Veterinary antiparasitic pharmaceuticals – effects on behaviour in fish larvae

Abstract: SUMMARY Antiparasitic pharmaceuticals are important groups of pharmaceuticals in veterinary medicine and are used in large quantities. Antiparasitic pharmaceuticals and their metabolites enter the environment via excretion in urine and/or faeces from treated animals and by run off to surface water these compounds reach aquatic environments and thereby present a risk for aquatic organisms. The goal of this master thesis is to investigate if veterinary antiparasitic pharmaceuticals have any effects on the behaviour of zebrafish larvae. In addition, adding of a behavioural assay could increase sensitivity in the fish embryo toxicity test. There are some studies of toxicity of veterinary pharmaceuticals in aquatic animals, but they focus mainly on general toxic effects. Several of the antiparasitic pharmaceuticals has a neurotoxic mechanism of action, and may therefore affect behaviour of zebrafish larvae, which in turn can affect its responses to stimuli and the survival of the larvae in its natural environment. In this study zebrafish larvae were exposed, from day 0 until day 6, to three different antiparasitic pharmaceuticals: doramectin, flumethrin and toltrazuril. An additional study was performed with doramectin, in order to study the effects of short time exposure. In the short term study the embryos were exposed to doramectin at day 6 for 1 h before behaviour recording. In the long time exposure experiments a behavioural assay was performed at day 6 using a video recording device (Viewpoint Zebrabox®) that recorded the swim activity of the larvae. Directly after the behavioural assay the larvae were assessed in stereo microscope for abnormalities and abnormal body posture. Only data from individuals that were considered as normal according to the macroscopic examination were included in the data set and used in the statistical analyses. Because of the large amount of data that were received from the recording device, four responses were chosen for statistical analysis: mean activity during the first period of darkness, mean activity during the first period of darkness, increase in activity during the first 10 seconds at the first period of darkness and difference between mean activity during the first and the last period of darkness. Significant results were found for doramectin and flumethrin. The activity in the first five minutes dark period and the first five minutes light period were both significantly reduced in larvae exposed to the high concentration (0.58 mg/L) of doramectin compared with the control. Larvae exposed to the high concentration (0.11 mg/L) of flumethrin had a significant higher reduction in activity from the first to the last dark period as compared with the reduction in control larvae. The conclusion is that it is possible to detect altered behavioural response, in individuals that could not be classified as affected based on visual features. This finding supports that behavioural assays are potential tools to increase the sensitivity in toxicological studies in zebrafish.