)
LSI appeared to be affected by BP-2 exposure; however statistical analyses were inconsistent
between genders due to non-monotonicity of the data in both cases. There was a significant
decrease in male LSI in the 6 mg/L treatment and a significant increase in female LSI in the 1.5
mg/L treatment (modified Dunn's, Table 8.4.4-4).
Juvenile plasma vitellogenin
There was significant induction of vitellogenin in all treatments compared to control and the
response was very consistent between genders (Jonckeere-Terpstra, Table 8.4.4-4). The
magnitude of induction is much higher than any of the other inter-laboratory studies and
emphasizes the responsiveness of this endpoint to estrogenic compounds.
Table 8.4.4-4
Juvenile growth and plasma vitellogenin titers following BP2 exposure at E (mean ± SD). Highlighted
cells are significantly different than control (a = 0,05),
Weight (g) Length (mm) LSI VTG (mg/mL)
Nominal BP2
concentration
Female
0 mg/L
1.5 mg/L
3 mg/L
6 mg/L
10.8 ±2.1
11.8 ± 3.1
9.5 ±4.8
4.1 ± 1.2
44.7 ±3.2
47.4 ±4.4
43.3 ±7.3
33.4 ±2.7
0.045 ± 0.006
0.052 ± 0.006
0.057 ±0.013
0.036 ± 0.009
Male
0.04 ±0.11
60.2 ±27.2
245.8 ±61.1
250.9 ± 75.5
0 mg/L
11.3 ± 2.7
45.2 ±3.3
0.046 ± 0.005
0.02 ± 0.03
1.5 mg/L
11.1 ±2.6
46.7 ±4.1
0.052 ± 0.006
59.9 ±23.5
3 mg/L
9.8 ±5.5
43.0 ±7.5
0.055 ±0.018
232.9 ±56.9
6 mg/L
4.4 ± 1.4
33.9 ±3.5
0.035 ± 0.007
259.4 ± 35.6
-------
127
Thyroid histopathology
There were significant treatment-related effects on thyroid tissue including increased prevalence
and severity of follicular cell hypertrophy and hyperplasia and gland hypertrophy. The 6 mg/L
treatment exhibited notable pathologies in 100% of the subjects (Table 8.4.4-5).
Table 8.4.4-5
Prevalance (%) and severity of larval thyroid gland histopathological
observations in BP2~exposed larvae (NR: not remarkable, highlighted
cells are significantly different than control based on RSCABS analysis).
NF62
Nominal BP2 concentration (mg/L)
0
1.5
3
6
n
20
20
20
18
replicates
4
4
4
4
Thyroid Gland, Hypertrophy
NR
100
95
25
0
Mild
0
5
75
0
Moderate
0
0
0
67
Severe
0
0
0
33
Follicular Cells, Hypertrophy
NR
50
30
5
0
Mild
50
70
50
0
Moderate
0
0
45
33
Severe
0
0
0
67
Follicular Cells, Hyperplasia
NR
80
50
15
0
Mild
20
50
65
0
Moderate
0
0
20
11
Severe
0
0
0
89
Liver histopathology
Treatment-related findings in the liver were limited to phenotypic females and genetic males
which showed mixed sex gonads (i.e. the 3.0 and 6.0 mg/L treatments). These included
concentration-dependent increases in the prevalence and severity of basophilia, decreased
hepatocellular vacuolation, and intravascular proteinaceous fluid. As compared to controls, BP-
2-treated frogs of the 3.0 and 6.0 mg/L treatment groups additionally had higher prevalences of
increased pigment, which also tended to be concentration-dependent. Furthermore, the few
findings of individual hepatocyte necrosis, and foci of hepatocellular alteration, were restricted to
frogs of the 3.0 and 6.0 mg/L concentration groups, and 6.0 mg/L treatment group, respectively
(Table 8.4.4-6). Overall, these liver changes are likely related to estrogenic activity of BP-2 and
significant up-regulation of vitellogenin production.
Table 8.4.4-6
Prevalence (%) and severity of juvenile liver histopathological observations following BP2 exposure (NR: not remarkable; highlighted cells are
significantly different than control based on RSCABS analysis),
Male Female
Nominal BP2 concentration (mg/L) 0 1J> 3 6_ 0 1J> 3 6_
n
20
20
18
14
20
20
20
16
replicates
4
4
4
4
4
4
4
4
NR
100
100
0
0
100
90
0
0
Minimal
0
0
28
0
0
10
25
0
Mild
0
0
33
0
0
0
45
25
Moderate
0
0
28
86
0
0
30
50
-------
128
Decreased Hepatocellular Vacuolation
Proteinaceous Fluid Intravascular
Individual Hepatocyte Necrosis/ Apoptosis
Severe
0
0
11
14
0
0
0
25
NR
100
100
0
0
100
90
0
0
Minimal
0
0
28
0
0
10
25
0
Mild
0
0
33
0
0
0
45
25
Moderate
0
0
28
86
0
0
30
50
Severe
0
0
11
14
0
0
0
25
NR
100
100
6
0
100
100
5
6
Minimal
0
0
28
0
0
0
30
0
Mild
0
0
22
0
0
0
30
19
Moderate
0
0
28
79
0
0
10
69
Severe
0
0
17
21
0
0
25
6
NR
100
100
94
93
100
100
100
88
Minimal
0
0
6
7
0
0
0
13
Mild
0
0
0
0
0
0
0
0
Moderate
0
0
0
0
0
0
0
0
Severe
0
0
0
0
0
0
0
0
Increased Pigment
Focus of Hepatocellular Alteration
NR
100
100
50
0
100
100
45
0
Minimal
0
0
50
100
0
0
55
100
Mild
0
0
0
0
0
0
0
0
Moderate
0
0
0
0
0
0
0
0
Severe
0
0
0
0
0
0
0
0
NR
100
100
100
79
100
100
100
88
Minimal
0
0
0
21
0
0
0
6
Mild
0
0
0
0
0
0
0
0
Moderate
0
0
0
0
0
0
0
6
Severe
0
0
0
0
0
0
0
0
Gonad histopathology
Genotypic males experienced feminization of the gonad resulting in a portion of individuals
presenting with mixed sex gonads and others with complete sex reversal in the 1.5 mg/L
treatment. 100% of genotypic males in the 3 and 6 mg/L treatments were functionally sex
reversed presenting only ovary tissue. In genotypic females, there was a significant treatment-
related delay in gonad development. Similarly, there was a treatment-related delay in ovary
development in sex-reversed genotypic males. Thinning of the germinal epithelium is specific to
testis tissue and significant increases in prevalence and severity were observed in the 1.5 mg/L
treatment. Mononuclear cellular infiltrate was observed in genotypic males and females across
all treatments and control, but prevalence and severity was significantly attenuated in the 3 and 6
mg/L treatments for genotypic females. Prevalence and severity of proteinaceous fluid infiltrate
significantly increased in both genders in the 3 and 6 mg/L treatments (Table 8.4.4-7).
Table 8.4.4-7
Prevalence (%) and severity of juvenile male gonad histopathological observations showing significant treatment-related effects following
BP2 exposure (NR: not remarkable; highlighted cells are significantly different than control based on RSCABS analysis ;),
Gonad phenotype
Gonad Stage
Male
Female
concentration (mg/L)
0
1.5
3
6
0
1.5
3
6
n
20
19
12
14
17
17
16
9
replicates
4
4
4
4
4
4
4
4
Testis 1
100
37
0
0
0
0
0
0
2
0
32
0
0
0
0
0
0
3
0
0
0
0
0
0
0
0
4
0
0
0
0
0
0
0
0
Ovary 5
0
32
100
100
100
100
100
100
1
0
0
0
0
0
6
6
0
-------
129
Testicular Oocytes
Germinal Epithelium,Thinning
Mononuclear cellular infiltrate
Proteinaceous Fluid Infiltrate
2
5
5
0
0
0
6
0
0
3
0
16
42
100
0
12
44
89
4
45
58
58
0
100
76
50
11
5
50
21
0
0
0
0
0
0
NR
100
53
0
0
_
_
_
_
Minimal
0
11
0
0
-
-
-
-
Mild
0
5
0
0
-
-
-
-
Moderate
0
0
0
0
-
-
-
-
Severe
0
0
0
0
-
-
-
-
NR
100
47
0
0
_
_
_
_
Minimal
0
11
0
0
-
-
-
-
Mild
0
5
0
0
-
-
-
-
Moderate
0
5
0
0
-
-
-
-
Severe
0
0
0
0
-
-
-
-
NR
90
79
83
86
47
35
88
100
Minimal
10
21
17
14
41
35
13
0
Mild
0
0
0
0
12
29
0
0
Moderate
0
0
0
0
0
0
0
0
Severe
0
0
0
0
0
0
0
0
NR
100
100
8
0
100
94
0
0
Minimal
0
0
67
21
0
6
63
33
Mild
0
0
25
79
0
0
31
44
Moderate
0
0
0
0
0
0
6
22
Severe
0
0
0
0
0
0
0
0
Kidney histopcithology
BP-2 exposure caused profound effects in both genotypic male and female kidneys. These
effects were very similar between genders and widespread in the 3 and 6 mg/L treatments.
However, these two treatments had 100% female phenotypes. Interestingly, many of the
pathologies have higher severity scores in the 3 mg/L treatment and are slightly less severe in the
6 mg/L treatment (Table 8.4.4-8).
Table 8.4.4-8
Prevalence (%) and severity of juvenile male kidney histopathological observations showing significant treatment-related effects following
BP2 exposure (NR: not remarkable; dash: not observed; highlighted cells are significantly different than control based on RSCABS
analysis).
Male Female
Nominal BP2 concentration (mg/L)
0
1.5
3
6
0
1.5
3
6
n
20
19
12
14
17
17
16
9
replicates
4
4
4
4
4
4
4
4
Glomerulomegaly
NR
100
100
0
0
100
88
6
22
Minimal
0
0
42
21
0
12
0
0
Mild
0
0
58
79
0
0
6
56
Moderate
0
0
0
0
0
0
38
22
Severe
0
0
0
0
0
0
50
0
Glomerular Protein
NR
100
100
17
0
100
82
0
0
Minimal
0
0
50
7
0
18
0
22
Mild
0
0
33
93
0
0
56
22
Moderate
0
0
0
0
0
0
31
56
Severe
0
0
0
0
0
0
13
0
Glomerulus, hypercellularity
NR
100
100
8
100
100
94
0
56
Minimal
0
0
67
0
0
6
13
33
Mild
0
0
25
0
0
0
25
11
Moderate
0
0
0
0
0
0
56
0
Severe
0
0
0
0
0
0
6
0
Tubules, Protein
-------
130
NR
100
100
0
43
100
100
6
11
Minimal
0
0
8
0
0
0
6
22
Mild
0
0
75
36
0
0
75
44
Moderate
0
0
17
21
0
0
13
0
Severe
0
0
0
0
0
0
0
22
Interstitial Fluid (Proteinaceous)
NR
100
100
0
0
100
71
0
0
Minimal
0
0
8
14
0
29
13
0
Mild
0
0
92
86
0
0
56
44
Moderate
0
0
0
0
0
0
19
56
Severe
0
0
0
0
0
0
13
0
Interstitial Fibrosis
NR
100
100
8
100
100
100
25
89
Minimal
0
0
33
0
0
0
19
11
Mild
0
0
50
0
0
0
50
0
Moderate
0
0
8
0
0
0
6
0
Severe
0
0
0
0
0
0
0
0
Regenerative blast cell hyperplasia
NR
-
-
-
-
100
100
25
44
Minimal
-
-
-
-
0
0
31
56
Mild
-
-
-
-
0
0
31
0
Moderate
-
-
-
-
0
0
13
0
Severe
-
-
-
-
0
0
0
0
Tubules, Dilation
NR
25
11
0
7
18
12
0
56
Minimal
55
79
0
36
71
47
13
22
Mild
20
11
92
50
12
41
50
0
Moderate
0
0
8
7
0
0
38
22
Severe
0
0
0
0
0
0
0
0
Tubules, Mineralization / Casts
NR
5
0
58
64
6
24
13
100
Minimal
30
42
8
29
35
24
25
0
Mild
65
53
33
7
59
53
63
0
Moderate
0
5
0
0
0
0
0
0
Severe
0
0
0
0
0
0
0
0
Reproductive duct histopathology
Oviduct development was rather variable within and between genders. In genotypic females,
there was a significant delay in oviduct development in all treatments. However, in both
genders, there are some individuals in the 1.5 and 3 mg/L treatments that are two stages more
advanced than the others. Both genders experienced advancement of Wolffian duct development
in the 3 and 6 mg/L treatments. Again, these were all phenotypic females (Table 8.4.4-9).
Table 8.4.4-9
Histological staging of juvenile reproductive ducts following BP2 exposure (NR: not remarkable; dash: not observed; highlighted cells are
significantly different than control based on RSCABS analysis),
Nominal BP2 c
Male
Female
oncentration (mg/L)
0
1.5
3
6
0
1.5
3
6
n
20
19
12
14
17
17
16
9
replicates
4
4
4
4
4
4
4
4
Oviduct Stage
Not present
5
0
0
0
0
0
0
0
1
95
89
92
93
6
35
81
89
2
0
0
0
7
88
0
0
0
3
0
0
0
0
6
0
0
0
4
0
11
8
0
0
65
19
0
Wolffian Stage
1
30
37
33
36
18
41
6
33
2
70
58
17
43
76
59
38
33
3
0
5
50
21
6
0
50
11
4
0
0
0
0
0
0
6
11
------- |