Eugenol induces body immobilization yet evoking an increased neuronal excitability in fish during short-term baths

Abstract

Several studies have suggested eugenol as a suitable anaesthetic for fish. However, it has also been regarded as a toxic and aversive substance to several aquatic organisms, including fish. This study sought to assess the eugenol-induced behavioural alterations and its seizurogenic potential to fish. Moreover, a distinctive methodology for an in vivo evaluation of the brain activity was also presented. Prior to the evaluation of eugenol-induced responses, fish were exposed to pentylenetetrazole (PTZ), to characterize any seizure-like patterns. Antagonizing responses to PTZ were assessed in fish receiving diazepam (BDZ) and subsequently exposed to PTZ. Tambaqui fish juveniles, Colossoma macropomum (15.8 ± 2.8 g) were used as models and assayed as follows: (i) fish exposed to PTZ (15 mM) and (ii) fish receiving a dose of BDZ (10 mg Kg^-1) and later exposed to PTZ (15 mM) (BDZ-PTZ group). Thereafter, fish were evaluated throughout (iii) eugenol exposure at 65 μL L^-1 (ethanolic solution) and recovery. Control fish and a vehicle control group (ethanol at 585 μL L^-1) were also established. PTZ baths elicited body immobilization preceded by hyperactivity in a stereotyped seizure-like behaviour with increased EEG wave amplitude and frequency. PTZ effects in the brain were attenuated by a pre-administration of BDZ. Upon eugenol exposure, tambaqui had an intense neuronal excitability, showing a clonus-like seizure behaviour, also corroborated by the EEG patterns, which were consistent with a seizure-like response. Responses of eugenol-exposed fish resembled those of the PZT-exposed animals, with epileptiform discharges. EMG was in line with the EEG modulation, showing increased tracing oscillations and higher mean amplitudes in PTZ-exposed fish whereas in BDZ-PTZ group muscle contraction was less frequent and powerful. Fish exposed to eugenol showed initially some muscle activity followed by a loss of muscle tonus over time. In summary, our results showed that upon eugenol exposure, although a time-dependent body immobilization was attained, fish presented an intense neuronal excitability comparable to that evoked by PTZ. Eugenol failed to promote depression of the CNS and therefore may be not suitable to be used for general anaesthesia of C. macropomum. As eugenol could be implicated in seizurogenesis and be potentially toxic to the fish brain, protocols suggesting the broad use of eugenol for short-term anaesthesia or euthanasia of fish should be carefully revised, as it raises important concerns in terms of ethics and fish welfare.

Keywords: Electroencephalography; Electromyography; Fish anaesthesia; Fish welfare; Seizures.