Developmental exposures to ethanol or dimethylsulfoxide at low concentrations alter locomotor activity in larval zebrafish: Implications for behavioral toxicity bioassays
Introduction
Carrier solvents are crucial to aquatic toxicity bioassays as delivery systems for lipophilic chemicals. Appropriate utilization of solvents for xenobiotic agents is a major concern in toxicological studies (Hallare et al., 2006). For instance, US.EPA has set a maximum acceptable limit of 0.05% solvents for acute toxicity tests and 0.01% for chronic toxicity tests (Eaton et al., 1975, Hallare et al., 2006). Ethanol and dimethylsulfoxide (DMSO) are frequently used solvents in toxicology tests (Castro et al., 1995). Ethanol is the most common delivery system in aquatic bioassays. DMSO, a by-product of the wood industry, has been used as a solvent since the 1950s due to their effectiveness for solubilizing a wide range of polar and nonpolar compounds (Hallare et al., 2006). Because of their extensive utilization in aquatic toxicology tests, it is necessary to elucidate potential toxic effects of these solvents on aquatic organisms such as fish.
Zebrafish (Danio rerio) is becoming a popular tool in aquatic toxicology (Hill et al., 2005, Nagel, 2002). A 96-h zebrafish embryonic bioassay suggested that ethanol and DMSO as carrier solvents at levels below 1% and 1.5%, respectively, are appropriate for zebrafish embryo bioassays using survival and malformation as endpoints (Hallare et al., 2006). Zebrafish is also emerging as an important neurobehavioral model in pharmacology, toxicology, and ecotoxicology (Chou et al., 2010, Creton, 2009, Irons et al., 2010). However, there are few reports on the neurobehavioral effects (e.g., locomotor effects) of these solvents in zebrafish. Indeed, zebrafish has been used as a behavioral model to study alcohol effects, and these studies showed that alcohol (i.e., ethanol) can cause hyperactivity at lower concentrations but hypoactivity at higher concentrations in both larval (Lockwood et al., 2004) or adult fish (Gerlai et al., 2000). Nevertheless, the concentrations of ethanol used in these studies (0.25–4%) were generally higher than that would be used for a carrier solvent in bioassays (e.g., 0.01–0.1%). As to DMSO, although its neurobehavioral toxicities have been published in a few rodent studies (Castro et al., 1995, Cavaletti et al., 2000), very limited behavioral data has been reported in fish. A recent study reported increased swimming activity in adult zebrafish acutely exposed to 0.05% DMSO for 4 min (Sackerman et al., 2010). These data suggested that ethanol and DMSO, when used as solvents in bioassays, may modify fish swimming behavior and consequently lead to under- or overestimation of the neurobehavioral effects of the tested compounds.
The objectives of the present study were to characterize the effects of developmental exposure to ethanol or DMSO at low concentrations (i.e., 0.01, 0.1, and 1%, v/v) in zebrafish, with an emphasis on the behavioral responses. Larval locomotor activity was quantitatively measured using an automated video-based animal movement analysis system. We hypothesized that behavioral endpoints are more sensitive to ethanol and DMSO exposure than other developmental endpoints, such as hatchability, survival, and deformity rate.
Section snippets
Animals
Adult ourbred wildtype zebrafish for broodstock were obtained from a local ornamental fish supplier. Male and female adult fish were maintained separately at 28.5 °C under a 12L:12D photoperiod, with a diet of commercial fish flakes and live Artemia nauplii. The fish had been maintained and spawned in our laboratory for more than six months. On the evening before spawning, two male and one female adult zebrafish were placed in each of ten hatching boxes, and spawning was triggered when the light
Hatchability, survival, and deformity rates
Hatchability was around 94, 96, and 89–92% in the control, ethanol, and DMSO groups, respectively. The embryos hatched between 48 and 72 dpf, and the hatchability was not significantly affected by either ethanol or DMSO. Survival of the hatched larvae in each group was nearly 100% until 108 hpf. However, during 108–120 hpf, survival in the 1% ethanol group rapidly dropped to 11.59%, which was significantly lower than that in the control (p < 0.001), while no mortality occurred in all the other
Discussion
Ethanol and DMSO are widely used in aquatic toxicology studies as carrier solvents for lipophilic chemicals. Although their embryotoxicity in fish has been previously reported, their behavioral effects at low concentrations commonly used for toxicity tests are not well characterized, especially for DMSO. In this present study, we demonstrated that developmental exposure to ethanol or DMSO at concentrations as low as 0.01–0.1% can affect locomotor activity of larval zebrafish without causing any
Acknowledgements
This study was supported by the National Science Council of Taiwan (Grant No. NSC99-2313-B-291-002) and the Thematic Research Grant from NMMBA (Grant No. 992003312). Y.-H. Wu was also funded by the Taiwan Tech Trek Project 2010 of the National Science Council of Taiwan.
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