SNAPSHOT

Event: 1009: Inhibition, Deiodinase 1

Short Name: Inhibition, Deiodinase 1

AOPs Including This Key Event

Stressors

Biological Context

Deiodination by DIO enzymes is known to exist in a wide range of vertebrates and invertebrates. Studies reporting DIO1 inhibition have used human liver (Kuiper et al., 2006), human recombinant DIO1 enzyme (Olker et al., 2019), rat (Rattus norvegicus) liver (Klaren et al., 2005; Freyberger and Ahr, 2006; Kuiper et al., 2006; Pavelka, 2010) and thyroid gland (Ferreira et al., 2002), mouse (Mus musculus) brain (hernandez et al., 2006), hog (Sus scrofa domesticus) liver (Stinckens et al., 2018), sheep (Ovis orientalis aries) fetal hepatic, renal and perirenal adipose tissue (Forhead et al., 2006), tadpole (Xenopus laevis) liver (Kuiper et al., 2006), fathead minnow (Pimephales promelas) whole fish (Noyes et al., 2011), Nile tilapia (Oreochromis niloticus) liver (Walpita et al., 2007), Gilthead Seabream (Sparus aurata) kidney (Klaren et al., 2005), and killifish (Fundulus heteroclitus) liver (Orozco et al., 2003) among others. The latter teleostean DIO1 enzymes as well as amphibian enzymes differ from other vertebrate DIO1 enzymes in their lower sensitivity to propylthiouracil (PTU), a typical DIO1 inhibitor in mammals.

Deiodinase 1 in liver is the main supplier of T3 to circulation in mammals (Marsili et al., 2011), and the same appears to be true for birds. By contrast, DIO1 function in teleostean and amphibian T3 plasma regulation is less clear (Finnson et al. 1999, Kuiper et al. 2006). The presence of DIO1 in the liver of teleosts has been a controversial issue, and both the high level of DIO2 activity and its expression in the liver of teleosts are unique among vertebrates (Orozco and Valverde, 2005). This could explain why DIO2 inhibition seems to be more important than DIO1 inhibition in determining the adverse outcome in zebrafish (Stinckens et al., 2018).

Deiodinase activity is important for all vertebrate life stages. Already during early embryonic development, deiodinase activity is needed to regulate thyroid hormone concentrations and coordinate developmental processes. However, the role of DIO1 and DIO2 seems to be distinct. The fact that DIO1 knockdown during zebrafish development only causes developmental defects when combined with DIO2 knockdown (Walpita et al., 2010), suggests that DIO1 is only important in cases of increased TH need during specific stages of development, as supported by increased expression during such stages (Vergauwen et al., 2018), and in cases of thyroid hormone depletion in fish.

Event: 1003: Decreased, Triiodothyronine (T3) in serum

Short Name: Decreased, Triiodothyronine (T3) in serum

AOPs Including This Key Event

Biological Context

Organ term

The overall evidence supporting taxonomic applicability is strong. With few exceptions vertebrate species have circulating T3 and T4 that are bound to transport proteins in blood. Clear species differences exist in transport proteins (Yamauchi and Isihara, 2009). Specifically, the majority of supporting data for TH decreases in serum come from rat studies, and the predominant iodothyronine binding protein in rat serum is transthyretin (TT4). TT4 demonstrates a reduced binding affinity for T4 when compared with thyroxine binding globulin (TBG), the predominant serum binding protein for T4 in humans. This difference in serum binding protein affinity for THs is thought to modulate serum half-life for T4; the half-life of T4 in rats is 12-24 hr, wherease the half-life in humans is 5-9 days (Capen, 1997). While these species differences impact hormone half-life, possibly regulatory feedback mechanisms, and quantitative dose-response relationships, measurement of serum THs is still regarded as a measurable key event causatively linked to downstream adverse outcomes.

THs are evolutionarily conserved molecules present in all vertebrate species (Hulbert, 2000; Yen, 2001). Moreover, their crucial role in amphibian and larbean metamorphoses is well established (Manzon and Youson, 1997; Yaoita and Brown, 1990). Their existence and importance has been also described in many differrent animal and plant kingdoms (Eales, 1997; Heyland and Moroz, 2005), while their role as environmental messenger via exogenous routes in echinoderms confirms the hypothesis that these molecules are widely distributed among the living organisms (Heyland and Hodin, 2004). However, the role of TH in the different species may differ depending on the expression or function of specific proteins (e.g receptors or enzymes) that are related to TH function, and therefore extrapolation between species should be done with cautious.

Event: 1007: Reduced, Anterior swim bladder inflation

Short Name: Reduced, Anterior swim bladder inflation

AOPs Including This Key Event

Biological Context

Organ term

The evidence for impaired inflation of the anterior chamber of the swim bladder currently comes from work on zebrafish and fathead minnow.

Event: 1005: Reduced, Swimming performance

Short Name: Reduced, Swimming performance

AOPs Including This Key Event

Biological Context

Importance of swimming performance for natural behaviour is generally applicable to fish.

Event: 1006: Reduced, Young of year survival

Short Name: Reduced, Young of year survival

AOPs Including This Key Event

Biological Context

Survival is important for all species.

Event: 360: Decrease, Population trajectory

Short Name: Decrease, Population trajectory

AOPs Including This Key Event

Biological Context

Consideration of population size and changes in population size over time is potentially relevant to all living organisms.

Relationship: 1037: Inhibition, Deiodinase 1 leads to Decreased, Triiodothyronine (T3) in serum

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

Mol et al. (1998) concluded that deiodinases in teleosts were more similar to mammalian deiodinases than had been generally accepted, based on the similarities in susceptibility to inhibition and the agreement of the Km values.

It is generally assumed that deiodinase 1 in liver is the main supplier of T3 to circulation in mammals (Leonard et al., 1986), and the same appears to be true for birds (Freeman et al., 1991), while DIO2 is assumed to regulate intracellular concentrations of T3. In contrast to the general assumptions however, Maia et al. (2005) determined that in a normal physiological situation in humans the contribution of DIO2 to plasma T3 levels is twice that of DIO1. By contrast, DIO1 function in teleostean and amphibian T3 plasma regulation is less clear (Finnson et al. 1999, Kuiper et al. 2006). The presence of DIO1 in the liver of teleosts has been a controversial issue, and both the high level of DIO2 activity and its expression in the liver of teleosts are unique among vertebrates (Orozco and Valverde, 2005). These differences make it difficult to exactly evaluate the importance of DIO1 in regulating serum/tissue T3 levels across vertebrates.

Key Event Relationship Description

The two major thyroid hormones are thyroxine (T4) and the more biologically active triiodothyronine (T3), both iodinated derivatives of tyrosine. Active and inactive THs are tightly regulated by enzymes called iodothyronine deiodinases (DIO). The activation occurs via outer ring deiodination (ORD), i.e. removing iodine from the outer, phenolic ring of T4 to form T3, while inactivation occurs via inner ring deiodination (IRD), i.e. removing iodine from the inner tyrosol ring of T4 or T3.

Three types of iodothyronine deiodinases (DIO1-3) have been described in vertebrates that activate or inactivate THs and are therefore important mediators of TH action. All deiodinases are integral membrane proteins of the thioredoxin superfamily that contain selenocysteine in their catalytic centre. Type I deiodinase is capable of converting T4 into T3, as well as to convert rT3 to the inactive thyroid hormone 3,3’ T2, through outer ring deiodination. rT3, rather than T4, is the preferred substrate for DIO1. furthermore, DIO1 has a very high Km (µM range, compared to nM range for DIO2) (Darras and Van Herck, 2012). Type II deiodinase (DIO2) is only capable of ORD activity with T4 as a preferred substrate (i.e., activation of T4 tot T3). DIO3 can inner ring deiodinate T4 and T3 to the inactive forms of THs, reverse T3, (rT3) and 3,3’-T2 respectively. DIO1 is a plasma membrane protein with its catalytic domain facing the cytosol.

Evidence Supporting this KER

Inhibition of DIO1 activity is widely accepted to directly decrease T3 levels, since the conversion of T4 to T3 is inhibited. The importance of DIO1 inhibition in altering serum T3 levels depends on the relative role of different deiodinases in regulating serum versus tissue T3 levels and in negative feedback within the HPT axis. Both aspects appear to differ to some extent among vertebrate taxa.

Inhibition of DIO1 activity is widely accepted to directly decrease T3 levels, since the conversion of T4 to T3 is inhibited.

• In the study of Cavallin et al. (2017) fathead minnow larvae were exposed to IOP, a model iodothyronine deiodinase inhibitor that is assumed to inhibit all three deiodinase enzymes (DIO1,2,3) and pronounced decreases of whole body T3 concentrations were observed.
• Stinckens et al. (submitted) showed that IOP reduced T3 levels in zebrafish in 21 and 32 day old larvae that had been exposed starting from fertilization.

In the study of Cavallin et al. (2017) fathead minnow larvae were exposed to IOP, a model iodothyronine deiodinase inhibitor that is assumed to inhibit all three deiodinase enzymes (DIO1,2,3). Transcriptional analysis showed that especially DIO2, but also DIO3 mRNA levels (in some treatments), were increased in 10 to 21 day old larvae exposed to IOP as of the age of 6 days. This suggests that IOP effectively inhibited DIO2 and DIO3 in the larvae and that mRNA levels increased as a compensatory response. The authors also observed pronounced decreases of whole body T3 concentrations and increases of whole body T4 concentrations. It is not clear whether inhibition of DIO1 also played a role in the decrease of T3 levels.

Quantitative Understanding of the Linkage

Thyroid hormone levels are regulated via negative feedback, influencing this KER. Additionally, deiodinases regulate the activity of thryoid hormones, not only in serum and target organs, but also in the thryoid gland. Deiodinases themselves are known to be involved in the negative feedback system that results in increased TSH levels when the levels of T4 (and also T3) in serum are low (Schneider et al., 2001), resulting in an even more complicated impact on this KER. Increased TSH levels then stimulate increased T4 release from the thyroid gland, resulting in a compensatory increase of serum T4 levels. In DIO2 knockout mice it seemed that the negative feedback system was blocked resulting in increased levels of T4 and TSH and in normal rather than decreased T3 levels compared to WT. By inhibiting DIO1 using a PTU exposure, Schneider et al. (2001) showed that DIO2 played a role in the increased TSH levels in response to T3 or T4 injection.

References

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.

Mol, K.A., Van der Geyten, S., Burel, C., Kuhn, E.R., Boujard, T., Darras, V.M., 1998. Comparative study of iodothyronine outer ring and inner ring deiodinase activities in five teleostean fishes. Fish Physiology and Biochemistry 18, 253-266.

Finnson, K.W., McLeese, J.M., Eales, J.G., 1999. Deiodination and deconjugation of thyroid hormone conjugates and type I deiodination in liver of rainbow trout, Oncorhynchus mykiss. General and Comparative Endocrinology 115, 387-397.

Kuiper, G., Klootwijk, W., Dubois, G.M., Destree, O., Darras, V.M., Van der Geyten, S., Demeneix, B., Visser, T.J., 2006. Characterization of recombinant Xenopus laevis type I iodothyronine deiodinase: substitution of a proline residue in the catalytic center by serine (Pro132Ser) restores sensitivity to 6-propyl-2-thiouracil. Endocrinology 147, 3519-3529.

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.

Schneider, M.J., Fiering, S.N., Pallud, S.E., Parlow, A.F., St Germain, D.L., Galton, V.A., 2001. Targeted disruption of the type 2 selenodeiodinase gene (D102) results in a phenotype of pituitary resistance to T-4. Molecular Endocrinology 15, 2137-2148.

Relationship: 1035: Decreased, Triiodothyronine (T3) in serum leads to Reduced, Anterior swim bladder inflation

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

This KER is probably not sex-dependent since both females and males rely on 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).

Key Event Relationship Description

Thyroid hormones are known to be involved in development, especially in metamorphosis in amphibians and in embryonic-to-larval transition and larval-to-juvenile transition in fish. Inflation of the anterior swim bladder chamber is part of the larval-to-juvenile transition in fish, together with the development of adult fins and fin rays, ossification of the axial skeleton, formation of an adult pigmentation pattern, scale formation, maturation and remodeling of organs including the lateral line, nervous system, gut and kidneys.

Evidence Supporting this KER

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. Inflation of the anterior swim bladder chamber is part of the larval-to-juvenile transition in fish, together with the development of adult fins and fin rays, ossification of the axial skeleton, formation of an adult pigmentation pattern, scale formation, maturation and remodeling of organs including the lateral line, nervous system, gut and kidneys (Brown, 1997; Liu and Chan, 2002; McMenamin and Parichy, 2013).

• In a study in which embryo-larval fathead minnows (Pimephales promelas) were exposed to the thyroid peroxidase inhibitor 2-mercaptobenzothiazole (MBT), T3 concentrations measured at 14dpf were reduced at the same concentration (1 mg/L) that significantly reduced anterior swim bladder inflation at the same time-point (Nelson et al. 2016).
• Maternal injection of T3, resulting in increased T3 concentrations in the eggs of striped bass (Morone saxatilis) lead to significant increases in both swim bladder inflation and survival (Brown et al., 1988).
• In the study of Cavallin et al. (2017) fathead minnow larvae were exposed to IOP, a model iodothyronine deiodinase inhibitor that is assumed to inhibit all three deiodinase enzymes (DIO1,2,3). The authors observed pronounced decreases of whole body T3 concentrations and increases of whole body T4 concentrations, together with impaired inflation of the anterior swim bladder chamber. More specifically, inflation was delayed and the size of the swim bladder chamber was reduced until the end of the exposure experiment. Since exposure was started after inflation of the posterior chamber, this study shows that DIO inhibition can directly affect anterior chamber inflation.
• In the study of Stinckens et al. (submitted) a strong correlation between reduced T3 levels and reduced anterior chamber inflation was observed in zebrafish exposed to iopanoic acid, a deiodinase inhibitor, as well as methimazole and propylthiouracil, both thyroperoxidase inhibitors, from fertilization until the age of 32 days. Anterior chamber inflation was delayed and a number of larvae did not manage to inflate the anterior chamber by the end of the 32 day exposure period. Additionally, exposed fish that had inflated the swim bladder had reduced anterior chamber sizes.

• The mechanism underlying the link between reduced T3 and reduced anterior chamber inflation remains unclear, but several hypotheses exist (Stinckens et al., submitted). For example, altered gas distribution between chambers could be the result of impaired development of smooth muscle fibers, delayed and/or impaired evagination of the anterior chamber, impaired anterior budding through altered Wnt and hedgehog signalling, etc.
• Increased T3 levels also seem to result in reduced swim bladder inflation. For example, Li et al. (2011) reported impairment of swim bladder inflation in Chinese rare minnows (Gobiocypris rarus) exposed to exogenous T3.

References

• 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. doi: 10.1002/jez.1402480207
• Brown, D.D., 1997. The role of thyroid hormone in zebrafish and axolotl development. Proceedings of the National Academy of Sciences of the United States of America 94, 13011-13016.
• 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.
• Li W, Zha J, Yang L, Li Z, Wang Z. Regulation of thyroid hormone related genes mRNA expression by exogenous T₃ in larvae and adult Chinese rare minnow (Gobiocypris rarus). Environ Toxicol Pharmacol. 2011 Jan;31(1):189-97. doi: 10.1016/j.etap.2010.10.007.
• Liu, Y.W., Chan, W.K., 2002. Thyroid hormones are important for embryonic to larval transitory phase in zebrafish. Differentiation 70, 36-45.
• McMenamin, S.K., Parichy, D.M., 2013. Metamorphosis in Teleosts. Animal Metamorphosis 103, 127-165.
• 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. Impaired anterior swim bladder inflation following exposure to the thyroid peroxidase inhibitor 2-mercaptobenzothiazole part I: Fathead minnow. Aquat Toxicol. 2016 Apr;173:192-203. doi: 10.1016/j.aquatox.2015.12.024.
• 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.
• 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.

Relationship: 1034: Reduced, Anterior swim bladder inflation leads to Reduced, Swimming performance

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

Importance of swimming performance for natural behaviour is generally applicable to fish.

Key Event Relationship Description

Effects on swim bladder inflation can alter swimming performance and buoyancy of fish, which is essential for predator avoidance, energy sparing, migration, reproduction and feeding behaviour, resulting in lower young-of-year survival.

Evidence Supporting this KER

The weight of evidence supporting a direct linkage between these two KEs, i.e. reduced anterior swim bladder inflation and reduced swimming performance, is weak.

The anterior chamber of the swim bladder has a function in regulating the buoyancy of fish, by altering the volume of the swim bladder (Roberston et al., 2007). Fish rely on the lipid and gas content in their body to regulate their position within the water column, with the latter being more efficient at increasing body buoyancy. Therefore, fish with functional swim bladders have no problem supporting their body (Brix 2002), while it is highly likely that impaired inflation severely impacts swimming performance. Fish with no functional swim bladder can survive, but are severely disadvantaged., making the likelihood of surviving smaller.

Several studies in zebrafish and fathead minnow showed that a smaller AC was associated with a larger posterior chamber (Nelson et al., 2016; Stinckens et al., 2016; Cavallin et al., 2017, Stinckens et al., submitted) suggesting a possible compensatory mechanism. As shown by Stoyek et al. (2011) however, the AC volume is highly dynamic under normal conditions due to a series of regular corrugations running along the chamber wall, and is in fact the main driver for adjusting buoyancy while the basic PC volume remains largely invariable. Therefore, it is plausible to assume that functionality of the swim bladder is affected when AC inflation is incomplete, even when the PC appears to fully compensate the gas volume of the swim bladder.

• After exposure to 2-mercaptobenzothiazole, a TPO inhibitor, from 0 to 32 days post fertilization (dpf) in zebrafish, the swimming activity of fish was impacted starting at 26 dpf if the inflation of the anterior chamber of the swim bladder was impaired or had no normal structure/size (Stinckens et al., 2016).
• Methimazole (MMI) and propylthiouracil (PTU), two thyroperoxidase inhibitors, and  iopanoic acid (IOP), a deiodinase inhibitor, each reduced both anterior chamber (AC) inflation and swimming distance in zebrafish exposed from fertlization until the age of 32 days (Stinckens et al., submitted). The current dataset provides evidence for a specific, direct link between AC inflation and reduced swimming performance. First, after 21 d of exposure to 111 mg/L PTU around 30% of ACs were not inflated and swimming distance was reduced, while by 32 dpf all larvae had inflated ACs and the effect on swimming distance had disappeared. The most direct way to assess the role of AC inflation in swimming performance, however, is to compare larvae with and without inflated AC at the same time point and within the same experimental treatment. Both in the PTU exposure at 21 dpf and in the IOP exposure at 21 and 32 dpf, swimming distance was clearly reduced in larvae lacking an inflated AC, while the swimming distance of larvae with inflated AC was equal to that of controls.
•  
• It has also been reported that larvae that fail to inflate their swim bladder use additional energy to maintain buoyancy (Lindsey et al., 2010, Goodsell et al. 1996), possibly contributing to reduced swimming activity. Furthermore, Chatain (1994) associated larvae with non-inflated swim bladders with numerous complications, such as spinal deformities and lordosis and reduced growth rates, adding to the impact on swimming behaviour.
• An increasing incidence of swim bladder non-inflation has also been reported in Atlantic salmon (Poppe et al. 1997). Affected fish had severely altered balance and buoyancy, observed through a specific swimming behaviour, as the affected fish were swimming upside down in an almost vertical position (Poppe et al. 1997).

After exposure to 100 mg/L MMI, 95% of the zebrafish larvae failed to inflate their AC at 32 dpf and swimming distance was reduced (Stinckens et al., submitted). On the other hand, there was no effect of impaired AC inflation on swimming distance in the MMI exposure of 50 mg/L. Also, inflated but smaller ACs did not result in a decreased swimming performance in the present study. A similar result, where non-inflated ACs did not consistently lead to reduced swimming performance, was previously found after exposure to MBT (Stinckens et al., 2016). In summary, the precise relationship between these two KEs is not easy to determine and may be different for different chemicals. Swimming capacity can be affected via other processes which may or may not depend on the HPT axis, such as decreased cardiorespiratory function, energy metabolism and growth.

As Robertson et al., (2007) reported, the swim bladder only starts regulating buoyancy actively from 32 dpf onward in zebrafish, possibly explaining the lack of effect on swimming capacity in some cases.

The anterior chamber is also important for producing and transducing sound through the Weberian Apparatus (Popper, 1974; Lechner and Ladich, 2008). It is highly plausible that impaired inflation or size of the anterior swim bladder could lead to a reduction in young-of-year survival as hearing loss would affect their ability to respond to their surrounding environment, thus impacting ecological relevant endpoints such as predator avoidance or prey seeking (Wisenden et al., 2008; Fay, 2009).

References

Brix O (2002) The physiology of living in water. In: Hart PJ, Reynolds J (eds) Handbook of Fish Biology and Fisheries, Vol. 1, pp. 70–96. Blackwell Publishing, Malden, USA.

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.

Chatain, B., 1994. Abnormal swimbladder development and lordosis in sea bass (Dicentrarchus labrax) and sea bream (Sparus auratus). Aquaculture 119:371–379.

Czesny, S.J., Graeb, B.D.S., Dettmersn, J.M., 2005. Ecological consequences of swimbladder noninflation for larval yellow perch. Trans. Am. Fish. Soc. 134,1011–1020, http://dx.doi.org/10.1577/T04-016.1.

Fay, R., 2009. Soundscapes and the sense of hearing of fishes. Integrative Zool. 4,26–32.

Goodsell, D.S., Morris, G.M., Olsen, A.J. 1996. Automated docking of fleixble ligands. Applications of Autodock. J. Mol. Recogonition, 9:1-5.

Kurata, M., Ishibashi, Y., Takii, K., Kumai, H., Miyashita, S., Sawada, Y., 2014.Influence of initial swimbladder inflation failure on survival of Pacific bluefintuna, Thuunus orientalis (Temminck and Schlegl) larvae. Aquacult. Res. 45,882–892.

Lechner, W., Ladich, F., 2008. Size matters: diversity in swimbladders andWeberian ossicles affects hearing in catfishes. J. Exp. Biol. 211, 1681–1689.

Lindsey, B.W., Smith, F.M., Croll, R.P., 2010. From inflation to flotation: contributionof the swimbladder to whole-body density and swimming depth duringdevelopment of the zebrafish (Danio rerio). Zebrafish 7, 85–96, http://dx.doi.org/10.1089/zeb.2009.0616.

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.

Poppe, T.T., Hellberg, H., Griffiths, D., Mendal, H. 1977. Swim bladder abnormality in farmed Atlantic salmon, Salmo salar. Diseases of aquatic organisms 30:73-76.

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, http://dx.doi.org/10.1002/jmor.

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.L., Maho, W., Blackwell, B., Witter, H.,Blust, R., Ankley, G.T., Covaci, A., Villenueve, D.L., Knapen, D., 2016. Disruption of thyroid hormone balance after 2-mercaptobenzothiazole exposure causes swim bladder inflation impairment—part II: zebrafish. Aquat. Toxicol. 173:204-17.

Stoyek, M.R., Smith, F.M., Croll, R.P., 2011. Effects of altered ambient pressure on the volume and distribution of gas within the swimbladder of the adult zebrafish, Danio rerio. Journal of Experimental Biology 214, 2962-2972.

Wisenden, B.D., Pogatschnik, J., Gibson, D., Bonacci, L., Schumacher, A., Willet, A.,2008. Sound the alarm: learned association of predation risk with novelauditory stimuli by fathead minnows (Pimephales promelas) and glowlighttetras (Hemigrammus erythrozonus) after single simultaneous pairings withconspecific chemical alarm cues. Environ. Biol. Fish 81, 141–147.

Woolley, L.D., Qin, J.G., 2010. Swimbladder inflation and its implication to theculture of marine finfish larvae. Rev. Aquac. 2, 181–190, http://dx.doi.org/10.1111/j.1753-5131.2010.01035.x.

Relationship: 1029: Reduced, Swimming performance leads to Reduced, Young of year survival

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

Importance of swimming performance on young of year survival is generally applicable to fish.

Evidence Supporting this KER

Reduced swimming performance is likely to affect essential endpoints such as predator avoidance, feeding behaviour and reproduction. These parameters are biologicaly plausible to affect young-of-year survival, especially in a non-laboratory environment where food is scarce and predators are abundant.

All zebrafish larvae that failed to inflate the posterior chamber after exposure to 2 mg/L iopanoic acid (IOP), died by the age of 9 dpf (Stinckens et al., unpublished). Since larvae from the same group that were able to inflate the posterior chamber survived, it is plausible to assume that the cause of death was the inability to swim and find food due to the failure to inflate the posterior swim bladder chamber.

Quantitative Understanding of the Linkage

Reduced swimming performance is not expected to immediately lead to mortality. Depending on the extent of the reduction in swimming performance and depending on the cause of death (e.g., starvation due to the inability to find food, being caught by a predator) the lag time may vary.

As an example, Stinckens et al. (unpublished) found that zebrafish larvae that failed to inflate the swim bladder at 5 dpf and did not manage to inflate it during the days afterwards died by the age of 9 dpf. Since zebrafish initiate exogenous feeding around day 5 when the yolk is almost completely depleted, there was a lag period of around 4 days after which reduced feeding resulted in mortality. Obviously, in a laboratory setup there is no increased risk of being caught by a predator.

All zebrafish larvae that failed to inflate the posterior chamber after exposure to 2 mg/L iopanoic acid (IOP), died by the age of 9 dpf (Stinckens et al., unpublished). Since larvae from the same group that were able to inflate the posterior chamber survived, it is plausible to assume that the cause of death was the inability to swim and find food due to the failure to inflate the posterior swim bladder chamber.

Relationship: 1030: Reduced, Young of year survival leads to Decrease, Population trajectory

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

Key Event Relationship Description

If young of year survival is reduced, ultimately the population trajectory will decrease.

Evidence Supporting this KER

It is widely accepted that if young of year survival is reduced, the population trajectory will eventually decrease.