This study aimed to evaluate the synergistic effect between eugenol and 1,8-cineole on anesthesia in female guppy fish (Poecilia reticulata). Experiment I evaluated the concentrations of 0, 12.5, 25, 50, and 75 mg/L of eugenol and 0, 100, 200, 300, and 400 mg/L of 1,8-cineole for times of induction and recovery from anesthesia. Experiment II divided fish into 16 study groups, combining eugenol and 1,8-cineole in pairs at varying concentrations, based on the dosage of the chemicals in experiment I. The results of the anesthesia showed that eugenol induced fish anesthesia at concentrations of 50 and 70 mg/L, with durations of 256.5 and 171.5 s, respectively. In contrast, 1,8-cineole did not induce fish anesthesia. In combination, using eugenol at 12.5 mg/L along with 1,8-cineole at 400 mg/L resulted in fish anesthesia at a time of 224.5 s. Increasing the eugenol concentration to 25 mg/L, combined with 1,8-cineole at 300 and 400 mg/L, induced fish anesthesia at times of 259.0 and 230.5 s, respectively. For treatments with eugenol at 50 mg/L combined with 1,8-cineole at 100 to 400 mg/L, fish exhibited anesthesia at times of 189.5, 181.5, 166.0, and 157.5 s. In the case of eugenol at 75 mg/L, fish showed anesthesia at times of 175.5, 156.5, 140.5, and 121.5 s, respectively. The testing results revealed that 1,8-cineole as a single treatment could not induce fish anesthesia. However, when supplementing 1,8-cineole in formulations containing eugenol, fish exhibited a significantly faster induction of anesthesia (p < 0.05). Furthermore, all fish that underwent anesthesia were able to fully recover without any mortality. However, the shorter anesthesia duration resulted in a significantly prolonged recovery time. In conclusion, eugenol and 1,8-cineole work better together as anesthetics than when used separately, and demonstrated the safety of using these anesthetic agents on guppy fish.

The objective of this research was to investigate the anesthetic properties of essential oil from Ocimum tenuiflorum L. (OTO) and Ocimum basilicum L. (OBO), which belong to the Lamiaceae family. They are used as flavoring agents, food preservatives, and traditional medicines. The serum biochemical responses were examined post-recovery to assess the impacts of anesthetics on stress levels in comet goldfish (Carassius auratus). The results revealed that OTO at concentrations of 80, 100, and 120 mg/L could induce deep anesthesia in 257 s, 170 s, and 136 s, and the recovery time was 293 s, 270 s, and 263 s, respectively. A greater concentration of OTO led to shortened induction times. OBO at 250 and 300 mg/L induced deep anesthesia in 212 s and 190 s, and the recovery time was significantly prolonged to 263 s and 554 s, respectively. Moreover, the effective concentrations (EC₅₀) within 3 min were 85.78 mg/L (OTO) and 306.49 mg/L (OBO). The blood parameters showed that the glucose and cortisol levels in only the OTO group remained significantly lower compared to all the other treatment groups. The ALT and ALP levels were elevated in the OBO group compared to those in the OTO group. In the OBO and MS-222 groups, albumin was significantly different from that in the OTO group. Moreover, the serum calcium, phosphorus, BUN, and creatinine levels were not significantly different in any of the groups. In conclusion, OTO and OBO have been shown to be efficient natural anesthetics in goldfish, with no mortality.

This study aimed to prepare and characterize nanoemulsions containing eugenol and 1,8-cineole using the emulsification method and to investigate their anesthetic effects on guppy fish. The optimized formulation comprised a 5–10% mixture of eugenol and 1,8-cineole in a 1:2 ratio, stabilized with 15–20% Tween 80. The selected formulations displayed mean particle sizes below 15 nm, a low polydispersity index (PDI) (<0.5), and a zeta potential that was more negative than −40 millivolts (mV), indicating stable emulsions. Their pH ranged from 6.50 to 6.63, indicating slight acidity. The formulations exhibited non-Newtonian rheology, as well as thinning under shear stress. Three formulations (F2, F6, and F12) remained stable after both accelerated and long-term stability testing. All nanoemulsions were able to induce guppy fish to the third stage of anesthesia. The nanoemulsions with concentrations of 50 mg/L and 100 mg/L eugenol effectively induced sedation and anesthesia in both sexes and reduced the induction and recovery times compared with the ethanol solution. In conclusion, this study highlights nanoemulsions as a promising drug delivery system for alternative anesthetics in aquaculture.