Developmental exposures to ethanol or dimethylsulfoxide at low concentrations alter locomotor activity in larval zebrafish: Implications for behavioral toxicity bioassays

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Abstract

Ethanol and dimethylsulfoxide (DMSO) are commonly used as carrier solvents for lipophilic chemicals in aquatic toxicity bioassays. However, very little information has been reported on the behavioral effects of these solvents. In this study, we examined the effects of ethanol and DMSO on development and locomotor activity by a zebrafish embryo-larval bioassay. The zebrafish were exposed to different concentrations (control, 0.01, 0.1, and 1%) of ethanol or DMSO from blastula stage to 144 hour-post-fertilization (hpf). Hatchability, survival, and abnormalities were monitored every 12 h, and locomotor activity of the larvae was analyzed at 144 hpf. Hatchability was not affected by the ethanol or DMSO treatments. No effect on survival was observed except the 1% ethanol group suffered 89% mortality during 108–120 hpf. No developmental defects were observed in any of the solvents at the 0.01 and 0.1% concentrations, but significantly higher deformity rates occurred with 1% ethanol and DMSO groups. Hyperactivity and less tortuous swimming paths were observed in all ethanol and DMSO concentrations. Based on this study, we suggest that data of behavioral toxicity bioassays using ethanol or DMSO as carrier solvents should be interpreted cautiously, because the solvents at low concentrations could alter locomotor activity of larval zebrafish without causing any observable developmental defects.

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.

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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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