DOI: 10.17033/data.00000184
title: The effects of zinc oxide and titanium dioxide on the morphology and behaviour of zebrafish Danio rerio

description: A range of impacts of TiO2 and ZnO nanoparticles are known to affect fish species, including Immunotoxicity (Wang et al, 2013), reproductive effects (Wang et al, 2011) brain development (Shaw and Handy, 2011) and cardiorespiratory effects (Bessemer et al, 2014). This research aims to determine the behavioural and developmental effects of TiO2 and ZnO on D. rerio.
description: Wildtype (AB strain) zebrafish (Danio rerio) embryos were obtained from the zebrafish facility at Kings College, London on 4th July 2016 and maintained at the Biosciences Research Facility at the University of Brighton, Hastings Campus. Embryos were supplied in batches of 50 and each batch was subjected to one of 10 treatments. Zinc oxide (ZnO) nanopowder with a particle size of &lt;50 nm and &gt;97% metal basis (Sigma Aldrich) and titanium dioxide (TiO2) nanopowder with a particle size of 32nm and a 99.9% metal basis (Alfa Aesar) were used. Stock solutions of TiO2 were made by adding 100mg of TiO2 to 100ml of dechlorinated tap water to make up a 10mg/ml-1 stock. ZnO stock solutions were made by adding 10mg of ZnO to 100ml of dechlorinated tap water to make a 1mg/ml-1 stock. Stock solutions of both ZnO and TiO2 were sonicated for 30 minutes to disperse the particles. The embryos were 24hpf at initial treatment. Embryos were dosed at 0.001 mg/ml-1, 0.01mg/ml-1 and 0.1mg/ml-1 of TiO2 as per Faria et al. (2014). Embryos were exposed to doses of 0.0001mg/ml-1, 0.001mg/ml-1 and 0.01mg/ml-1 of ZnO as per Brun et al. (2014). Exposures to a mix of both compounds at low, medium and high doses was also carried out. Eggs were placed in beakers with 200ml of treatment water (2ml, 0.2ml and 0.02ml of each stock solution for the high, medium and low doses respectively). Each treatment, including a control was carried out in triplicate. Water was changed daily. At 48hpf a 90% water change was made and dosing altered accordingly (i.e. 1.8ml of stock solution for the high dose). At 72hpf and due to the motile nature of the embryos a 75% water change was made, again, adjusting the dosing (i.e. 1.5ml of stock solution for the high dose). Beakers were suspended from polystyrene tiles which were housed in a 30cm2 tank. Water was maintained at 28±0.°C by recirculating heated water through the system. pH, conductivity, DO. Beakers were randomly distributed amongst 6 tanks on a rack system. At 96hpf fish were transferred from the beakers with a 3ml disposable Pasteur pipette to a flat bottomed 96 well plate with round wells in preparation for behavioural experimentation. A Daniovision observation chamber and EthoVision software (Noldus Information and Technology, Wageningen, The Netherlands) were used for tracking. Treatments were randomly allocated across the well plate with 12 fish per row from each treatment. Forty eight fish from each treatment were used for the behavioural study. The embryos were exposed to a light:dark challenge as per Hua et al. (2014). This involved an adaptation period of 2 minutes with the light on, followed by a basal phase of 4 minutes with the light on. The challenge phase entailed the light being off for 4 minutes with the onset of sudden darkness being the stressor. The recovery phase consisted of 4 minutes with the light on. During the filming swimming velocity, distance and direction were recorded. After the behavioural testing fish were anaesthetised and placed into 10% neutrally buffered saline in order to preserve the samples for morphometric analysis. Each fish was then imaged at 20x Motic SMZ-168 stereo microscope which was linked to a 5 megapixel Moticam camera. ImageJ image analysis software was used to determine body length, yolk sac circumference, caudal fin height, head height and eye circumference.