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Relationship: 1039


The title of the KER should clearly define the two KEs being considered and the sequential relationship between them (i.e., which is upstream and which is downstream). Consequently all KER titles take the form “upstream KE leads to downstream KE”.  More help

T4 in serum, Decreased leads to Reduced, Anterior swim bladder inflation

Upstream event
Upstream event in the Key Event Relationship. On the KER page, clicking on the Event name under Upstream Relationship will bring the user to that individual KE page. More help
Downstream event
Downstream event in the Key Event Relationship. On the KER page, clicking on the Event name under Upstream Relationship will bring the user to that individual KE page. More help

Key Event Relationship Overview

The utility of AOPs for regulatory application is defined, to a large extent, by the confidence and precision with which they facilitate extrapolation of data measured at low levels of biological organisation to predicted outcomes at higher levels of organisation and the extent to which they can link biological effect measurements to their specific causes. Within the AOP framework, the predictive relationships that facilitate extrapolation are represented by the KERs. Consequently, the overall WoE for an AOP is a reflection in part, of the level of confidence in the underlying series of KERs it encompasses. Therefore, describing the KERs in an AOP involves assembling and organising the types of information and evidence that defines the scientific basis for inferring the probable change in, or state of, a downstream KE from the known or measured state of an upstream KE. More help

AOPs Referencing Relationship

This table is automatically generated upon addition of a KER to an AOP. All of the AOPs that are linked to this KER will automatically be listed in this subsection. Clicking on the name of the AOP in the table will bring you to the individual page for that AOP. More help
AOP Name Adjacency Weight of Evidence Quantitative Understanding Point of Contact Author Status OECD Status
Thyroperoxidase inhibition leading to increased mortality via reduced anterior swim bladder inflation non-adjacent Moderate Moderate Evgeniia Kazymova (send email) Open for adoption EAGMST Under Review

Taxonomic Applicability

Select one or more structured terms that help to define the biological applicability domain of the KER. In general, this will be dictated by the more restrictive of the two KEs being linked together by the KER. Authors can indicate the relevant taxa for this KER in this subsection. The process is similar to what is described for KEs (see pages 30-31 and 37-38 of User Handbook) More help
Term Scientific Term Evidence Link
zebrafish Danio rerio NCBI
fathead minnow Pimephales promelas NCBI

Sex Applicability

Authors can indicate the relevant sex for this KER in this subsection. The process is similar to what is described for KEs (see pages 31-32 of the User Handbook). More help
Sex Evidence
Unspecific High

Life Stage Applicability

Authors can indicate the relevant life stage for this KER in this subsection. The process is similar to what is described for KEs (see pages 31-32 of User Handbook). More help

Key Event Relationship Description

Provide a brief, descriptive summation of the KER. While the title itself is fairly descriptive, this section can provide details that aren’t inherent in the description of the KEs themselves (see page 39 of the User Handbook). This description section can be viewed as providing the increased specificity in the nature of upstream perturbation (KEupstream) that leads to a particular downstream perturbation (KEdownstream), while allowing the KE descriptions to remain generalised so they can be linked to different AOPs. The description is also intended to provide a concise overview for readers who may want a brief summation, without needing to read through the detailed support for the relationship (covered below). Careful attention should be taken to avoid reference to other KEs that are not part of this KER, other KERs or other AOPs. This will ensure that the KER is modular and can be used by other AOPs. More help

Reduced T4 levels in serum prohibit local production of active T3 hormone by deiodinases expressed in the target tissues. There is evidence suggesting that anterior swim bladder inflation relies on increased thyroid hormone levels at this specific developmental time point.

Evidence Supporting this KER

Assembly and description of the scientific evidence supporting KERs in an AOP is an important step in the AOP development process that sets the stage for overall assessment of the AOP (see pages 49-56 of the User Handbook). To do this, biological plausibility, empirical support, and the current quantitative understanding of the KER are evaluated with regard to the predictive relationships/associations between defined pairs of KEs as a basis for considering WoE (page 55 of User Handbook). In addition, uncertainties and inconsistencies are considered. More help
Biological Plausibility
Define, in free text, the biological rationale for a connection between KEupstream and KEdownstream. What are the structural or functional relationships between the KEs? For example, there is a functional relationship between an enzyme’s activity and the product of a reaction it catalyses. Supporting references should be included. However, it is recognised that there may be cases where the biological relationship between two KEs is very well established, to the extent that it is widely accepted and consistently supported by so much literature that it is unnecessary and impractical to cite the relevant primary literature. Citation of review articles or other secondary sources, like text books, may be reasonable in such cases. The primary intent is to provide scientifically credible support for the structural and/or functional relationship between the pair of KEs if one is known. The description of biological plausibility can also incorporate additional mechanistic details that help inform the relationship between KEs, this is useful when it is not practical/pragmatic to represent these details as separate KEs due to the difficulty or relative infrequency with which it is likely to be measured (see page 40 of the User Handbook for further information).   More help

Thyroid hormones are known to be involved in development, especially in metamorphosis in amphibians and in embryonic-to-larval transition (Liu and Chan, 2002) and larval-to-juvenile transition (Brown et al., 1997) in fish. The formation of the anterior chamber coincides with the second transition phase (Winata et al., 2009) and with a peak in T4 synthesis (Chang et al., 2012) suggesting that anterior inflation is under thyroid hormone regulation. Since most of the more biologically active T3 originates from the conversion of T4, decreased circulatory T4 levels are plausibly linked to reduced anterior chamber inflation.

Uncertainties and Inconsistencies
In addition to outlining the evidence supporting a particular linkage, it is also important to identify inconsistencies or uncertainties in the relationship. Additionally, while there are expected patterns of concordance that support a causal linkage between the KEs in the pair, it is also helpful to identify experimental details that may explain apparent deviations from the expected patterns of concordance. Identification of uncertainties and inconsistencies contribute to evaluation of the overall WoE supporting the AOPs that contain a given KER and to the identification of research gaps that warrant investigation (seep pages 41-42 of the User Handbook).Given that AOPs are intended to support regulatory applications, AOP developers should focus on those inconsistencies or gaps that would have a direct bearing or impact on the confidence in the KER and its use as a basis for inference or extrapolation in a regulatory setting. Uncertainties that may be of academic interest but would have little impact on regulatory application don’t need to be described. In general, this section details evidence that may raise questions regarding the overall validity and predictive utility of the KER (including consideration of both biological plausibility and empirical support). It also contributes along with several other elements to the overall evaluation of the WoE for the KER (see Section 4 of the User Handbook).  More help

The mechanism through which reduced T4 hormone concentrations in serum result in anterior chamber inflation impairment is not yet understood. The anterior chamber is formed by evagination from the cranial end of the posterior chamber (Robertson et al., 2007, Winata et al., 2009). Several hypotheses could explain effects on anterior chamber inflation due to reduced T4 levels:

  • Evagination from the posterior chamber could be impaired. Villeneuve et al. (unpublished results) showed that although the anterior bud was present after exposure to a deiodinase 2 inhibitor, the anterior chamber did not inflate.
  • The formation of the tissue layers of the anterior swim bladder could be affected, although Villeneuve et al. (unpublished results) observed intact tissue layers of the anterior swim bladder after exposure to a deiodinase 2 inhibitor.
  • The anterior chamber is inflated with gas from the posterior chamber through the communicating duct. Impaired gas exchange between the two chambers could be at the basis of impaired anterior inflation. Both Nelson et al. (2016) and Stinckens et al. (2016) found that posterior chambers were larger when anterior chambers were smaller or not inflated at all. The sum of the areas of the posterior and anterior chambers remained constant independent of inflation of the anterior chamber (Stinkens et al., 2016). These results suggest retention of the gas in the posterior chamber.
  • Since gas exchange relies on a functional communicating duct between the posterior and anterior chamber, and the communicating duct is known to progressively narrow and eventually close during development, a dysfunctional communicating duct or a closure prior to anterior inflation could inhibit inflation. However, Villeneuve et al. (unpublished results) showed that the communicating duct was anatomically intact and open after exposure to iopanoic acid (a deiodinase 2 inhibitor), still leading to impaired anterior inflation.
  • Lactic acid production which is essential for producing gas to fill the swim bladder could be affected, although the observation that the total amount of gas in both chambers is not affected when anterior inflation is impaired seems to contradict this (Stinckens et al., 2016).
  • Possibly there is an effect on the production of surfactant, which is crucial to maintain the surface tension necessary for swim bladder inflation.
Response-response Relationship
This subsection should be used to define sources of data that define the response-response relationships between the KEs. In particular, information regarding the general form of the relationship (e.g., linear, exponential, sigmoidal, threshold, etc.) should be captured if possible. If there are specific mathematical functions or computational models relevant to the KER in question that have been defined, those should also be cited and/or described where possible, along with information concerning the approximate range of certainty with which the state of the KEdownstream can be predicted based on the measured state of the KEupstream (i.e., can it be predicted within a factor of two, or within three orders of magnitude?). For example, a regression equation may reasonably describe the response-response relationship between the two KERs, but that relationship may have only been validated/tested in a single species under steady state exposure conditions. Those types of details would be useful to capture.  More help
This sub-section should be used to provide information regarding the approximate time-scale of the changes in KEdownstream relative to changes in KEupstream (i.e., do effects on KEdownstream lag those on KEupstream by seconds, minutes, hours, or days?). This can be useful information both in terms of modelling the KER, as well as for analyzing the critical or dominant paths through an AOP network (e.g., identification of an AO that could kill an organism in a matter of hours will generally be of higher priority than other potential AOs that take weeks or months to develop). Identification of time-scale can also aid the assessment of temporal concordance. For example, for a KER that operates on a time-scale of days, measurement of both KEs after just hours of exposure in a short-term experiment could lead to incorrect conclusions regarding dose-response or temporal concordance if the time-scale of the upstream to downstream transition was not considered. More help
Known modulating factors
This sub-section presents information regarding modulating factors/variables known to alter the shape of the response-response function that describes the quantitative relationship between the two KEs (for example, an iodine deficient diet causes a significant increase in the slope of the relationship; a particular genotype doubles the sensitivity of KEdownstream to changes in KEupstream). Information on these known modulating factors should be listed in this subsection, along with relevant information regarding the manner in which the modulating factor can be expected to alter the relationship (if known). Note, this section should focus on those modulating factors for which solid evidence supported by relevant data and literature is available. It should NOT list all possible/plausible modulating factors. In this regard, it is useful to bear in mind that many risk assessments conducted through conventional apical guideline testing-based approaches generally consider few if any modulating factors. More help
Known Feedforward/Feedback loops influencing this KER
This subsection should define whether there are known positive or negative feedback mechanisms involved and what is understood about their time-course and homeostatic limits? In some cases where feedback processes are measurable and causally linked to the outcome, they should be represented as KEs. However, in most cases these features are expected to predominantly influence the shape of the response-response, time-course, behaviours between selected KEs. For example, if a feedback loop acts as compensatory mechanism that aims to restore homeostasis following initial perturbation of a KE, the feedback loop will directly shape the response-response relationship between the KERs. Given interest in formally identifying these positive or negative feedback, it is recommended that a graphical annotation (page 44) indicating a positive or negative feedback loop is involved in a particular upstream to downstream KE transition (KER) be added to the graphical representation, and that details be provided in this subsection of the KER description (see pages 44-45 of the User Handbook).  More help

Reduced anterior chamber inflation upon disruption of the thyroid hormone system is consistently accompanied by reduced whole body T4 levels when fish are exposed to thyroid hormone synthesis inhibitors. however, when fish are exposed to deiodinase inhibitors, in the absence of feedback processes, stable T4 levels would be expected. Stable T4 levels were indeed observed in 14, 21 and 32 day old zebrafish exposed to iopanoic acid, a deiodinase inhibitor. While Cavallin et al. (2017) found a consistent relationship between reduced whole body T3 levels, and reduced anterior chamber inflation after exposure of fathead minnows to iopanoic acid, they observed increased T4 levels. Possibly, the inhibition of the conversion of T4 to T3 resulted in a compensatory mechanism that increased T4 levels. This was accompanied by increases of deiodinase 2 and 3 mRNA levels, which also indicate a compensatory response to deiodinase inhibition.

Domain of Applicability

As for the KEs, there is also a free-text section of the KER description that the developer can use to explain his/her rationale for the structured terms selected with regard to taxonomic, life stage, or sex applicability, or provide a more generalizable or nuanced description of the applicability domain than may be feasible using standardized terms. More help

The evidence for a relationship between circulating T4 levels and inflation of the anterior chamber of the swim bladder currently comes from work on zebrafish and fathead minnow.

This KER is probably not sex-dependent since both females and males rely on synthesis of THs for regulation of vital processes. Additionally, zebrafish are undifferentiated gonochorists, and gonad differentiation starts only around 23-25 dpf (Uchida et al., 2002), after the time point of anterior chamber inflation (around 21 dpf).


List of the literature that was cited for this KER description using the appropriate format. Ideally, the list of references should conform, to the extent possible, with the OECD Style Guide (OECD, 2015). More help
  • Alt, B., Reibe, S., Feitosa, N.M., Elsalini, O.A., Wendl, T., Rohr, K.B., 2006. Analysis of origin and growth of the thyroid gland in zebrafish. Dev. Dyn. 235, 1872–1883,
  • Brown, C.L., Doroshov, S.I., Nunez, J.M., Hadley, C., Vaneenennaam, J., Nishioka, R.S. and Bern, H.A. 1988. Maternal triiodothyronine injections cause increases in swimbladder inflation and survival rates in larval striped bass, Morone saxatilis. J. Exp. Zool. 248: 168–176.
  • Brown, C.L., Sullivan, C.V., Bern, H.A. and Dickhoff, W.W. 1987. Occurrence of thyroid hormones in early developmental stages of teleost fish. Trans. Am. Fish. Soc. Symp. 2: 144–150.
  • Brown, D.D., 1997. The role of thyroid hormone in zebrafish and axolotl development. Proc. Natl. Acad. Sci. U. S. A. 94, 13011–13016, 10.1073/pnas.94.24.13011.
  • Campinho, M.A., Saraiva, J., Florindo, C., Power, D.M., 2014. Maternal Thyroid Hormones Are Essential for Neural Development in Zebrafish. Molecular Endocrinology 28, 1136-1149.
  • Cavallin, J.E., Ankley, G.T., Blackwell, B.R., Blanksma, C.A., Fay, K.A., Jensen, K.M., Kahl, M.D., Knapen, D., Kosian, P.A., Poole, S.T., Randolph, E.C., Schroeder, A.L., Vergauwen, L., Villeneuve, D.L., 2017. Impaired swim bladder inflation in early life stage fathead minnows exposed to a deiodinase inhibitor, iopanoic acid. Environmental Toxicology and Chemistry 36, 2942-2952.
  • Chang, J., Wang, M., Gui, W., Zhao, Y., Yu, L., Zhu, G., 2012. Changes in thyroid hormone levels during zebrafish development. Zool. Sci. 29, 181–184, http://
  • Chopra, K., Ishibashi, S., Amaya, E., 2019. Zebrafish duox mutations provide a model for human congenital hypothyroidism. Biology Open 8(2):bio.037655, DOI:10.1242/bio.037655.
  • Elsalini, O.A., Rohr, K.B., 2003. Phenylthiourea disrupts thyroid function in developing zebrafish. Dev. Genes Evol. 212, 593–598, 1007/s00427-002-0279-3.
  • Godfrey, A., Hooser, B., Abdelmoneim, A., Horzmann, K.A., Freemanc, J.L., Sepulveda, M.S., 2017. Thyroid disrupting effects of halogenated and next generation chemicals on the swim bladder development of zebrafish. Aquatic Toxicology 193, 228-235.
  • Hsu, C.W., Tsai, S.C., Shen, S.C., Wu, S.M., 2014. Profiles of thyrotropin, thyroid hormones, follicular cells and type I deiodinase gene expression during ontogenetic development of tilapia larvae and juveniles. Fish Physiology and Biochemistry 40, 1587-1599.
  • Liu, Y.W., Chan, W.K., 2002. Thyroid hormones are important for embryonic to larval transitory phase in zebrafish. Differentiation 70, 36–45, http://dx.doi. org/10.1046/j.1432-0436.2002.700104.x.
  • Nelson KR, Schroeder AL, Ankley GT, Blackwell BR, Blanksma C, Degitz SJ, Flynn KM, Jensen KM, Johnson RD, Kahl MD, Knapen D, Kosian PA, Milsk RY, Randolph EC, Saari T, Stinckens E, Vergauwen L, Villeneuve DL. 2016. Impaired anterior swim bladder inflation following exposure to the thyroid peroxidase inhibitor 2-mercaptobenzothiazole – Part I: fathead minnow. Aquatic Toxicology 173: 192-203.
  • Power DM, Llewellyn L, Faustino M, Nowell MA, Björnsson BT, Einarsdottir IE, Canario AV, Sweeney GE. Thyroid hormones in growth and development of fish. Comp Biochem Physiol C Toxicol Pharmacol. 2001 Dec; 130(4):447-59.
  • Reider, M., Connaughton, V.P., 2014. Effects of Low-Dose Embryonic Thyroid Disruption and Rearing Temperature on the Development of the Eye and Retina in Zebrafish. Birth Defects Res. Part B Dev. Reprod. Toxicol. 101, 347–354,
  • Roberston, G.N., McGee, C.A.S., Dumbarton, T.C., Croll, R.P., Smith, F.M., 2007. Development of the swim bladder and its innervation in the zebrafish, Danio rerio. J. Morphol. 268, 967–985,
  • Ruuskanen, S., Hsu, B.Y., 2018. Maternal Thyroid Hormones: An Unexplored Mechanism Underlying Maternal Effects in an Ecological Framework. Physiological and Biochemical Zoology 91, 904-916.
  • Stinckens E, Vergauwen L, Schroeder AL, Maho W, Blackwell BR, Witters H, Blust R, Ankley GT, Covaci A, Villeneuve DL, Knapen D. 2016. Impaired anterior swim bladder inflation following exposure to the thyroid peroxidase inhibitor 2-mercaptobenzothiazole – Part II: zebrafish. Aquatic Toxicology 173:204-217.
  • Stinckens, E., Vergauwen, L., Ankley, G.T., Blust, R., Darras, V.M., Villeneuve, D.L., Witters, H., Volz, D.C., Knapen, D., 2018. An AOP-based alternative testing strategy to predict the impact of thyroid hormone disruption on swim bladder inflation in zebrafish. Aquatic Toxicology 200, 1-12.
  • Stinckens, E., Vergauwen, L., Blackwell, B.R., Anldey, G.T., Villeneuve, D.L., Knapen, D., 2020. Effect of Thyroperoxidase and Deiodinase Inhibition on Anterior Swim Bladder Inflation in the Zebrafish. Environmental Science & Technology 54, 6213-6223.
  • Uchida, D., Yamashita, M., Kitano, T., Iguchi, T., 2002. Oocyte apoptosis during the transition from ovary-like tissue to testes during sex differentiation of juvenile zebrafish. Journal of Experimental Biology 205, 711-718.
  • Walpita, C.N., Van der Geyten, S., Rurangwa, E., Darras, V.M., 2007. The effect of 3,5,3′-triiodothyronine supplementation on zebrafish (Danio rerio) embryonic development and expression of iodothyronine deiodinases and thyroid hormone receptors. Gen. Comp. Endocrinol. 152, 206–214, 10.1016/j.ygcen.2007.02.020.
  • Winata, C.L., Korzh, S., Kondrychyn, I., Zheng, W., Korzh, V., Gong, Z., 2009. Development of zebrafish swimbladder: the requirement of Hedgehog signaling in specification and organization of the three tissue layers. Dev. Biol. 331, 222–236,