Influence of triphenyltin on morphologic abnormalities and the thyroid hormone system in early-stage zebrafish (Danio rerio)
Graphical abstract
Introduction
Zebrafish (D. rerio) are widely used for ecotoxicity testing since they are small (<5 cm in length), easy to keep, and have a short generation cycle (~3 months). Numerous studies have used zebrafish to investigate the influence of chemicals on mortality (Horie et al., 2017a; Wang et al., 2019), embryonic development (Horie et al., 2017c; Sarasquete et al., 2018), growth (Horie et al., 2020; Kovács et al., 2016; Memmert et al., 2013), reproduction (Kwon et al., 2016; Oliveira et al., 2020), sexual differentiation (Baumann et al., 2013), and effect on multigenerations (Constantine et al., 2020). Recently, attention of animal welfare consideration for current ecotoxicity testing is increasing. One such ecotoxicity testing is short-term toxicity test using embryo and sac-fry stages (OECD TG 212). Test animals typically are not fed throughout the test when the effects of chemical substances are examined in this toxicity test (OECD, 1998). Therefore, this test has been termed by some as the “fish starvation test” (ENV/JM/MONO (2012)). This suggests that the short-term toxicity test should include feeding from the view of animal welfare. However, whether ecotoxicity values differ between fed and fasted zebrafish is unknown.
The organotin compound triphenyltin (TPT) is used worldwide as a biocide in marine anti-fouling coatings to prevent the attachment and growth of marine organisms (Blunden and Evans, 1989; Snoeij et al., 1987). However, many adverse effects of TPT on aquatic organisms have been reported, including inhibition of reproduction in freshwater gastropods (Lymnaea stagnalis; Giusti et al., 2013) and Japanese medaka (O. latipes; Zhang et al., 2008); growth inhibition in marine medaka (O. melastigma; Yi and Leung, 2017) and Japanese medaka (O. latipes; Horie et al., 2017b); and abnormal sexual development in false kelpfish (Sebastiscus marmoratus; Sun et al., 2011) and calenoid copepods (Acartia tonsa; Watermann et al., 2013). Therefore, the use of TPT has been limited in various countries, including Japan.
The potential for thyroid hormone-disrupting chemical contamination in ecosystems is garnering increased attention worldwide, owing to thyroid hormone's key roles in maintaining normal physiology and in brain development, metabolism, and growth (Bernal, 2005; Casals-Casas and Desvergne, 2011). In a previous study, TPT induced increased expression of thyroid stimulating hormone β subunit (tshβ) but inhibited thyroid hormone receptor α (trα) and thyroid hormone receptor β (trβ) expression in zebrafish larvae at 7 dah (Li et al., 2019), whereas another group reported upregulation of both tshβ and trβ expression in zebrafish larvae at 7 and 14 days after fertilization (daf) (Yao et al., 2020). Together these data suggest that TPT disrupts the thyroid hormone system in zebrafish, but the precise effects of TPT exposure on the expression of tshβ or tr remain unclear because results differ between studies.
Recently, swim bladder inflation has become a potential biomarker for the detection of thyroid hormone disruptors because swim bladder development in fish is controlled by thyroid hormone systems (Spaan et al., 2019). In fact, perfluorooctanoic acid (PFOA), perfluorobutyric acid (PFBA), and tris(1,3-dichloro-2-propyl) phosphate (TDCPP), all of which are thought to act as thyroid disruptors, induced delays in or the absence of swim bladder inflation in zebrafish (Godfrey et al., 2017). However, it is still unclear whether TPT exposure induces delays in or the absence of swim bladder inflation.
Therefore, we addressed the following questions in the present study. First, does the ecotoxicity of TPT on zebrafish larvae differ between fed and fasted group during testing? Second, does TPT exposure affect expression of TSH-related genes (tshβ and tr)? Third, does TPT exposure induce delays in or the absence of swim bladder inflation?
Section snippets
Test fish and chemicals
The present study used zebrafish (NIES-R strain) from a population bred since 2017 at Akita Prefectural University, Japan, under a room temperature of 25 ± 2 °C and a 16:8 h light:dark photocycle. The zebrafish were handled humanely in accordance with the guidelines of the National Institute for Environmental Studies and of Akita Prefectural University. Triphenyltin (TPT) (CAS no. 639–58-7; purity >98.0%) was obtained from Wako Pure Chemical Industries (Osaka, Japan).
Short-term toxicity test
Short-term toxicity testing
Results of water analysis
During short-term toxicity testing (OECD TG 212), the concentrations of TPT to which groups of fed and fasted zebrafish eggs, embryos, and larvae were exposed remained relatively stable, showing similar tendencies, throughout the test period (Table 2). For the long-term toxicity test (OECD TG 210), the TPT concentration remained stable throughout the test period (Table 3). We did not detect TPT in any of the samples from the control group.
Effects of feeding and fasting on TPT toxicity
Among the groups that were fed during the test period,
Discussion
In the current study, we examined whether the toxicity of TPT varied depending on whether zebrafish larvae were fed compared with fasted group during testing. We found that feeding did not alter the effect concentration of TPT, as indicated by average number of days until hatching and hatching rate. Consequently we conclude that the presence or absence of food does not affect toxicity parameters in the early larval stages (i.e., until 5 dah), although the lethal concentration in the fed group
Conclusions
We evaluated the influence of TPT on mortality, larval development, growth, and the expression of thyroid-related genes in zebrafish. First, we confirmed that whether larvae were fed or fasted during toxicity testing did not affect the test results obtained. Second, the exposure of TPT to early life stages of zebrafish did not alter their growth. In contrast, TPT exposure prevented swim bladder inflation, and affected larvae died soon after hatching. These findings suggest that morphologic
Declaration of competing interest
The authors have no conflicts of interest related to this research.
Acknowledgments
This study was supported in part by the Steel Foundation for Environmental Protection Technology, Japan (Y.H.), and a grant from the Ministry of Education, Culture, Sports, Science and Technology, Japan (Grant-in-Aid for Scientific Research [B] grant no. 19H04294) (Y.H.), (Grant-in-Aid for Scientific Research [A] grant no. 19H01166) (N.T.). This project is a part of the European Union's Horizon 2020 research and innovation program under grant agreement No. 825753 (ERGO). This output reflects
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