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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
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 Do not cite||Under Development||1.35||Included in OECD Work Plan|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 June 09, 2020 12:19
|Inhibition, Deiodinase 2||August 20, 2019 09:52|
|Decrease, Population trajectory||September 26, 2017 11:33|
|Decreased, Triiodothyronine (T3) in serum||September 26, 2017 11:03|
|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 leads to Reduced, Anterior swim bladder inflation||February 25, 2020 11:48|
|Inhibition, Deiodinase 2 leads to Decreased, Triiodothyronine (T3) in serum||February 25, 2020 11:02|
|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|
The AOP describes the effects of inhibition of deiodinase 2 (DIO2) on anterior swim bladder inflation leading to reduced young of year survival and population trajectory decline. The inhibition of DIO2 is the molecular-initiating event (MIE), which results in decreased circulating concentrations of triiodothyronine (T3) in serum. Disruption of the thyroid hormone (TH) system is increasingly being recognized as an important MoA 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 conversion of T4 to T3 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||1002||Inhibition, Deiodinase 2||Inhibition, Deiodinase 2|
|2||KE||1003||Decreased, Triiodothyronine (T3) in serum||Decreased, Triiodothyronine (T3) in serum|
|3||KE||1007||Reduced, Anterior swim bladder inflation||Reduced, Anterior swim bladder inflation|
|5||KE||1005||Reduced, Swimming performance||Reduced, Swimming performance|
|6||KE||1006||Reduced, Young of year survival||Reduced, Young of year survival|
|7||AO||360||Decrease, Population trajectory||Decrease, Population trajectory|
Relationships Between Two Key Events
(Including MIEs and AOs)
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.
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)
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.
Jomaa, B., Hermsen, S.A.B., Kessels, M.Y., van den Berg, J.H.J., Peijnenburg, A.A.C.M., Aarts, J.M.M.J.G., Piersma, A.H., Rietjens, I.M.C.M., 2014. Developmental Toxicity of Thyroid-Active Compounds in a Zebrafish Embryotoxicity Test. Altex-Alternatives to Animal Experimentation 31, 303-317.
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.
Liu, Y.W., Chan, W.K., 2002. Thyroid hormones are important for embryonic to larval transitory phase in zebrafish. Differentiation 70, 36-45.
Maack, G., Segner, H., 2003. Morphological development of the gonads in zebrafish. Journal of Fish Biology 62, 895-906.
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.
Power, D.M., Llewellyn, L., Faustino, M., Nowell, M.A., Bjornsson, B.T., Einarsdottir, I.E., Canario, A.V., Sweeney, G.E., 2001. Thyroid hormones in growth and development of fish. Comp Biochem Physiol C Toxicol Pharmacol 130, 447-459.
Robertson, G.N., McGee, C.A.S., Dumbarton, T.C., Croll, R.P., Smith, F.M., 2007. Development of the swimbladder and its innervation in the zebrafish, Danio rerio. Journal of Morphology 268, 967-985.
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.
Villeneuve, D., Angrish, M., Fortin, M., Katsiadaki, I., Leonard, M., Margiotta-Casaluci, L., Munn, S., O'Brien, J., Pollesch, N., Smith, L., Zhang, X., Knapen, D., 2018. Adverse Outcome Pathway Networks II: Network Analytics. Environ Toxicol Chem doi: 10.1002/etc.4124.
Villeneuve, D., Volz, D.C., Embry, M.R., Ankley, G.T., Belanger, S.E., Leonard, M., Schirmer, K., Tanguay, R., Truong, L., Wehmas, L., 2014. Investigating alternatives to the fish early-life stage test: a strategy for discovering and annotating adverse outcome pathways for early fish development. Environmental Toxicology and Chemistry 33, 158-169.
Walpita, C.N., Crawford, A.D., Janssens, E.D., Van der Geyten, S., Darras, V.M., 2009. Type 2 iodothyronine deiodinase is essential for thyroid hormone-dependent embryonic development and pigmentation in zebrafish. Endocrinology 150, 530-539.
Woolley, L.D., Qin, J.G., 2010. Swimbladder inflation and its implication to the culture of marine finfish larvae. Reviews in Aquaculture 2, 181-190.