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AOP: 201


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

Juvenile hormone receptor agonism leading to male offspring induction associated population decline

Short name
A name that succinctly summarises the information from the title. This name should not exceed 90 characters. More help
JHR agonism leading to population decline
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 v1.0

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

You Song1 and Knut Erik Tollefsen1,2

1 Norwegian Institute for Water Research (NIVA), Section of Ecotoxicology and Risk Assessment, Gaustadalléen 21, N-0349 Oslo, Norway

2 Norwegian University of Life Sciences (NMBU), Faculty of Environmental Science and Technology, Department of Environmental Sciences (IMV). P.O. Box 5003, N-1432 Ås, Norway


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
Knut Erik Tollefsen   (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
  • Knut Erik Tollefsen
  • You Song


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
This AOP was last modified on April 29, 2023 16:02

Revision dates for related pages

Page Revision Date/Time
Activation, Juvenile hormone receptor September 16, 2017 10:17
Induction, Doublesex1 gene September 16, 2017 10:17
Increased, Male offspring December 03, 2016 16:37
Induction, Male reproductive tract September 16, 2017 10:17
Decline, Population December 03, 2016 16:33
Alteration, Food-web structures December 03, 2016 16:37
Activation, Juvenile hormone receptor leads to Induction, Doublesex1 gene December 03, 2016 16:38
Induction, Doublesex1 gene leads to Induction, Male reproductive tract December 03, 2016 16:38
Induction, Male reproductive tract leads to Increased, Male offspring December 03, 2016 16:38
Increased, Male offspring leads to Decline, Population December 03, 2016 16:38
Decline, Population leads to Alteration, Food-web structures December 03, 2016 16:38
fenoxycarb November 29, 2016 18:42
DIOFENOLAN November 29, 2016 18:42
pyriproxyfen November 29, 2016 18:42
(7S)-Hydroprene November 29, 2016 18:42
kinoprene 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

Endocrine disruption (ED) associated with environmental sex determination (ESD) and reproduction effects has been frequently reported in various ecotoxicologcail studies with crustaceans, such as daphniids (reviewed in (LeBlanc and Medlock, 2015)). Daphniids normally go through parthenogenic reproduction cycles in which only female offspring are produced. When under environmental stress (e.g. chemical exposure, short length of daylight, food shortage and high population density), a switch of reproductive strategy from asexual to sexual reproduction occurs and male offspring are produced. As an indicator of ED, the induction of male offspring is used as an important endpoint in daphnia toxicity tests. It has been widely recognized that analogs of the crustacean juvenile hormone (JH), methyl farnesoate (MF) are able to bind and activate the crustacean JH receptor, methoprene tolerant (Met), thus inducing the putative sex determination genes, doublesex genes, and causing male offspring formation. The induction of male neonates is usually associated with reduced fecundity and population decline as adverse ED effects. The existing evidences from different studies currently support the causal relationships between the biological events occurred in daphniids after exposure to JH analogs and subsequently form an adverse outcome pathway (AOP) as proposed herein.

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


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

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 1205 Activation, Juvenile hormone receptor Activation, Juvenile hormone receptor
KE 1206 Induction, Doublesex1 gene Induction, Doublesex1 gene
KE 1209 Induction, Male reproductive tract Induction, Male reproductive tract
AO 1208 Increased, Male offspring Increased, Male offspring
AO 361 Decline, Population Decline, Population
AO 1210 Alteration, Food-web structures Alteration, Food-web structures

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

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
Daphnia magna Daphnia magna NCBI
Daphnia pulex Daphnia pulex NCBI

Sex Applicability

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

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

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 Applicability This AOP applies to: juvenile (<24h old) exposed to the MF analogs during a chronic (>14d) exposure (Abe et al., 2014a); and sex-mature adult (>10d old) exposed to MF analogs in the middle of a reproduction cycle for at least three days (Abe et al., 2014a).

Taxonomic Applicability This AOP can be potentially applied to other crustacean species and insects. The ligand binding domain of Met is considered to be conserved across different daphnia species and insects (Miyakawa et al., 2013). The juvenile hormone III (methyl farnesoate) signaling pathway to exert hormonal actions are thought to be similar between daphnids and insects with only two amino acids being different, which enhances the responsiveness of the Met receptor to various juvenile hormone III analogs (Miyakawa et al., 2013).

Sex Applicability This AOP applies to females.

Elaborate on the domains of applicability listed in the summary section above. Specifically, provide the literature supporting, or excluding, certain domains.

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

Molecular Initiating Event Summary

The support for essentiality for MiE is considered to be strong. It has been shown that MF and the MF analog pyriproxyfen activated the cloned Daphnia pulex Met when expressed with the mosquito SRC ortholog (LeBlanc et al., 2013). A two-hybrid in vitro screening bioassay has been developed using transfected Chinese hamster ovary (CHO) cells containing the transcriptional activity of Met/SRC for screening of Met activation by MF analogs (Miyakawa et al., 2013). The application of this screening assay by different studies supported that chemical with MF activities, including MF, juvenile hormone III, fenoxycarb (Miyakawa et al., 2013) and diofenolan (Abe et al., 2015a) were able to activate the Met receptor and initiate downstream transcriptional regulation in a concentration-dependent manner. Evidences generated from this screening assay quantitatively demonstrated the causal relationship between chemical exposure and transcriptional activation of Met.

Key Event Summary The dsx1 gene has shown to be highly responsible for the regulation of male traits development in the embryos of D. magna after exposure to the MF analog fenoxycarb (Kato et al., 2011). The same study has also shown that lack of dsx1 gene expression in male embryos of D. magna resulted in all female phenotypes. A high number of studies have quantitatively assessed the effects of MF-like chemicals on the induction of male offspring in daphnids (Abe et al., 2015b; Abe et al., 2014b; Ginjupalli and Baldwin, 2013; Kim et al., 2011; Lampert et al., 2012; Matsumoto et al., 2008; Oda et al., 2007; Oda et al., 2005; Olmstead and LeBlanc, 2001a, 2003; Olmstead and LeBlanc, 2001b; Tatarazako et al., 2003; Wang et al., 2005). These studies strongly support the induction of male offspring as a results of MF-related ED effects. Therefore, it is evident that the support for essentiality of Met receptor activation by MF analogs leading to the induction of dsx1, male traits and male offspring is strong.

Provide an overall assessment of the essentiality for the key events in the AOP. Support calls for individual key events can be included in the molecular initiating event, key event, and adverse outcome tables above.

Evidence Assessment

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

Summary Table The overall weight of evidence (WoE) for this AOP is considered to be moderate. The WoE for each individual KER is summarized in the overview table above. The MiE of Met receptor activation leading to the induction of dsx1 gene is considered to be strong. It has been demonstrated in insects such as Drosophila that the direct target gene following the MF receptor activation was the sex lethal gene (sxl) (Zhang et al., 2014). The sxl protein as an RNA splicing factor then leads to the expression of the transformer (TRA) gene and protein to regulate the doublesex (dsx) genes (Kopp, 2012). However, studies have shown that the tra gene was not involved in the sex determination in daphnids (Kato et al., 2010). Therefore, intermediate targets between the Met receptor activation and the induction of dsx genes still need to be found. Nevertheless, the dsx1 gene has shown to be highly responsible for the regulation of male traits development in the embryos of D. magna after exposure to the MF analog fenoxycarb (Kato et al., 2011). The same study has also shown that lack of dsx1 gene expression in male embryos of D. magna resulted in all female phenotypes. Therefore, it is evident that the support for essentiality of Met receptor activation by MF analogs leading to the induction of dsx1 and male traits formation is strong. The induction of dsx1 genes and male traits development leading to the induction of male offspring is considered to be strong. A study by (Miyakawa et al., 2013) reported good correlation between the EC50s of male offspring induction and EC50s of Met receptor activation for MF-like chemicals, including MF, fenoxycarb, JH III and pyriproxyfen. The induction of male offspring leading to reduction of fecundity is considered to be moderate. A number of studies have shown that the reduction of fecundity often occurred with the increased male:female ratio of offspring in daphnia after exposure to MF analogs (Abe et al., 2015b; Abe et al., 2014b; Ginjupalli and Baldwin, 2013; Kim et al., 2011; Lampert et al., 2012; Matsumoto et al., 2008; Oda et al., 2007; Oda et al., 2005; Olmstead and LeBlanc, 2001a, 2003; Olmstead and LeBlanc, 2001b; Tatarazako et al., 2003; Wang et al., 2005). Although the underlying mechanism and quantitative causal relationship between male formation and reduced reproduction has not been well-characterized, these evidences support the hypothesis that reduction of fecundity may be caused by disrupted sex determination pathway. The reduction of fecundity and induction of male offspring leading to the decline of population size is considered to be weak. Although lack of direct experimental evidences, it may be deduced that the most efficient way of reproduction, parthenogenesis is disrupted, thus the population size in the next generations may decrease. The decline of population size leading to the disturbance of food-web structure is considered to be weak. Although lack of experimental evidences, it may deduced that the food-web structure may be disrupted due to the decline of daphnia population.

Provide an overall summary of the weight of evidence based on the evaluations of the individual linkages from the Key Event Relationship pages.

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

Quantitative Understanding

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

Summary Table The mechanistic evidences between the MiE of Met activation and induction of dsx1 gene have been clearly shown by various studies. However, the concentration-response relationship still needs to be obtained. A combination of the two-hybrid screening assay and quantitative real-time reverse transcriptase polymerase chain reaction (qPCR) would greatly facilitate the quantification of this KER. So this KER is considered to be moderate in terms of quantitative considerations. Demonstrations of the KER between the induction of dsx1 gene and male traits formation in the embryos using a combination of qPCR and in situ hybridization have been clearly provided (Kato et al., 2010). However, concentration-response relationship for this KER has not been well determined. So this KER is considered to be moderate in terms of quantitative considerations. The mechanism underlying the induction of male offspring leading to fecundity reduction has not been well characterized, and not quantitative assessment has been performed. So this KER is considered to be weak in terms of quantitative considerations. Linkages between the fecundity reduction, population decline and food-web disturbance have not been well studied. So these KERs are considered to be weak in terms of quantitative considerations.

Provide an overall discussion of the quantitative information available for this AOP. Support calls for the individual relationships can be included in the Key Event Relationship table above.

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


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

Abe, R., Toyota, K., Miyakawa, H., Watanabe, H., Oka, T., Miyagawa, S., Nishide, H., Uchiyama, I., Tollefsen, K.E., Iguchi, T., Tatarazako, N., 2014. Diofenolan induces male offspring production through binding to the juvenile hormone receptor in Daphnia magna. Aquatic toxicology 159C, 44-51.

Abe, R., Watanabe, H., Yamamuro, M., Iguchi, T., Tatarazako, N., 2014b. Establishment of a short-term, in vivo screening method for detecting chemicals with juvenile hormone activity using adult Daphnia magna. Journal of applied toxicology : JAT.

Ginjupalli, G.K., Baldwin, W.S., 2013. The time- and age-dependent effects of the juvenile hormone analog pesticide, pyriproxyfen on Daphnia magna reproduction. Chemosphere 92, 1260-1266. Kato, Y., Kobayashi, K., Oda, S., Tatarazako, N., Watanabe, H., Iguchi, T., 2010. Sequence divergence and expression of a transformer gene in the branchiopod crustacean, Daphnia magna. Genomics 95, 160-165.

Kato, Y., Kobayashi, K., Watanabe, H., Iguchi, T., 2011. Environmental sex determination in the branchiopod crustacean Daphnia magna: deep conservation of a Doublesex gene in the sex-determining pathway. PLoS genetics 7, e1001345.

Kim, J., Kim, Y., Lee, S., Kwak, K., Chung, W.J., Choi, K., 2011. Determination of mRNA expression of DMRT93B, vitellogenin, and cuticle 12 in Daphnia magna and their biomarker potential for endocrine disruption. Ecotoxicology 20, 1741-1748.

Kopp, A., 2012. Dmrt genes in the development and evolution of sexual dimorphism. Trends Genet 28, 175-184.

Lampert, W., Lampert, K.P., Larsson, P., 2012. Induction of male production in clones of Daphnia pulex by the juvenoid hormone methyl farnesoate under short photoperiod. Comparative biochemistry and physiology. Toxicology & pharmacology : CBP 156, 130-133.

LeBlanc, G.A., Medlock, E.K., 2015. Males on demand: the environmental-neuro-endocrine control of male sex determination in daphnids. FEBS J 282, 4080-4093.

LeBlanc, G.A., Wang, Y.H., Holmes, C.N., Kwon, G., Medlock, E.K., 2013. A transgenerational endocrine signaling pathway in Crustacea. PloS one 8, e61715.

Matsumoto, T., Ikuno, E., Itoi, S., Sugita, H., 2008. Chemical sensitivity of the male daphnid, Daphnia magna, induced by exposure to juvenile hormone and its analogs. Chemosphere 72, 451-456.

Miyakawa, H., Toyota, K., Hirakawa, I., Ogino, Y., Miyagawa, S., Oda, S., Tatarazako, N., Miura, T., Colbourne, J.K., Iguchi, T., 2013. A mutation in the receptor Methoprene-tolerant alters juvenile hormone response in insects and crustaceans. Nature communications 4, 1856.

Oda, S., Tatarazako, N., Dorgerloh, M., Johnson, R.D., Ole Kusk, K., Leverett, D., Marchini, S., Nakari, T., Williams, T., Iguchi, T., 2007. Strain difference in sensitivity to 3,4-dichloroaniline and insect growth regulator, fenoxycarb, in Daphnia magna. Ecotoxicology and environmental safety 67, 399-405.

Oda, S., Tatarazako, N., Watanabe, H., Morita, M., Iguchi, T., 2005. Production of male neonates in Daphnia magna (Cladocera, Crustacea) exposed to juvenile hormones and their analogs. Chemosphere 61, 1168-1174.

Olmstead, A.W., LeBlanc, G.A., 2001a. Temporal and quantitative changes in sexual reproductive cycling of the cladoceran Daphnia magna by a juvenile hormone analog. The Journal of experimental zoology 290, 148-155.

Olmstead, A.W., LeBlanc, G.A., 2003. Insecticidal juvenile hormone analogs stimulate the production of male offspring in the crustacean Daphnia magna. Environmental health perspectives 111, 919-924.

Olmstead, A.W., LeBlanc, G.L., 2001b. Low exposure concentration effects of methoprene on endocrine-regulated processes in the crustacean Daphnia magna. Toxicological sciences : an official journal of the Society of Toxicology 62, 268-273.

Tatarazako, N., Oda, S., Watanabe, H., Morita, M., Iguchi, T., 2003. Juvenile hormone agonists affect the occurrence of male Daphnia. Chemosphere 53, 827-833.

Wang, H.Y., Olmstead, A.W., Li, H., Leblanc, G.A., 2005. The screening of chemicals for juvenoid-related endocrine activity using the water flea Daphnia magna. Aquatic toxicology 74, 193-204.

Zhang, Z., Klein, J., Nei, M., 2014. Evolution of the sex-lethal gene in insects and origin of the sex-determination system in Drosophila. Journal of molecular evolution 78, 50-65.