Graphical RepresentationClick to download graphical representation template
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
Dries Knapen (email point of contact)
- Dries Knapen
- Lucia Vergauwen
|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 Do not cite||Under Development||1.35||Included in OECD Work Plan|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 August 28, 2020 05:47
|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||August 26, 2020 11:53|
|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||September 02, 2020 16:32|
|TH synthesis, Decreased leads to T4 in serum, Decreased||September 03, 2020 09:39|
|T4 in serum, Decreased leads to Decreased, Triiodothyronine (T3) in serum||August 28, 2020 03:51|
|Decreased, Triiodothyronine (T3) in serum leads to Reduced, Anterior swim bladder inflation||August 28, 2020 04:13|
|Reduced, Anterior swim bladder inflation leads to Reduced, Swimming performance||August 28, 2020 05:04|
|Reduced, Swimming performance leads to Reduced, Young of year survival||August 28, 2020 05:11|
|Reduced, Young of year survival leads to Decrease, Population trajectory||August 25, 2020 06:21|
|Thyroperoxidase, Inhibition leads to T4 in serum, Decreased||August 26, 2020 12:38|
|T4 in serum, Decreased leads to Reduced, Anterior swim bladder inflation||August 28, 2020 06:11|
|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) and triiodothyronine (T3) in serum. 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 embryonic development. In fish, many different adverse effects during early development resulting from disruption of the TH endocrine system have been reported (e.g., effects on body and eye size, head-to-trunk angle, heartbeat, otolith formation, pigmentation index, swim bladder inflation, hatching time, somite formation, escape response and photoreceptor development). As in amphibians, the transition in fish between the different developmental phases, including maturation and inflation of the swim bladder, have been shown to be
mediated by THs. Chemicals interfering with the synthesis of T4 have the potential to inhibit anterior chamber inflation which may result in reduced auditory capacity and reduced swimming capacity of the fish, a relevant adverse outcome that can affect feeding behaviour and predator avoidance, resulting in lower survival probability and ultimately population trajectory decline (Czesny et al., 2005; Woolley and Qin, 2010).
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
Life stage applicability: While evidence supports a link between reduced serum T4 levels and reduced anterior swim bladder inflation in late larvae, experimental evidence suggests that inhibition of T4 synthesis does not result in reduced posterior swim bladder inflation in early larvae. The absence of effects on posterior chamber inflation is possibly due to maternal transfer of T4 into the eggs. These maternally derived THs are depleted at 21 dpf and cannot offset TPO inhibition, resulting in impaired anterior chamber inflation. This has been previously suggested by Reider and Connaughton (2014) with specific reference to eye development.
Maternal thyroid hormone levels in embryos have been demonstrated in zebrafish, fathead minnow, brown trout, striped bass, tilapia, rabbitfish, conger eel, sea bream and different species of salmon (Ruuskanen and Hsu, 2018; Walpita et al., 2007; Chang et al., 2012; Hsu et al., 2014; Power et al., 2001; Brown et al., 1987, 1988). Campinho et al. (2014) confirmed that maternal thyroid hormones are essential for normal brain development in zebrafish by knocking down MCT8, responsable for transporting thyroid hormones into the cells. Alt et al. (2006) found a first differentiated thyroid follicle in zebrafish at 55 hours post fertilization. Elsalini et al. (2003) used immunohistochemistry to show the development of the first thyroid follicles producing thyroid hormone at 72 hours post fertilization in zebrafish. During further larval development, the number of follicles increases. Therefore early developmental processes (before thyroid activation) that are dependent on T4, such as posterior swim bladder inflation, might not be affected by chemicals reducing T4 synthesis. Nelson et al. (2016) and Stinckens et al. (2016) indeed found that MBT (a thyroperoxidase inhibitor) decreased T4 levels in both zebrafish (5 days post fertilization) and fathead minnow (6 days post fertilization), which is after activation of the thyroid gland for both species, while it did not affect posterior inflation. Godfrey et al. (2017) also reported normal posterior chamber inflation after exposure of zebrafish embryos to methimazole, a TPO inhibitor. In the latter study, only perfluorooctanoic acid (a deiodinase inhibitor in pig, Stinckens et al., 2018) affects posterior chamber inflation.
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)
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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., 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.
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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.
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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.
Elsalini, O.A., Rohr, K.B., 2003. Phenylthiourea disrupts thyroid function in developing zebrafish. Dev. Genes Evol. 212, 593–598, http://dx.doi.org/10. 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.
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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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.
Knapen, D., Angrish, M.M., Fortin, M.C., Katsiadaki, I., Leonard, M., Margiotta-Casaluci, L., Munn, S., O'Brien, J.M., Pollesch, N., Smith, L.C., Zhang, X.W., Villeneuve, D.L., 2018. Adverse outcome pathway networks I: Development and applications. Environmental Toxicology and Chemistry 37, 1723-1733.
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Nelson, K., Schroeder, A., Ankley, G., Blackwell, B., Blanksma, C., Degitz, S., Flynn, K., Jensen, K., Johnson, R., Kahl, M., Knapen, D., Kosian, P., Milsk, R., Randolph, E., Saari, T., Stinckens, E., Vergauwen, L., Villeneuve, D., 2016. Impaired anterior swim bladder inflation following exposure to the thyroid peroxidase inhibitor 2-mercaptobenzothiazole part I: Fathead minnow. Aquatic Toxicology 173, 192-203.
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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., 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.
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