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Dries Knapen , [dries.knapen (at)uantwerpen.be]
Lucia Vergauwen , [lucia.vergauwen(at)uantwerpen.be]
Evelyn Stinckens , [evelyn.stinckens(at)uantwerpen.be]
Dan Villeneuve , [villeneuve.dan*(at)epa.gov]
 Zebrafishlab, Veterinary Physiology and Biochemistry, Department of Veterinary Sciences, University of Antwerp, Universiteitsplein 1, 2610 Wilrijk, Belgium
 United States Environmental Protection Agency, Mid-Continent Ecology Division, 6201 Congdon Blvd, Duluth, MN, USA.
Point of Contact
Evgeniia Kazymova (email point of contact)
- Dries Knapen
- Lucia Vergauwen
- Evgeniia Kazymova
|Author status||OECD status||OECD project||SAAOP status|
|Under development: Not open for comment. Do not cite||Under Development||1.35||Included in OECD Work Plan|
This AOP was last modified on May 10, 2020 21:23
|Thyroperoxidase, Inhibition||August 07, 2018 15:09|
|Decrease, Population trajectory||September 26, 2017 11:33|
|Thyroid hormone synthesis, Decreased||August 11, 2018 13:21|
|Thyroxine (T4) in serum, Decreased||April 04, 2019 09:05|
|Reduced, Anterior swim bladder inflation||October 31, 2019 04:54|
|Reduced, Swimming performance||April 24, 2020 15:05|
|Reduced, Young of year survival||November 29, 2016 19:36|
|Decreased, Triiodothyronine (T3) in serum||September 26, 2017 11:03|
|Thyroperoxidase, Inhibition leads to TH synthesis, Decreased||August 11, 2018 19:01|
|TH synthesis, Decreased leads to T4 in serum, Decreased||April 04, 2019 10:47|
|T4 in serum, Decreased leads to Decreased, Triiodothyronine (T3) in serum||February 25, 2020 09:58|
|Decreased, Triiodothyronine (T3) in serum leads to Reduced, Anterior swim bladder inflation||February 25, 2020 11:48|
|Reduced, Anterior swim bladder inflation leads to Reduced, Swimming performance||February 25, 2020 12:15|
|Reduced, Swimming performance leads to Reduced, Young of year survival||September 17, 2019 12:00|
|Reduced, Young of year survival leads to Decrease, Population trajectory||December 03, 2016 16:38|
|Thyroperoxidase, Inhibition leads to T4 in serum, Decreased||March 01, 2019 08:19|
|T4 in serum, Decreased leads to Reduced, Anterior swim bladder inflation||August 13, 2019 09:47|
|Methimazole||November 29, 2016 18:42|
|Mercaptobenzothiazole||November 29, 2016 18:42|
|Propylthiouracil||November 29, 2016 18:42|
The AOP describes the effects of inhibition of thyroperoxidase (TPO) on anterior swim bladder inflation leading to reduced young of year survival and population trajectory decline. The inhibition of TPO is the molecular-initiating event (MIE), which results in decreased circulating concentrations of thyroxine (T4) in serum. Chemicals interfering with the synthesis of T4 cause reduced availability of T4 for activation to the more biologically active triiodothyronine (T3). As in amphibians, the transition between the different developmental phases in fish, including maturation and inflation of the swim bladder, has been shown to be mediated by THs (Brown et al., 1988; Liu and Chan, 2002). Impaired swim bladder inflation results in reduced swimming performance (Stinckens et al. submitted; Hagenaars et al., 2014; Stinckens et al., 2016; Stinckens et al., 2018), an adverse outcome that can affect feeding behavior and predator avoidance, ultimately leading to lower survival probability and population trajectory decline (Czesny et al., 2005; Woolley and Qin, 2010; Villeneuve et al., 2014).
Disruption of the thyroid hormone (TH) system is increasingly being recognized as an important mode of action that can lead to adverse outcomes, especially during development. The description of AOPs may help to select or develop assays to identify TH disrupting compounds and add such assays to existing test guidelines using fish.
This AOP is part of a larger AOP network describing how decreased synthesis and/or decreased biological activation of THs leads to incomplete or improper inflation of the swim bladder, leading to reduced swimming performance and ultimately to reduced survival. (Knapen et al., 2018; Villeneuve et al., 2018). Specific parts of the AOP network are relevant to different life stages. The swim bladder is an internal gas-filled organ found in many bony fish species and typically consists of two gas-filled chambers. The posterior chamber inflates during early development and contributes to the ability of fish to control their buoyancy, while the anterior chamber inflates during late development and has an additional role as a resonating chamber to produce or receive sound (Robertson et al., 2007). The earliest life stages of teleost fish rely on maternally transferred THs to regulate certain developmental processes until embryonic TH synthesis is active (Power et al., 2001). As a result, early developmental processes that are dependent on THs, such as posterior swim bladder chamber inflation, appear to be less sensitive to inhibition of TH synthesis. On the other hand, when maternally derived THs are depleted during late development (larval stage), endogenous TH synthesis becomes more important and inhibition of TPO interferes with proper inflation of the anterior swim bladder chamber (Stinckens et al. submitted; Nelson et al., 2016; Stinckens et al., 2016; Godfrey et al., 2017). In all life stages however, the conversion of T4 into T3 is essential. Inhibition of deiodinase (DIO) therefore impacts swim bladder inflation in both early and late developmental life stages (Stinckens et al. submitted; Jomaa et al., 2014; Cavallin et al., 2017; Godfrey et al., 2017; Stinckens et al., 2018). In addition to evidence from chemical exposure summarized above, data from knockdowns, knockouts and TH supplementation has been instrumental in supporting the AOP network (Walpita et al., 2009, 2010; Heijlen et al., 2013, 2014; Bagci et al., 2015; Houbrechts et al., 2016; Chopra et al., 2019).
Although there is strong evidence for the link between TH and swim bladder inflation, the exact underlying mechanism (e.g., impairment of development and/or inflation process) is not understood. Another uncertainty relates to serum versus tissue TH levels. Since collecting blood from early life stages of fish is not feasible, whole body TH measurements are typically used as a proxy for serum TH levels.
Summary of the AOP
Events: Molecular Initiating Events (MIE)
|Sequence||Type||Event ID||Title||Short name|
|1||MIE||279||Thyroperoxidase, Inhibition||Thyroperoxidase, Inhibition|
|2||KE||277||Thyroid hormone synthesis, Decreased||TH synthesis, Decreased|
|3||KE||281||Thyroxine (T4) in serum, Decreased||T4 in serum, Decreased|
|4||KE||1003||Decreased, Triiodothyronine (T3) in serum||Decreased, Triiodothyronine (T3) in serum|
|5||KE||1007||Reduced, Anterior swim bladder inflation||Reduced, Anterior swim bladder inflation|
|6||KE||1005||Reduced, Swimming performance||Reduced, Swimming performance|
|7||KE||1006||Reduced, Young of year survival||Reduced, Young of year survival|
|8||AO||360||Decrease, Population trajectory||Decrease, Population trajectory|
Relationships Between Two Key Events
(Including MIEs and AOs)
|Thyroperoxidase, Inhibition leads to T4 in serum, Decreased||non-adjacent|
|T4 in serum, Decreased leads to Reduced, Anterior swim bladder inflation||non-adjacent|
Life Stage Applicability
|fathead minnow||Pimephales promelas||NCBI|
Overall Assessment of the AOP
Overall, the weight of evidence for the sequence of key events laid out in the AOP is moderate to high. Nonetheless, the exact underlying mechanism of TH disruption leading to impaired swim bladder inflation is not understood. The current domain of applicability is larval life stages of zebrafish and fathead minnow pending future research in other fish species such as medaka.
Domain of Applicability
The current AOP is only applicable to larval development, which is the period where the anterior swim bladder chamber inflates. The earliest life stages of teleost fish rely on maternally transferred THs to regulate certain developmental processes until embryonic TH synthesis is active (Power et al., 2001). As a result, early developmental processes that are dependent on THs, such as posterior swim bladder chamber inflation, appear to be less sensitive to inhibition of TH synthesis. When maternally derived THs are depleted during late development (larval stage), endogenous TH synthesis becomes more important and inhibition of TPO interferes with proper inflation of the anterior swim bladder chamber (Stinckens et al. submitted; Nelson et al., 2016; Stinckens et al., 2016; Godfrey et al., 2017).
The AOP is currently mainly based on experimental evidence from studies on zebrafish and fathead minnow. A first logical step in expanding the applicability of the AOP network is to assess its relevance to other species that are frequently used in existing fish test guidelines, such as the Japanese rice fish (medaka), three-spined stickleback and rainbow trout.
Sex differences are typically not investigated in tests using early life stages of fish and it is currently unclear whether sex-related differences are important in this AOP. Zebrafish are undifferentiated gonochorists since both sexes initially develop an immature ovary (Maack and Segner, 2003). Immature ovary development progresses until approximately the onset of the third week. Later, in female fish immature ovaries continue to develop further, while male fish undergo transformation of ovaries into testes. Final transformation into testes varies among male individuals, however finishes usually around 6 weeks post fertilization. Since the anterior chamber inflates around 20 days post fertilization, when sex differentiation is still in its early stages, sex differences are expected to play a minor role in the current AOP.
Essentiality of the Key Events
Overall, the confidence in the supporting data for essentiality of KEs within the AOP is high since there is direct evidence from specifically designed experimental studies (knockdown and knockout studies) illustrating that the impact on downstream KEs corresponds to what is predicted by the AOP.
Overall, the weight of evidence for the biological plausibility of the KERs in the AOP is moderate since there is empirical support for an association between the sets of KEs and the KERs are plausible based on analogy to accepted biological relationships, but scientific understanding is not completely established. Especially for some of the upstream KERs biological plausibility is high.
Overall, the empirical support for the KERs in the AOP is moderate since dependent changes in sets of KEs following exposure to a small number of specific stressors has been demonstrated, but there are still some data gaps.
There is some level of quantitative understanding that can form the basis for development of a quantitative AOP. Quantitative relationships between reduced T4 and reduced T3, and between reduced T3 and reduced anterior chamber inflation were established. The latter is particularly critical for linking impaired swim bladder inflation to TH disruption.
Considerations for Potential Applications of the AOP (optional)
A growing number of environmental pollutants are known to adversely affect the thyroid hormone system, and major gaps have been identified in the tools available for the identification, and the hazard and risk assessment of these thyroid hormone disrupting chemicals. Knapen et al. (submitted) provides an example of how the adverse outcome pathway (AOP) framework and associated data generation can address current testing challenges in the context of fish early-life stage tests, and fish tests in general. A suite of assays covering all the essential biological processes involved in the underlying toxicological pathways can be implemented in a tiered screening and testing approach for thyroid hormone disruption, using the levels of assessment of the OECD’s Conceptual Framework for the Testing and Assessment of Endocrine Disrupting Chemicals as a guide.
Bagci, E., Heijlen, M., Vergauwen, L., Hagenaars, A., Houbrechts, A.M., Esguerra, C.V., Blust, R., Darras, V.M., Knapen, D., 2015. Deiodinase knockdown during early zebrafish development affects growth, development, energy metabolism, motility and phototransduction. PLOS One 10, e0123285.
Brown, C.L., Doroshov, S.I., Nunez, J.M., Hadley, C., Vaneenennaam, J., Nishioka, R.S., Bern, H.A., 1988. MATERNAL TRIIODOTHYRONINE INJECTIONS CAUSE INCREASES IN SWIMBLADDER INFLATION AND SURVIVAL RATES IN LARVAL STRIPED BASS, MORONE-SAXATILIS. Journal of Experimental Zoology 248, 168-176.
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.
Chopra, K., Ishibashi, S., Amaya, E., 2019. Zebrafish duox mutations provide a model for human congenital hypothyroidism. Biology Open 8.
Czesny, S.J., Graeb, B.D.S., Dettmers, J.M., 2005. Ecological consequences of swim bladder noninflation for larval yellow perch. Transactions of the American Fisheries Society 134, 1011-1020.
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.
Hagenaars, A., Stinckens, E., Vergauwen, L., Bervoets, L., Knapen, D., 2014. PFOS affects posterior swim bladder chamber inflation and swimming performance of zebrafish larvae. Aquatic Toxicology 157, 225-235.
Heijlen, M., Houbrechts, A., Bagci, E., Van Herck, S., Kersseboom, S., Esguerra, C., Blust, R., Visser, T., Knapen, D., Darras, V., 2014. Knockdown of type 3 iodothyronine deiodinase severely perturbs both embryonic and early larval development in zebrafish. Endocrinology 155, 1547-1559.
Houbrechts, A.M., Delarue, J., Gabriels, I.J., Sourbron, J., Darras, V.M., 2016. Permanent Deiodinase Type 2 Deficiency Strongly Perturbs Zebrafish Development, Growth, and Fertility. Endocrinology 157, 3668-3681.
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Knapen, D., Stinckens, E., Cavallin, J., Ankley, G., Holbech, H., Villeneuve, D., Vergauwen, L., Toward an AOP network-based tiered testing strategy for the assessment of thyroid hormone disruption. Environmental Science & Technology submitted.
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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., Ankley, G.T., Villeneuve, D.L., Knapen, D., The effect of thyroperoxidase and deiodinase inhibition on anterior swim bladder inflation in the zebrafish. Environmental Science & Technology submitted.
Stinckens, E., Vergauwen, L., Schroeder, A., Maho, W., Blackwell, B., Witters, H., Blust, R., Ankley, G., Covaci, A., Villeneuve, D., 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.
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