Chemosphere 189 (2017) 498e506 Contents lists available at ScienceDirect Chemosphere journal homepage: www.elsevier.com/locate/chemosphere Tributyltin induces premature hatching and reduces locomotor activity in zebrafish (Danio rerio) embryos/larvae at environmentally relevant levels * Xuefang Liang a, b, Christopher L. Souders II b, Jiliang Zhang c, Christopher J. Martyniuk b, a School of Ecology and Environment, Inner Mongolia University, Hohhot, 010021, China b Center for Environmental and Human Toxicology, Department of Physiological Sciences, College of Veterinary Medicine, UF Genetics Institute, Interdisciplinary Program in Biomedical Sciences Neuroscience, University of Florida, Gainesville, FL, 32611, USA c Henan Open Laboratory of Key Subjects of Environmental and Animal Products Safety, College of Animal Science and Technology, Henan University of Science and Technology, Henan, China highlights Exposure to 1 nM TBT induced premature hatching in zebrafish. TBT at 1 nM inhibited locomotor behaviors of zebrafish larvae. Mitochondrial bioenergetics were not affected by TBT. Genes related to muscle function and dopamine signaling were not altered by TBT. article info abstract Article history: Tributyltin (TBT) is an organotin compound that is the active ingredient of many biocides and antifouling Received 27 July 2017 agents. In addition to its well established role as an endocrine disruptor, TBT is also associated with Received in revised form adverse effects on the nervous system and behavior. In this study, zebrafish (Danio rerio) embryos were 18 September 2017 exposed to environmentally relevant concentrations of TBT (0.01, 0.1, 1 nM) to determine how low levels Accepted 19 September 2017 affected development and behavior. Fish exposed to 1 nM TBT hatched earlier when compared to con- Available online 20 September 2017 trols. Following a 96-h exposure, total swimming distance, velocity, and activity of zebrafish larvae were Handling Editor: David Volz reduced compared to controls. To identify putative mechanisms for these altered endpoints, we assessed embryo bioenergetics and gene expression. We reasoned that the accelerated hatch time could be related Keywords: to ATP production and energy, thus embryos were exposed to TBT for 24 and 48-h exposure prior to Tributyltin hatch. There were no differences among groups for endpoints related to bioenergetics (i.e. basal, ATP- Mitochondrial bioenergetics dependent, and maximal respiration). To address mechanisms related to changes in behavioral activ- Locomotion ity, we measured transcripts associated with muscle function (myf6, myoD, and myoG) and dopamine Dopaminergic signaling signaling (th, dat, dopamine receptors) as dopamine regulates behavior. No transcript was altered in Premature hatching expression by TBT in larvae, suggesting that other mechanisms exist that may explain changes in higher level endpoints. These results suggest that endpoints related to the whole animal (i.e. timing of hatch and locomotor behavior) are more sensitive to environmentally-relevant concentrations of TBT compared to the molecular and metabolic endpoints examined here. © 2017 Elsevier Ltd. All rights reserved. 1. Introduction The organotin compound tributyltin (TBT) has been widely applied as a biocide in antifouling coatings for the shipping in- * Corresponding author. Center for Environmental and Human Toxicology & dustry since the 1970s (Novelli et al., 2002). TBT is a documented Department of Physiological Sciences, College of Veterinary Medicine, University of endocrine disruptor that induces imposex in mollusks and in- Florida, 2187, Mowry Rd. Bldg 471, Gainesville, FL, 32611, USA. terferes with reproductive processes in bivalves and fish (Ruiz et al., E-mail address: cmartyn@ufl.edu (C.J. Martyniuk). https://doi.org/10.1016/j.chemosphere.2017.09.093 0045-6535/© 2017 Elsevier Ltd. All rights reserved. X. Liang et al. / Chemosphere 189 (2017) 498e506 499 1995; McAllister and Kime, 2003; Shimasaki et al., 2003, 2006; 2015). However, the full scope of TBT-induced nervous and Zhang et al., 2007, 2009; Matthiessen, 2008). Studies also show behavior dysfunction, as well as its role in regulating mitochondrial that TBT can exert adverse effects on non-target aquatic organisms bioenergetics in fish, remains unclear. at very low concentrations in the water. For example, exposure to In this study, zebrafish (Danio rerio) embryos were exposed to 0.1 ng/L TBT for 70 days induced masculinization and sperm ab- environmentally relevant concentrations of TBT (0.01, 0.1, 1 nM). normality in zebrafish (Danio rerio)(McAllister and Kime, 2003). As We first assessed endpoints that were ecologically relevant, and a result, TBT was banned globally by the international community this included survival, presence of deformity, hatch rates, and in 2008 (Gipperth, 2009). As of 2009, the reduction of TBT levels in locomotion. To address potential mechanisms of toxicity, we also seawater and biota on a global scale has been more than 50% since measured oxygen consumption rates (bioenergetics), and the the ban (Kim et al., 2014). Tributyltin in seawater in Jinhae Bay, expression of genes related to muscle function and dopamine South Korea, for example decreased more than 50-fold from ~1995 signaling, the rationale being that these systems are involved in to 97 (mean 11.3 ± 8.2 ng/L) to 2008e09 (mean 0.2 ± 0.3 ng/L) after regulating locomotor activity and there is evidence that neuro- the total ban (Kim et al., 2014). However, due to its persistence and transmitter systems are perturbed in fish with TBT exposure. We widespread use, TBT residues in aquatic environments continue to hypothesized that TBT impairs locomotion in zebrafish larvae, and remain a concern in many regions. For example, in China, TBT levels that this is related to lower ATP production and suppressed in water varied from non-detectable to 93.8 ng/L (measured as Sn) expression of genes associated with muscle and the dopamine between the years of 2000e03 (Cao et al., 2009). In water, TBT also system, which is a dominant neurotransmitter involved in regu- ranged between 0.1 and 103 ng Sn/L in different ports of India from lating behavior. 2007 to 2008 (Garg et al., 2010, 2011) and levels in the seawater along the Croatian Adriatic Coast in 2009 and 2010 ranged 2. Materials and methods 10.1e72.4 ng/L as Sn (Furdek et al., 2012). In addition, TBT in sedi- ment can reach concentrations >1000 ng Sn/g, and this can be one 2.1. Chemicals of the leading sources of TBT contamination for aquatic ecosystems (Kim et al., 2015). Thus, despite a significant reduction in global TBT Tributyltin (TBT, CAS no. 7486-35-3, purity >97%) was pur- levels over the past several years, the biological effects of TBT at low chased from Sigma-Aldrich Co. LLC (USA). Stock solutions of 1, 10 concentrations, and its environmental persistence, ensures that and 100 mM TBT were prepared by dilution in ethanol (EtOH, CAS TBT remains a concern for the environment. no. 64-17-5, purity >99.5þ%, Acros Organics). The exposure solu- In addition to reproductive effects as an anti-androgen, TBT af- tions were prepared by adding 1 ml of each stock solution into fects the nervous system of fish by altering neurotransmitter levels. 100 mL of embryo rearing medium (ERM; 8 g NaCl, 0.4 g KCl, TBT alters monoamine concentrations in the brain and studies 0.035 g Na2HPO4, 0.6 g KH2PO4, 0.14 g CaCl2, 0.12 g MgSO4, 0.35 g report that the concentration of dopamine (DA) and 5- NaHCO3, into 1 L, pH 7.2) yielding the final concentrations of 0.01, hydroxytryptamine (5-HT) of male and female medaka (Oryzias 0.1 and 1 nM TBT (equal 3.17, 31.7, and 317 ng/L). A vehicle treat- latipes) are significantly decreased with exposure at 125 mg TBT/g ment of EtOH served as the control. The ratio of vehicle to ERM was diet/d for 21 days (Nakayama et al., 2007). Conversely, in another 1:100,000 (vol/vol). study, DA was increased in the brain of rockfish Sebastiscus mar- moratus following exposure to 10e1000 ng/L TBT while levels of 5- 2.2. Experimental design HT were decreased with treatment (Yu et al., 2013). In addition, the levels of the neurotransmitter glutamate were increased following Adult zebrafish (ZF, Wild type AB) were raised at the University exposure to 500 mg/kg TBT (Zuo et al., 2009) and the N-methyl-d- of Florida (Gainesville) in the Cancer Genetics Research Center aspartate receptor (NMDAR) signaling pathway was inhibited by (CGRC). The mean water pH was 7.2 ± 1.0 and mean temperature TBT in the brains of rockfish (Zuo et al., 2009; Yu et al., 2013). Thus, was 28.1 ± 1.0 C, monitored daily. Dissolved oxygen was measured ® there is good evidence for neurotransmitter alterations by TBT at 6.6 ppm using a LeMotte Freshwater Fish Farm test kit (Pentair). which underlie the scientific premise that this compound may Fish were exposed to 14 h light and 10 h dark per day. ZF were affect behavior. This is an important component to investigate as randomly selected from a breeding stock and placed in a shallow behavioral endpoints can be used to detect neuronal effects of water breeding tank the night before embryo collection. The ratio environmental stressors (Robinson, 2009). Previous studies was two males to two females per tank and there were two tanks demonstrate that predatory behavior (Yu et al., 2013), antipredator used to generate the fertilized embryos. A divider separated the behavior (Wibe et al., 2001), shoaling and anxiety behavior (Zhang males and females overnight. These dividers were removed at 8:00 et al., 2016b), swimming distances, speed and orientation am when the facility lights turned on and spawning occurred. Both (Triebskorn et al., 1994), as well as feeding behavior of fish, are tanks yielded viable fertilized embryos. These embryos were disrupted by TBT (Zhang et al., 2016a).