This AOP is licensed under the BY-SA license. This license allows reusers to distribute, remix, adapt, and build upon the material in any medium or format, so long as attribution is given to the creator. The license allows for commercial use. If you remix, adapt, or build upon the material, you must license the modified material under identical terms.

AOP: 346


A descriptive phrase which references both the Molecular Initiating Event and Adverse Outcome.It should take the form “MIE leading to AO”. For example, “Aromatase inhibition leading to reproductive dysfunction” where Aromatase inhibition is the MIE and reproductive dysfunction the AO. In cases where the MIE is unknown or undefined, the earliest known KE in the chain (i.e., furthest upstream) should be used in lieu of the MIE and it should be made clear that the stated event is a KE and not the MIE.  More help

Aromatase inhibition leads to male-biased sex ratio via impacts on gonad differentiation

Short name
A name that succinctly summarises the information from the title. This name should not exceed 90 characters. More help
Aromatase inhibition leads to male-biased sex ratio via impacts on gonad differentiation
The current version of the Developer's Handbook will be automatically populated into the Handbook Version field when a new AOP page is created.Authors have the option to switch to a newer (but not older) Handbook version any time thereafter. More help
Handbook Version v2.0

Graphical Representation

A graphical representation of the AOP.This graphic should list all KEs in sequence, including the MIE (if known) and AO, and the pair-wise relationships (links or KERs) between those KEs. More help
Click to download graphical representation template Explore AOP in a Third Party Tool


The names and affiliations of the individual(s)/organisation(s) that created/developed the AOP. More help

Kelvin J. Santana Rodriguez, Oak Ridge Institute for Science and Education, U.S. Environmental Protection Agency, Great Lakes Toxicology and Ecology Divison, Duluth, MN, USA

Daniel L. Villeneuve, Kathleen M. Jensen, Gerald T. Ankley, US Environmental Protection Agency, Great Lakes Toxicology and Ecology Division, Duluth, MN, USA

David H. Miller, US Environmental Protection Agency, Great Lakes Toxicology and Ecology Division, Ann Arbor, MI, USA

Point of Contact

The user responsible for managing the AOP entry in the AOP-KB and controlling write access to the page by defining the contributors as described in the next section.   More help
Kelvin Santana Rodriguez   (email point of contact)


Users with write access to the AOP page.  Entries in this field are controlled by the Point of Contact. More help
  • Kelvin Santana Rodriguez
  • Dan Villeneuve
  • Gerald Ankley


This field is used to identify coaches who supported the development of the AOP.Each coach selected must be a registered author. More help

OECD Information Table

Provides users with information concerning how actively the AOP page is being developed and whether it is part of the OECD Workplan and has been reviewed and/or endorsed. OECD Project: Assigned upon acceptance onto OECD workplan. This project ID is managed and updated (if needed) by the OECD. OECD Status: For AOPs included on the OECD workplan, ‘OECD status’ tracks the level of review/endorsement of the AOP . This designation is managed and updated by the OECD. Journal-format Article: The OECD is developing co-operation with Scientific Journals for the review and publication of AOPs, via the signature of a Memorandum of Understanding. When the scientific review of an AOP is conducted by these Journals, the journal review panel will review the content of the Wiki. In addition, the Journal may ask the AOP authors to develop a separate manuscript (i.e. Journal Format Article) using a format determined by the Journal for Journal publication. In that case, the journal review panel will be required to review both the Wiki content and the Journal Format Article. The Journal will publish the AOP reviewed through the Journal Format Article. OECD iLibrary published version: OECD iLibrary is the online library of the OECD. The version of the AOP that is published there has been endorsed by the OECD. The purpose of publication on iLibrary is to provide a stable version over time, i.e. the version which has been reviewed and revised based on the outcome of the review. AOPs are viewed as living documents and may continue to evolve on the AOP-Wiki after their OECD endorsement and publication.   More help
OECD Project # OECD Status Reviewer's Reports Journal-format Article OECD iLibrary Published Version
1.12 WPHA/WNT Endorsed
This AOP was last modified on October 17, 2023 04:51

Revision dates for related pages

Page Revision Date/Time
Inhibition, Aromatase March 14, 2022 08:43
Reduction, 17beta-estradiol synthesis by the undifferentiated gonad July 13, 2022 10:25
Increased, Differentiation to Testis December 28, 2022 10:15
Increased, Male Biased Sex Ratio January 03, 2023 08:22
Decrease, Population growth rate January 03, 2023 09:09
Inhibition, Aromatase leads to Increased, Differentiation to Testis December 28, 2022 15:28
Inhibition, Aromatase leads to Reduction, E2 Synthesis by the undifferentiated gonad July 11, 2022 14:23
Inhibition, Aromatase leads to Increased, Male Biased Sex Ratio January 10, 2023 11:55
Reduction, E2 Synthesis by the undifferentiated gonad leads to Increased, Differentiation to Testis July 11, 2022 15:17
Increased, Differentiation to Testis leads to Increased, Male Biased Sex Ratio January 03, 2023 08:35
Increased, Male Biased Sex Ratio leads to Decrease, Population growth rate January 03, 2023 08:37
Fadrozole November 29, 2016 18:42
Letrozole November 29, 2016 18:42
Exemestane November 12, 2020 01:53
Stressor:292 Clotrimazole November 12, 2020 01:55
Prochloraz November 29, 2016 18:42


A concise and informative summation of the AOP under development that can stand-alone from the AOP page. The aim is to capture the highlights of the AOP and its potential scientific and regulatory relevance. More help

This adverse outcome pathway links inhibition of aromatase activity in teleost fish during gonadogenesis to increased differentiation to testis resulting in a male-biased sex ratio in the population, and ultimately, reduced population sustainability.  Most gonochoristic fish species develop either as males or females and do not change sex throughout their life span. However, in species where sexual differentiation is controlled at least to some degree by environmental factors, there can be a window of development during gonadal differentiation that is sensitive to a variety of exogenous conditions, including exposure to some chemicals. For example, treatment with sex steroids in conjunction with the period of sexual differentiation has been showed to favor ovary or testis development in fish exposed to estrogens or androgens, respectively. Altered synthesis and regulation of endogenous steroids can also affect sexual differentiation in fish. In most vertebrate taxa, aromatase (cytochrome P450 [CYP]19a1) is the rate-limiting enzyme for the conversion of 17β-estradiol (E2) from testosterone (T). Endocrine-active chemicals such as fadrozole, letrozole and exemestane (pharmaceuticals) or prochloraz and propiconazole (fungicides) inhibit aromatase activity. Exposure of some  fish species to aromatase inhibitors during sex differentiation can reduce endogenous E2 synthesis, thereby resulting in phenotypic males, the default sex in the absence of estrogen signaling during gonadal differentiation. Given the critical role of female fecundity in determining total numbers of offspring, the resultant male-biased sex ratio can reduce population size, especially if sustained over multiple generations.

AOP Development Strategy


Used to provide background information for AOP reviewers and users that is considered helpful in understanding the biology underlying the AOP and the motivation for its development.The background should NOT provide an overview of the AOP, its KEs or KERs, which are captured in more detail below. More help

In fish sexual differentiation occurs post hatch and can be influenced by exogenous factors such as chemicals, temperature, pH, population density, social cues and more. As a result, the gonadal sex phenotype in oviparous fish can be altered by environmental conditions experienced during development, particularly in conjunction with sexual differentiation (Scholz and Klüver, 2009). At this stage, the bipotential gonad can differentiate to either testes or ovaries depending both on genetic and environmental factors (Strüssmann and Nakamura, 2002). Sex steroids are among the factors that influence sex differentiation in non-mammalian vertebrates; in many fish species exogenous androgens and estrogens act, respectively, to enhance the development of testes and ovaries in exposed animals (Nakamura 2010). In teleost fish, the relative balance between endogenous estrogens and androgens during sexual differentiation is critical to ensuring normal sex ratios and, ultimately, viable populations. Various homeostatic mechanisms ensure that steroid biosynthesis is appropriately controlled during development. A key biosynthetic enzyme is CYP19a1 (aromatase), which is responsible for the conversion of C19 androgens (e.g., T) to C18 estrogens (e.g., E2) in brain and gonadal tissues of vertebrates (Payne and Hales, 2004; Simpson et al. 1994).  In fish, there are two CYP19a1 isoforms, with CYP19a1a mostly expressed in the gonads and CYP19a1b largely expressed in the brain (Callard et al. 2001).

Since the mid-90s, there has been concern about the potential impacts of endocrine disrupting chemicals (EDCs) in fish and wildlife. Many  EDCs can exert effects in early life stages that can lead to potential impacts at the population level. For example, some chemicals have been shown to alter the sexual phenotype of fish by affecting steroidogenic enzymes such as aromatase. Inhibition of CYP19a1 expression or activity can alter the production  of estrogens in  developing gonads, affecting processes such as gonadal differentiation. In many fish species the “default” gonad type is testes, so when estrogen signaling is reduced there is a resultant bias toward male-biased sex ratios (Guiguen et al. 2010).   When male biased sex ratios occur, the number of breeding females can decrease over time and have negative impacts on population growth and sustainability. The present AOP provides the evidence framework of the negative impacts of aromatase inhibition at early developmental stages of teleost fish  during the critical period of sexual differentiation and how this could lead to  population-level effects.



Provides a description of the approaches to the identification, screening and quality assessment of the data relevant to identification of the key events and key event relationships included in the AOP or AOP network.This information is important as a basis to support the objective/envisaged application of the AOP by the regulatory community and to facilitate the reuse of its components.  Suggested content includes a rationale for and description of the scope and focus of the data search and identification strategy/ies including the nature of preliminary scoping and/or expert input, the overall literature screening strategy and more focused literature surveys to identify additional information (including e.g., key search terms, databases and time period searched, any tools used). More help

Work on this AOP initially was conducted by Mr. Santana-Rodriguez under the supervision of Dr. Daniel Villeneuve, a leader in the field of AOP development. Dr. Gerald Ankley also contributed to AOP development, particularly at later stages of the effort. Dr. Ankley is experienced with AOP development, and is a widely-recognized international expert concerning the effects of EDCs on fish. The AOP is based on published peer-reviewed literature derived from focused searches guided by the expertise of the authors. Ms. Kathleen Jensen, who also has worked for many years on EDCs/fish endocrinology, provided a secondary QA review of key papers supporting the AOP.

Summary of the AOP

This section is for information that describes the overall AOP.The information described in section 1 is entered on the upper portion of an AOP page within the AOP-Wiki. This is where some background information may be provided, the structure of the AOP is described, and the KEs and KERs are listed. More help


Molecular Initiating Events (MIE)
An MIE is a specialised KE that represents the beginning (point of interaction between a prototypical stressor and the biological system) of an AOP. More help
Key Events (KE)
A measurable event within a specific biological level of organisation. More help
Adverse Outcomes (AO)
An AO is a specialized KE that represents the end (an adverse outcome of regulatory significance) of an AOP. More help
Type Event ID Title Short name
MIE 36 Inhibition, Aromatase Inhibition, Aromatase
KE 1789 Reduction, 17beta-estradiol synthesis by the undifferentiated gonad Reduction, E2 Synthesis by the undifferentiated gonad
KE 1790 Increased, Differentiation to Testis Increased, Differentiation to Testis
KE 1791 Increased, Male Biased Sex Ratio Increased, Male Biased Sex Ratio
AO 360 Decrease, Population growth rate Decrease, Population growth rate

Relationships Between Two Key Events (Including MIEs and AOs)

This table summarizes all of the KERs of the AOP and is populated in the AOP-Wiki as KERs are added to the AOP.Each table entry acts as a link to the individual KER description page. More help

Network View

This network graphic is automatically generated based on the information provided in the MIE(s), KEs, AO(s), KERs and Weight of Evidence (WoE) summary tables. The width of the edges representing the KERs is determined by its WoE confidence level, with thicker lines representing higher degrees of confidence. This network view also shows which KEs are shared with other AOPs. More help

Prototypical Stressors

A structured data field that can be used to identify one or more “prototypical” stressors that act through this AOP. Prototypical stressors are stressors for which responses at multiple key events have been well documented. More help

Life Stage Applicability

The life stage for which the AOP is known to be applicable. More help
Life stage Evidence
Development High

Taxonomic Applicability

Latin or common names of a species or broader taxonomic grouping (e.g., class, order, family) can be selected.In many cases, individual species identified in these structured fields will be those for which the strongest evidence used in constructing the AOP was available. More help
Term Scientific Term Evidence Link
zebrafish Danio rerio High NCBI
Oreochromis niloticus Oreochromis niloticus High NCBI
Chinook salmon Oncorhynchus tshawytscha Low NCBI
fathead minnow Pimephales promelas Low NCBI
European sea bass Dicentrarchus labrax Low NCBI

Sex Applicability

The sex for which the AOP is known to be applicable. More help
Sex Evidence
Unspecific High

Overall Assessment of the AOP

Addressess the relevant biological domain of applicability (i.e., in terms of taxa, sex, life stage, etc.) and Weight of Evidence (WoE) for the overall AOP as a basis to consider appropriate regulatory application (e.g., priority setting, testing strategies or risk assessment). More help

See details below.

Domain of Applicability

Addressess the relevant biological domain(s) of applicability in terms of sex, life-stage, taxa, and other aspects of biological context. More help

Life Stage

The life stage to which this AOP applies is developing embryos/juveniles during gonadal differentiation. Since the sexually dimorphic expression of aromatase has been shown to play a crucial role in the differentiation to testis vs ovary of the undifferentiated bipotential gonad (Guiguen et al. 2010), the AOP is applicable to the stage of development during which aromatase might influence this process. The precise timing of the sensitive period relevant to this AOP will vary by species, but the AOP is not applicable to differentiated juveniles or to adults.

Studies with zebrafish (Danio rerio) have shown that both brain and gonadal aromatase expression can be observed at 20 days post-fertilization (dpf) with an increase in expression at 25 dpf in fish destined to become females, coinciding with the onset of gonadal differentiation period (Lau et al. 2016). In Nile tilapia (Oreochromis niloticus), aromatase expression can be observed as early as 3-4 dpf with an increase in expression starting at 11 dpf in genetic females (Kwon et al. 2001). Additionally, it has been shown that the period of 7-14 dpf is the most sensitive to chemical inhibition of CYP19a1 activity, and a continuous exposure of 2-3 weeks is sufficient for the masculinization of the majority of genetic female tilapia (Kwon et al. 2000). This clearly indicates alteration of differentiation from ovary to testis results during sex differentiation (OECD 2011). 


The molecular initiating event for this AOP occurs during gonad differentiation. Therefore, the AOP is only applicable to sexually undifferentiated individuals.  


Most evidence for the taxonomic applicability of this AOP comes from species in the class Osteichthyes. Aromatase itself is well conserved among vertebrates (e.g., Wilson et al. 2005; LaLone et al. 2018).  However, the degree to which aromatase and subsequent production of endogenous estrogens such as E2 are involved in sex determination or sexual differentiation varies with species. Many fish, amphibian, and reptile species have environmental sex determination, and regulation of aromatase expression and sex steroids profiles are closely tied to sex-determining environmental factors (Angelopoulou et al. 2012). Alternatively, vertebrates that largely rely on genetic sex determination (birds, mammals) would be anticipated to be less vulnerable to effects of aromatase inhibitors during gonad differentiation, although there remains compelling evidence for an important role of steroid signaling during the process (Angelopoulou et al. 2012).  Overall, regardless of differing roles for aromatase in sexual differentiation, expression appears universal among vertebrates during this life stage (Angelopoulou et al. 2012; Sarre et al. 2004; Uller and Helantera, 2011; Ramsey and Crews, 2009).  Thus, in principle, components of the present AOP may have some degree of applicability to all vertebrates. Given the substantial diversity of sex determination and differentiation strategies in fish, amphibians and reptiles (including those from closely related phylogenetic groups; Sarre et al. 2004; Angelopoulou et al. 2012), quantiative sensitivity, and taxonomic domain of appicability of the present AOP are hard to generalize, although there is reason to believe it should have broad applicability in bony fishes.

Essentiality of the Key Events

The essentiality of KEs can only be assessed relative to the impact of manipulation of a given KE (e.g., experimentally blocking or exacerbating the event) on the downstream sequence of KEs defined for the AOP. Consequently, evidence supporting essentiality is assembled on the AOP page, rather than on the independent KE pages that are meant to stand-alone as modular units without reference to other KEs in the sequence. The nature of experimental evidence that is relevant to assessing essentiality relates to the impact on downstream KEs and the AO if upstream KEs are prevented or modified. This includes: Direct evidence: directly measured experimental support that blocking or preventing a KE prevents or impacts downstream KEs in the pathway in the expected fashion. Indirect evidence: evidence that modulation or attenuation in the magnitude of impact on a specific KE (increased effect or decreased effect) is associated with corresponding changes (increases or decreases) in the magnitude or frequency of one or more downstream KEs. More help

Direct support for the essentiality of several of the key events in the AOP has been provided by gene modification/knockout studies of the cyp19a1 gene in zebrafish and Nile tilapia. Specifically:

  1. Lau et al. (2016) generated insertion/deletion mutations in the zebrafish cyp19a1a gene using TALEN (transcription activator-like effector nuclease) and CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 approaches. All mutant cyp19a1a-/- fish developed as males. Histological examination (at 120 dpf) of the cyp1a1a-/- mutants showed that they exhibited normal spermatogenesis in the testis with no observable difference between the wild type (+/+) and heterozygous (+/-) males. To confirm the necessity of E2 synthesis for ovarian differentiation, they performed an experiment to "rescue" the phenotype of cyp19a1a mutants by E2 treatment (0.05, 0.50 and 5.00 nM) encampassing the period of gonadal differentiation (15–30 days pdf). Treatment with the estrogen resulted in normal functioning ovaries with fully developed perinucleolar oocytes and small amount of stromal tissue, even in some individuals at the lowest E2 concentration (0.05 nM). This supports the essentiality of aromatase inhibition relative to E2 synthesis reduction as a critical step for testis differentiation.
  2. In a similar study also with zebrafish, Muth-Köhne et al. (2016) generated cyp19a1a and cyp19a1b gene mutant lines and a cyp19a1a;cyp19a1b double-knockout line using TALENs. All cyp19a1a mutants and cyp19a1a;cyp19a1b double mutants developed as males, whereas cyp1a1b double mutant (-/-) had a 1:1 sex ratio similar to the wild type controls. This again supports the essentiality of gonadal aromatase inhibition for testis differentiation that would lead to a male biased sex ratio. Additionally, a small rescue experiment performed using E2 on all male mutant cyp1a1a-/-  indicated that E2 treatment could restore a near normal sex ratio (9 females among 14 fish).
  3. Studies in Nile tilapia similar to those conducted in zebrafish were described by Zhang et al. (2017), who worked with genetic female mutants for cypa19a and cyp19a1b. Results showed that all cyp19a1a+/- XX and cyp19a1a+/+ XX fish developed as females, whereas all cyp19a1a-/- XX and cyp19a1a-/- XY fish developed as males, based on gonad differentiation. The cyp19a1a-/- XX tilapia shifted to the male pathway as early as 5 dph and ultimately were fertile. This again provides strong support for the critical role of gonadal aromatase relative to ovarian development. 

Key Event



Inhibition, Aromatase


There is good evidence from gene knockout experiments of the two different isoforms of aromatase that support the specificity of gonadal aromatase inhibition for the subsequent key events to occur.

E2 Synthesis by the undifferentiated gonad


There is evidence from a stop (by cyp19a1 knockout) and recovery (through compensation) experiment where E2 can rescue the sex ratio altered due to the gonadal aromatase gene knockout suggesting that E2 depletion is necessary for the subsequent key events to occur.

Differentiation to Testis


By definition, differentiation to testis is required for a male reproductive phenotype. 

Male Biased Sex Ratio


Breeding females (and both sexes) are necessary for population sustainability. A male biased sex population suggests a reduced offspring production and consequentially reduced population sustainability.

Population Sustainability


This is the terminal key event in the AOP.  Its essentiality for progression to downstream events in the sequence cannot be evaluated.


Evidence Assessment

Addressess the biological plausibility, empirical support, and quantitative understanding from each KER in an AOP. More help

Biological Plausibility

Aromatase catalyzes the conversion of T to E2, so the biological plausibility of aromatase inhibition leading to reductions in available E2 is clear. Additionally, the role of E2 as a major regulator of normal female gonad development is well documented (Gorelick et al. 2011; Guiguen et al. 2010). The link between E2 reductions leading to increased differentiation of the bipotential gonad to testis is highly plausible. As E2 signaling is reduced, ER responsive genes required for ovarian differentiation will be downregulated in the bipotential gonad resulting in a default development of testes (Yin et al. 2017; Zhang et al. 2017). Therefore, it is plausible that E2 reduction in the undifferentiated gonad at the onset of sexual differentiation would promote testis formation. The direct link between increased differentiation to testis leading to a male biased sex ratio is also well supported by biological plausibility. If the conditions that favor a male producing phenotype (in this case, the aromatase inhibitor) overlap with the critical period of sex differentiation in a given population, it is reasonable that relatively more male offspring will be produced (D'Cotta et al., 2001, Kwon et al., 2000; Luzio et al. 2016). Therefore, exposure of sensitive species to aromatase inhibition for an extended period of time during reproducitve development plausibly would result in a male-biased population. Empirical evidence supporting the direct link between male biased cohorts and a reduced population sustainability in fish species is limited. However, biased sex ratios can definitely impact fish populations (Marty et al. 2017). For example, a male-biased sex ratio would logically lead to a reduction in the number of breeding females such that over time decreases in offspring would result in population declines (Brown et al. 2015; Grayson et al. 2014). Miller et al. (2022) recently developed a model specifically designed to capture the effects of male-biased sex ratios on population trajectories in fathead minnows (Pimephales promelas). 

Concordance of Dose Response Relationships

There have been a number of in vitro and in vivo studies, primarily in fish, that have examined the effects of known aromatase inhibitors on different key events in the AOP.  Most of these studies only measured one key event in the AOP so cannot be directly used to explore dose-response concordance between key events.

The differential sensitivity to inhibition of aromatase is most easily measured in vitro. Doering et al. (2019b) determined the effects of different concentrations of several known aromatase inhibitors (e.g., fadrozole, prochloraz) on brain aromatase activity in a taxonomically-diverse set of fish species, and found that while absolute potency of the chemicals varied across species, rank order potency of the test chemicals was generally similar. Importantly, relative potencies measured in vitro reflected those observed in in vivo studies such as those described below, thus providing indirect evidence of dose concordance between the MIE and downstream Key Events.

There have been several in vivo studies evaluating the effects of varying degrees of aromatase inhibition on different key events in the AOP. However, there are limitations to these studies in the context of determining dose-dependency across all key events in the AOP. For example, E2 levels typically have not been or measured or determined at a time relevant to gonadal differentiation. However, a few have measured multiple key events, although typically only at one time point. One study assessed dose-reponse relationships between different concentrations of the model aromatase inhibitor exemestane and expression of the enzyme. Immunohistochemical analyses revealed that gonad tissue of Nile tilapia (Oreochromis niloticus) exposed from 9-35 days post-hatch (dph) to 100, 500, 1000 and 2000 μg/g feed had no cross-reaction with P450arom at the three highest doses, but gonad tissue samples exhibited a strong immunopositive responses against P450arom at a lower dose of exemestane (100 μg/g feed), similar to the differentiating ovaries of the control fish (Ruksana et al. 2010). No ovarian development was noted in fish in the 500, 1000 and 2000 mg/kg treatments, and the 1000 and 2000 treatments resulted in 100% phenotypic males.

Uchida et al. (2004) evaluated two key events in the AOP in an experiment with fadrozole using zebrafish genetic females exposed from 15-40 dph via the diet. They observed ovarian transition to testis in all exposed animals, culminating in 62.5, 100 and 100% males in 10, 100 and 1000 mg/kg treatments, respectively.

Another study showed a dose-dependent rate of increased differentiation to testes in zebrafish exposed from 0-63 dph to different concentrations of fadrozole (10, 32, 100 ug/L) via the water (Muth-Köhne et al. 2016).

The most commonly reported dose response relationship for this AOP was for the non-adjacent relationship between aromatase inhibition and an increased male biased sex ratio. For example, Nile tilapia, zebrafish, fathead minnow (Pimephales promelas), bluegill (Lepomis macrochirus), yellow catfish (Pelteobagrus fulvidraco) and Japanese flounder (Paralichthys olivaceus) exposed to different concentrations of known aromatase inhibitors (exemestane, fadrozole, letrozole, prochloraz) via the diet or water reported dose-dependent increases in the relative number of males (Kwon et al. 2000; Kitano et al. 2000; Thorpe et al. 2011;  Holbech et al. 2012; Gao et al. 2010; Shen et al. 2013).

Finally, there are models that demonstrate a dose-dependent decrease in population size corresponding with an increasing proportion of males in zebrafish and fathead minnows (Brown et al. 2015; Miller et al. 2022).

Temporal Concordance

Because this AOP involves actions during a specific development transition from an undifferentiated to differentiated gonad, the temporal concordance of the events is implicit. A male biased sex ratio cannot be observed until the population has undergone sexual differentiation. Likewise, reproduction and associated population growth rate cannot be assessed until the animals achieve sexual maturity.


There have been a number of in vitro and in vivo studies, primarily in fish, that have examined the effects of known aromatase inhibitors on different key events in the AOP. Some of these studies measured only one key event in the AOP and/or employed just a single dose of a given stressor, so cannot be directly used to explore dose-response concordance. However, even with these limitations, they demonstrate that the overall AOP is consistent with expectations in a variety of species exposed to known chemical inhibitors of aromatase (see Dose Concordance table). For example, studies with chinook salmon (Oncorhynchus tshawytscha), Japanese fugu (Takifugu rubripes), Japanese medaka (Oryzias latipes), Nile tilapia, zebrafish, fathead minnow, bluegill, yellow catfish and Japanese flounder exposed to known aromatase inhibitors (exemestane, fadrozole, letrozole, prochloraz) via the diet or water during sexual differentiation have reported increases in differentiation to testis and/or the relative number of males (Piferrer et al. 1994; Kwon et al. 2000; Rashid et al. 2007; Kitano et al. 2000; Thorpe et al. 2011; Thresher et al. 2011; Holbech et al. 2012; Gao et al. 2010; Shen et al. 2013).

Male-biased sex ratios are not specific to this AOP. Many of the key events included overlap with another AOP (#376) linking activation of the androgen receptor to male biased sex ratios.

Uncertainties, inconsistencies, and data gaps

Currently the major uncertainty in this AOP is the biological linkage between E2 synthesis reduction by the undifferentiated gonad leading to an increased, differentiation to testis. Biological plausibility connections have been established, but experimental measurements of E2 during the particular period of differentiation are lacking. Also, as noted in the Domain of Applicability section, the taxonomic range of applicability of the AOP is uncertain.

Known Modulating Factors

Modulating factors (MFs) may alter the shape of the response-response function that describes the quantitative relationship between two KES, thus having an impact on the progression of the pathway or the severity of the AO.The evidence supporting the influence of various modulating factors is assembled within the individual KERs. More help
Modulating Factor (MF) Influence or Outcome KER(s) involved

Quantitative Understanding

Optional field to provide quantitative weight of evidence descriptors.  More help

There is not yet a sufficient quantitative understanding of this overall AOP to predict the degree to which aromatase inhibition would result in population-level impacts. That said, there are models available suitable for the quantitative prediction of changes in E2 levels caused by degree of aromatase inhibition in some small fish species (Conolly et al. 2018; Doering et al. 2019a), as well as the effects of different (male-biased) sex ratios on fathead minnow population size (Miller et al. 2022).  However, there currently are no quantitative data/models relating reductions in E2 to the degree of (increased) differentiation to male gonads and/or male-biased cohorts of fish.  

Considerations for Potential Applications of the AOP (optional)

Addressess potential applications of an AOP to support regulatory decision-making.This may include, for example, possible utility for test guideline development or refinement, development of integrated testing and assessment approaches, development of (Q)SARs / or chemical profilers to facilitate the grouping of chemicals for subsequent read-across, screening level hazard assessments or even risk assessment. More help

Altered sex ratios in fish can be a useful diagnostic endpoint for identifying EDCs both in field and lab settings. For example, the Fish Sexual Development Test (FSDT) has formally been adopted by the Organisation of Economic Cooperation and Development (OECD) as a test guideline (No. 234) for the detecting EDCs (OECD, 2011b). The FDST is conducted in zebrafish during early development, including sexual differentiation, and uses gonadal differentiation and skewed sex ratios to detect estrogen, androgen and steroidogenesis activity of test chemicals (Dang & Kienzler 2019). This AOP directly supports the mechanistic basis for assays such as the FDST. The AOP also supports the use of in vitro assays that measure aromatase inhibition by test chemicals as a basis for predicting apical impacts on fish (e.g., Conolly et al. 2018; Doering et al. 2019a; 2019b). 


List of the literature that was cited for this AOP. More help


Angelopoulou, R., Lavranos, G., & Manolakou, P. (2012). Sex determination strategies in 2012: towards a common regulatory model?. Reproductive biology and endocrinology : RB&E10, 13.

Brown, A. R., Owen, S. F., Peters, J., Zhang, Y., Soffker, M., Paull, G. C., Hosken, D. J., Wahab, M. A., & Tyler, C. R. (2015). Climate change and pollution speed declines in zebrafish populations. Proceedings of the National Academy of Sciences of the United States of America112(11), E1237–E1246.

Conolly, R.B., G.T. Ankley, W.-Y. Cheng, M.L. Mayo, D.H. Miller, E.J. Perkins, D.L. Villeneuve and K.H. Watanabe. 2017. Quantitative adverse outcome pathways and their application to predictive toxicology. Environ. Sci. Technol. 51, 4661-4672.

D'Cotta, H., Fostier, A., Guiguen, Y., Govoroun, M., & Baroiller, J. F. (2001). Aromatase plays a key role during normal and temperature-induced sex differentiation of tilapia Oreochromis niloticus. Molecular reproduction and development59(3), 265–276.

Dang, Z., & Kienzler, A. (2019). Changes in fish sex ratio as a basis for regulating endocrine disruptors. Environment international130, 104928.

Doering, J.A., D.L. Villeneuve, K.A. Fay, E.C. Randolph, K.M. Jensen, M.D. Kahl, C.A. LaLone and G.T. Ankley. (2019b). Differential sensitivity to in vitro inhibition of cytochrome P450 aromatase (CYP19) activity among 18 freshwater fishes. Toxicol. Sci. 170, 394-403.

Doering, J.A., D.L. Villeneuve, S.T. Poole, B.R. Blackwell, K.M. Jensen, M.D. Kahl, A.R. Kittelson, D.J. Feifarek, C.B. Tilton, C.A. LaLone and G.T. Ankley. (2019a). Quantitative response-response relationships linking aromatase inhibition to decreased fecundity are conserved across three fishes with asynchronous oocyte development. Environ. Sci. Technol. 53, 10470-10578.

Gao, Z.X., Wang H.P., Wallat, G., Yao, H., Rapp, D. , O ’ Bryant, P., MacDonald, R. & Wang, W. (2010). Effects of a non-steroidal aromatase inhibitor on gonadal differentiation of bluegill sunfish Lepomis macrochirus . Aquacult Res , 41 , 1282 – 9 .

Gorelick, D. A., & Halpern, M. E. (2011). Visualization of estrogen receptor transcriptional activation in zebrafish. Endocrinology, 152(7), 2690–2703.

Grayson, K. L., Mitchell, N. J., Monks, J. M., Keall, S. N., Wilson, J. N., & Nelson, N. J. (2014). Sex ratio bias and extinction risk in an isolated population of Tuatara (Sphenodon punctatus). PloS one, 9(4), e94214.

Guiguen, Y., Fostier, A., Piferrer, F., & Chang, C. F. (2010). Ovarian aromatase and estrogens: a pivotal role for gonadal sex differentiation and sex change in fish. General and comparative endocrinology, 165(3), 352–366.

Holbech, H., Kinnberg, K. L., Brande-Lavridsen, N., Bjerregaard, P., Petersen, G. I., Norrgren, L., Orn, S., Braunbeck, T., Baumann, L., Bomke, C., Dorgerloh, M., Bruns, E., Ruehl-Fehlert, C., Green, J. W., Springer, T. A., & Gourmelon, A. (2012). Comparison of zebrafish (Danio rerio) and fathead minnow (Pimephales promelas) as test species in the Fish Sexual Development Test (FSDT). Comparative biochemistry and physiology. Toxicology & pharmacology : CBP155(2), 407–415.

Kitano, T., Takamune, K., Nagahama, Y., & Abe, S. I. (2000). Aromatase inhibitor and 17alpha-methyltestosterone cause sex-reversal from genetical females to phenotypic males and suppression of P450 aromatase gene expression in Japanese flounder (Paralichthys olivaceus). Molecular reproduction and development, 56(1), 1–5.<1::AID-MRD1>3.0.CO;2-3

Kwon, J. Y., Haghpanah, V., Kogson-Hurtado, L. M., McAndrew, B. J., & Penman, D. J. (2000). Masculinization of genetic female nile tilapia (Oreochromis niloticus) by dietary administration of an aromatase inhibitor during sexual differentiation. The Journal of experimental zoology287(1), 46–53.

Kwon, J. Y., McAndrew, B. J., & Penman, D. J. (2001). Cloning of brain aromatase gene and expression of brain and ovarian aromatase genes during sexual differentiation in genetic male and female Nile tilapia Oreochromis niloticus. Molecular reproduction and development, 59(4), 359–370.

Lau, E. S., Zhang, Z., Qin, M., & Ge, W. (2016). Knockout of Zebrafish Ovarian Aromatase Gene (cyp19a1a) by TALEN and CRISPR/Cas9 Leads to All-male Offspring Due to Failed Ovarian Differentiation. Scientific reports, 6, 37357.

LaLone, C.A., D.L. Villeneuve, J.A. Doering, B.R. Blackwell, T.R. Transue, C.W. Simmons, J. Swintek, S.J. Degitz, A.J. Williams and G.T. Ankley. 2018. Evidence for cross-species extrapolation of mammalian-based high-throughput screening assay results. Environ. Sci. Technol. 52, 13960-13971.

Luzio, A., Matos, M., Santos, D., Fontaínhas-Fernandes, A. A., Monteiro, S. M., & Coimbra, A. M. (2016). Disruption of apoptosis pathways involved in zebrafish gonad differentiation by 17α-ethinylestradiol and fadrozole exposures. Aquatic toxicology (Amsterdam, Netherlands), 177, 269–284.

Luzio, A., Monteiro, S. M., Rocha, E., Fontaínhas-Fernandes, A. A., & Coimbra, A. M. (2016). Development and recovery of histopathological alterations in the gonads of zebrafish (Danio rerio) after single and combined exposure to endocrine disruptors (17α-ethinylestradiol and fadrozole). Aquatic toxicology (Amsterdam, Netherlands), 175, 90–105.

Marty, M. S., Blankinship, A., Chambers, J., Constantine, L., Kloas, W., Kumar, A., Lagadic, L., Meador, J., Pickford, D., Schwarz, T., & Verslycke, T. (2017). Population-relevant endpoints in the evaluation of endocrine-active substances (EAS) for ecotoxicological hazard and risk assessment. Integrated environmental assessment and management13(2), 317–330.

Miller, D.H., D.L. Villeneuve, K.J. Santana-Rodriguez and G.T. Ankley. 2022. A multi-dimensional matrix model for predicting the effects of male-biased sex ratios on fish populations. Environmental Toxicology and Chemistry. 41, 1066-1077.

Muth-Köhne, E., Westphal-Settele, K., Brückner, J., Konradi, S., Schiller, V., Schäfers, C., Teigeler, M., & Fenske, M. (2016). Linking the response of endocrine regulated genes to adverse effects on sex differentiation improves comprehension of aromatase inhibition in a Fish Sexual Development Test. Aquatic toxicology (Amsterdam, Netherlands), 176, 116–127.

Nakamura M. (2010). The mechanism of sex determination in vertebrates-are sex steroids the key-factor?. Journal of experimental zoology. Part A, Ecological genetics and physiology313(7), 381–398.

Payne, A. H., & Hales, D. B. (2004). Overview of steroidogenic enzymes in the pathway from cholesterol to active steroid hormones. Endocrine reviews25(6), 947–970.

Piferrer, F., Zanuy, S., Carrillo, M., Solar, I. I., Devlin, R. H., & Donaldson, E. M. (1994). Brief treatment with an aromatase inhibitor during sex differentiation causes chromosomally female salmon to develop as normal, functional males. Journal of Experimental Zoology, 270(3), 255–262.

Ramsey, M., & Crews, D. (2009). Steroid signaling and temperature-dependent sex determination-Reviewing the evidence for early action of estrogen during ovarian determination in turtles. Seminars in cell & developmental biology20(3), 283–292.

Rashid, H., Kitano, H., Lee, K. H., Nii, S., Shigematsu, T., Kadomura, K., Yamaguchi, A., & Matsuyama, M. (2007). Fugu (Takifugu rubripes) sexual differentiation: CYP19 regulation and aromatase inhibitor induced testicular development. Sexual development : genetics, molecular biology, evolution, endocrinology, embryology, and pathology of sex determination and differentiation, 1(5), 311–322.

Ruksana, S., Pandit, N. P., & Nakamura, M. (2010). Efficacy of exemestane, a new generation of aromatase inhibitor, on sex differentiation in a gonochoristic fish. Comparative biochemistry and physiology. Toxicology & Pharmacology : CBP, 152(1), 69–74.

Sarre, S. D., Georges, A., & Quinn, A. (2004). The ends of a continuum: genetic and temperature-dependent sex determination in reptiles. BioEssays : news and reviews in molecular, cellular and developmental biology26(6), 639–645.

Scholz, S., & Klüver, N. (2009). Effects of endocrine disrupters on sexual, gonadal development in fish. Sexual development : genetics, molecular biology, evolution, endocrinology, embryology, and pathology of sex determination and differentiation3(2-3), 136–151.

Shen, Z. G., Fan, Q. X., Yang, W., Zhang, Y. L., Hu, P. P., & Xie, C. X. (2013). Effects of non-steroidal aromatase inhibitor letrozole on sex inversion and spermatogenesis in yellow catfish Pelteobagrus fulvidraco. The Biological bulletin, 225(1), 18–23.

Simpson, E. R., Mahendroo, M. S., Means, G. D., Kilgore, M. W., Hinshelwood, M. M., Graham-Lorence, S., Amarneh, B., Ito, Y., Fisher, C. R., & Michael, M. D. (1994). Aromatase cytochrome P450, the enzyme responsible for estrogen biosynthesis. Endocrine reviews15(3), 342–355.

Strüssmann, C.A. & Nakamura, M. (2002). Morphology, endocrinology, and environmental modulation of gonadal sex differentiation in teleost fishes. Fish Physiology and Biochemistry, 26, 13–29.

Thorpe, K. L., Marca Pereira, M. L., Schiffer, H., Burkhardt-Holm, P., Weber, K., & Wheeler, J. R. (2011). Mode of sexual differentiation and its influence on the relative sensitivity of the fathead minnow and zebrafish in the fish sexual development test. Aquatic Toxicology, 105(3–4), 412–420.

Thresher, R., Gurney, R., & Canning, M. (2011). Effects of lifetime chemical inhibition of aromatase on the sexual differentiation, sperm characteristics and fertility of medaka (Oryzias latipes) and zebrafish (Danio rerio). Aquatic toxicology (Amsterdam, Netherlands), 105(3-4), 355–360.

Uchida, D., Yamashita, M., Kitano, T., & Iguchi, T. (2004). An aromatase inhibitor or high water temperature induce oocyte apoptosis and depletion of P450 aromatase activity in the gonads of genetic female zebrafish during sex-reversal. Comparative biochemistry and physiology. Part A, Molecular & integrative physiology, 137(1), 11–20.

Uller, T., & Helanterä, H. (2011). From the origin of sex-determining factors to the evolution of sex-determining systems. The Quarterly review of biology86(3), 163–180.

Wilson, J. Y., McArthur, A. G., & Stegeman, J. J. (2005). Characterization of a cetacean aromatase (CYP19) and the phylogeny and functional conservation of vertebrate aromatase. General and comparative endocrinology140(1), 74–83.

Yin, Y., Tang, H., Liu, Y., Chen, Y., Li, G., Liu, X., & Lin, H. (2017). Targeted Disruption of Aromatase Reveals Dual Functions of cyp19a1a During Sex Differentiation in Zebrafish. Endocrinology, 158(9), 3030–3041.

Zhang, X., Li, M., Ma, H., Liu, X., Shi, H., Li, M., & Wang, D. (2017). Mutation of foxl2 or cyp19a1a Results in Female to Male Sex Reversal in XX Nile Tilapia. Endocrinology158(8), 2634–2647.