Anesthesia and simulated transport of two Amazonian fish with three different basil (Ocimum sp.) essential oils

Anestesia e transporte simulado de dois peixes amazônicos com três diferentes óleos essenciais de manjericão (Ocimum sp.)

ABSTRACT

This study aimed to evaluate the anesthetic induction and transportation of freshwater angelfish (Pterophyllum scalare) and tambaqui (Colossoma macropomum) (both Amazonian fish) juveniles using three basil essential oils (EO): Ocimum sanctum (EOOSE) (Eugenol 58.94%), Ocimum canum (EOOCL) (Linalool 68.64%), and Ocimum basilicum (EOOBM) (Methyl chavicol 68.67%). The concentrations used were: 0 (control), 10, 25, 50, 100, 200, 300, 400 (only for EOOCL and EOOBM), 600 and 800 (both only for EOOBM) µL L^-1. In another experiment, concentrations of 0 (control), 10, and 20 µL L⁻¹ were tested for simulated transport during 6 h. There was only sedation for the fish with the three EO tested in concentrations up to 25 µL L^-1. In this order, for freshwater angelfish, anesthesia and recovery times of less than 3 and 10 min, respectively, were found from 200, 400, and 800 µL L^-1 for EOOSE, EOOCL, and EOOBM. For tambaqui, following these same assumptions, the concentrations indicated for EOOSE, EOOCL, and EOOBM were 200, 300, and 400 µL L^-1, respectively. It is concluded that for sedation and transport of fish, 10 and 20 µL L^-1 can be used (regardless of the EO), and for rapid anesthesia, 200 µL L^-1 of EOOSE is indicated.

Keywords
Colossoma macropomum ; eugenol; linalool; methyl chavicol; Pterophyllum scalare

RESUMO

Este estudo objetivou avaliar a indução anestésica e o transporte de juvenis de acará bandeira (Pterophyllum scalare) and tambaqui (Colossoma macropomum) (ambos peixes Amazônicos) com a utilização de três óleos essenciais (EO) de manjericão: Ocimum sanctum (EOOSE) (Eugenol 58,94%), Ocimum canum (EOOCL) (Linalol 68,64%), e Ocimum basilicum (EOOBM) (Metilo chavicol 68,67%). As concentrações utilizadas foram de 0 (controle), 10, 25, 50, 100, 200, 300, 400 (somente para EOOCL e EOOBM), 600 e 800 (ambas somente para EOOBM) µL LL^-1. Em outro experimento, concentrações de 0 (controle), 10, e 20 µL L⁻¹ foram testadas para transporte simulado de 6 h. Houve apenas sedação para os peixes com os três EO testados em concentrações inferiores a 25 µL LL^-1. Para acará bandeira, tempos de anestesia e de recuperação inferiores a, respectivamente, 3 e 10 min foram encontrados a partir de 200, 400, e 800 µL LL^-1 para EOOSE, EOOCL, e EOOBM, nesta ordem. Para tambaqui, seguindo estes mesmos pressupostos, as concentrações indicadas para EOOSE, EOOCL, e EOOBM foram 200, 300, e 400 µL LL^-1, respectivamente. Conclui-se que para a sedação e transporte dos peixes pode-se usar 10 e 20 µL LL^-1 (independente do EO) e para rápida anestesia indica-se 200 µL EOOSE LL^-1.

Palavras-chave
Colossoma macropomum ; eugenol; linalool; methyl chavicol; Pterophyllum scalare

1. Introduction

Freshwater angelfish (P. scalare) is an essential ornamental fish native to the Amazon basin, which can be produced in different production systems. Its coloration is striking, increasing the interest in its commercialization by aquarists. Despite its commercial potential, this species is sensitive to handling and transportation, which can cause diseases, opacity, and even deaths, resulting in economic losses for producers (De Oliveira et al., 2019; De Oliveira et al., 2022; Castro Neto et al., 2024).

Tambaqui (C. macropomum) also is a fish native to the Amazon basin. Unlike freshwater angelfish, fish farmers target it to produce fillets and slices for marketing. However, the production and marketing of fish intended for consumption add stress factors, mainly resulting from their transportation and handling (Stevens et al., 2017; Marques et al., 2021).

These fish species are commonly transported to distinct locations from one region to another in Brazil, especially when they are in the juvenile phase (Ventura et al., 2021; De Oliveira et al., 2022). Thus, sedative substances, which can reduce reactive responses to external stimuli without altering the balance and swimming capacity, are associated with transport stress (Copatti et al., 2024). As a result, fish could consume less oxygen and excrete fewer nitrogen compounds, which could increase post-transport survival (Aydin & Barbas, 2020).

Sedation (ideal for transport) is achieved when anesthetics are used in very low concentrations (Sena et al., 2016). In addition, natural anesthetics are a safer and more economical alternative than synthetic anesthetics (Mirghaed et al., 2018), which have the advantages of easy access, low toxicity, and biodegradability ability (Sena et al., 2016). Among natural anesthetics, essential oils (EO) stand out. EO from Ocimum sp. (Lamiaceae), in addition to folk medicine (Piras et al., 2018), are also highly valued for use as sedatives and anesthetics in fish, having proven action in silver catfish (Rhamdia quelen) (Silva et al., 2012; Silva et al., 2015), tambacu (Piaractus mesopotamicus × C. macropomum) (Limma-Neto et al., 2016), Nile tilapia (Oreochromis niloticus) (Limma-Neto et al., 2017), and rainbow trout (Oncorhynchus mykiss) (Yigit et al., 2022).

This study aimed to evaluate the sedative and anesthetic potential of three different basil EO: O. sanctum (chemotype eugenol) (EOOSE), O. canum (EOOCL), and O. basilicum (chemotype methyl chavicol) (EOOBM) in freshwater angelfish and tambaqui juveniles, as well as survival and water quality parameters after simulated transport.

2. Material and methods

The three EOs used in this study were purchased from Terra Flor Aromaterapia^® (Alto Paraíso de Goias, Brazil). The major chemical constituents of EOOSE were eugenol (58.94%) and caryophyllene (30.82%), of EOOCL were linalool (68.64%) and caryophyllene (6.55%), and of EOOBM were methyl chavicol (68.67%) and linalool (18.40%).

The Ethical Committee of the Institution approved procedures for handling animals and collecting biological samples under protocol numbers 01/2023 and 01/2024. Two experiments were conducted, one for freshwater angelfish (gold-lineage) (n = 192; 2.64 ± 0.25 g; 5.75 ± 0.13 cm) and another for tambaqui (n = 186; 1.81 ± 0.12 g; 5.31 ± 0.15 cm) obtained from commercial fish farms in Salvador – BA were used. The animals were transported to the laboratory and kept in two tanks with a capacity of 250 L and aeration and filters (chemical and biological). They remained fed with commercial feed (Alcon Guard Herbal (Camboriú, Brazil), with 50% crude protein for freshwater angelfish and Supra (São Leopoldo, Brazil), with 32% crude protein for tambaqui) for 15 days until the beginning of the experiment. Feeding was carried out once a day and was suspended 24 h before the start of the experiment.

Juveniles (n = 144 and 138 for freshwater angelfish and tambaqui, respectively) were exposed to concentrations (n = 6 per group) of 10, 25, 50, 100, 200, and 300 μL L^-1 for EOOSE, with an additional 400 μL L^-1 for EOOCL and 400, 600, and 800 μL L^-1 for EOOBM. For freshwater angelfish was also used the 800 μL L^-1 for EOOBM. The EO was diluted 1:10 with ethanol. Two control treatments (to the two experiments) were conducted: one containing only water and another containing water and ethanol at the highest concentration used in the dilution (7,200 and 5,400 μL L^-1 for freshwater angelfish and tambaqui, respectively).

To evaluate anesthetic induction times, two fish were placed in a 1 L aquarium without aeration to optimize the time to execute the experiment and the amount of EO used. After 30 min, the dissolved oxygen level remained above 5.0 mg O₂ L^-1. The animals remained in the aquarium until they were considered anesthetized or after 30 min had elapsed. Animals were considered sedated when they showed a reduced reaction to external stimuli and anesthetized when they presented a total loss of balance and no response to stimuli (Limma-Neto et al., 2016). After this stage, the animals were transferred to 5 L aquariums containing only water without aeration to assess anesthetic recovery. They remained in these conditions until full recovery, with a maximum duration of 8 min for this test. Each animal was transferred to this aquarium as it underwent anesthesia, and the two fish that received the anesthetic dosage simultaneously shared the same recovery aquarium. The animals partially recovered when they regained balance and fully recovered when they regained swimming behavior similar to the control groups (Hikasa et al., 1986). At the end of each evaluation of a set of two fish, the water was changed in both the anesthesia and recovery aquarium.

For the simulated transport experiment (n total = 48 for each fish species), two fish (density of 5.28 and 3.62 g L^-1 for freshwater angelfish and tambaqui, respectively) were placed per plastic bag (50 x 30 cm) containing 1 L of water. The remaining space (2 L) was filled with oxygen. The following treatments were defined: 10 and 20 μL L^-1 of each EO (diluted in ethanol in a proportion of 1:10) and a control group containing only water. Another group (pre-transport) was utilized. In this group, water was collected 1 min after placing the fish in the packaging. The plastic bags were kept for 6 h under these conditions in the Laboratory and handled every 20 min, simulating conventional transport. The treatments were in triplicate.

After this period (6 h), all fish from the same treatment were placed in 20 L aquariums with aeration, and their survival was verified for up to 72 h. Additionally, before and after transportation, water quality parameters (dissolved oxygen, temperature, pH, nitrite, ammonia, hardness, and alkalinity) were measured using a water quality kit (Kit do aquicultor, Alfatecnoquímica, Florianópolis, Brazil).

The results are expressed as the mean ± standard error of the mean. Levene's test verified the homoscedasticity of the variances. Anesthetic induction and recovery data were evaluated using power regression (p < 0.05). Water quality data were analyzed by ANOVA, followed by Tukey's test, and comparisons were made with non-transported fish using Dunnett's test. The minimum significance level was 95% (p < 0.05).

3. Results and Discussion

No mortality occurred during or up to 72 h after the evaluations performed in this study. No sedation or anesthesia was observed in the control groups. There was a regression of sedation and anesthesia in all EO evaluated (Figures 1 and 2); however, no regression was verified for recovery (partial and total) (Table 1).

Figure 1
Time (s) required to reach sedation (A, C, and E) and anesthesia (B, D, and F) in freshwater angelfish (P. scalare) using essential oils from O. sanctum (EOOSE), O. canum (EOOCL), and O. basilicum (EOOBM). (n = 6 fish per concentration).

Figure 2
Time (s) required to reach sedation (A, C, and E) and anesthesia (B, D, and F) in tambaqui (C. macropomum) using essential oils from O. sanctum (EOOSE), O. canum (EOOCL), and O. basilicum (EOOBM). (n = 6 fish per concentration).

Table 1
Time (in seconds) required to reach partial recovery (PR) and total recovery (TR) in freshwater angelfish (P. scalare) and tambaqui (C. macropomum) using essential oils.

Concentration (mL L-1)	EOOSE		EOOCL		EOOBM
	PR	TR	PR	TR	PR	TR
Freshwater angelfish
50	255.66±30.53	340.50±45.93	-	-	-	-
100	210.50±42.40	294.50±40.66	-	-	-	-
200	286.33±21.84	411.50±30.15	92.60±13.06	172.00±11.97	192.25±49.00	280.00±39.80
300	381.00±45.06	433.50±27.15	160.00±38.73	244.50±57.22	175.18±44.38	253.53±56.33
400	-	-	195.16±53.40	280.83±51.76	214.67±74.96	309.83±59.69
600	-	-	-	-	145.50±26.27	318.83±29.93
800	-	-	-	-	190.67±32.71	349.33±41.78
Tambaqui
50	189.00±30.36	348.33±43.53	-	-	-	-
100	217.33±36.98	360.50±28.72	176.00±28.02	292.00±17.68	-	-
200	259.83±32.14	368.33±36.52	340.33±33.69	357.33±23.92	122.00±22.07	312.17±44.03
300	311.67±44.27	438.67±43.20	188.67±16.68	302.67±16.67	207.17±19.81	319.50±51.23
400	-	-	214.83±23.03	301.50±31.48	113.50±13.84	253.33±23.28
600	-	-	-	-	131.50±15.06	312.00±44.06

• Ocimum sanctum chemotype eugenol = EOOSE, Ocimum canum chemotype linalool = EOOCL, and Ocimum basilicum chemotype methyl chavicol = EOOBM). N = 6 fish per concentration (mean ± SEM).

Freshwater angelfish exposed to EOOSE and EOOCL reached only the sedation, respectively, at 10-25 and 10-100 μL L⁻¹. The anesthesia to EOOSE and EOOCL was recorded at concentrations starting from 50 and 200 μL L⁻¹, respectively. The concentration of 200 and 400 μL L⁻¹, respectively, for EOOSE for EOOCL, promoted rapid anesthesia (less than 3 min) and total anesthetic recovery with average times of 165.00 and 411.50 and 121.83 and 280.83 s, respectively. For EOOBM, concentrations between 10 and 100 μL L⁻¹ caused only sedation. The anesthesia for EOOBM started with 200 μL L⁻¹, where the best concentration was 800 μL L⁻¹, with average anesthesia and total recovery times of 153.83 and 349.33 s (Figure 1 and Table 1).

Tambaqui exposed to 10 and 25 μL L⁻¹ for EOOSE had only sedation, with anesthesia starting from 50 μL L⁻¹, where anesthesia and total recovery times for 200 μL L⁻¹ were 165.00 and 411.50 s. Concentrations between 10 and 100 μL L⁻¹ only caused EOOCL and EOOBM sedation. Anesthesia lasting less than 3 min was found with 300 and 400 μL L⁻¹ concentrations for EOOCL and EOOBM, respectively. In these concentrations, anesthesia and total recovery times for EOOCL were 151.67 and 302.67 s, and for EOOBM, were 163.67 and 253.33 s (Figure 2 and Table 1).

For freshwater angelfish, water ammonia levels in the treatments with simulated transport were significantly lower than in the control group (p < 0.05). For tambaqui, except for 10 μL L^-1 of EOOCL, the transported fish showed water ammonia significantly higher than the non-transported fish (p < 0.05). The other water quality parameters did not show statistical differences (Table 2).

Table 2
Water quality parameters after simulated transport (6 h) of freshwater angelfish (P. scalare) and tambaqui (C. macropomum) with or without the addition of the essential oils.

Treatments	Water quality parameters1
	Alk	Hardness	DO	TAM	Nitrite	pH	Temp
Freshwater angelfish
Pre-transport	20.00±0.00	90.00±0.00	7.00±0.50	0.27±0.05b	0.02±0.01	7.00±0.00	26.00±0.00
Control	20.00±0.00	105.00±5.00	7.00±0.00	1.00±0.05a	0.02±0.01	7.00±0.00	26.00±0.00
EOOSE
10 mL L-1	20.00±0.00	105.00±5.00	7.50±0.50	0.40±0.05b	0.02±0.01	6.50±0.50	26.00±0.00
20 mL L-1	20.00±0.00	105.00±5.00	7.50±0.50	0.20±0.05b	0.03±0.01	7.00±0.00	26.00±0.00
EOOCL
10 mL L-1	20.00±0.00	110.00±0.00	7.00±0.00	0.30±0.05b	0.03±0.01	7.00±0.00	26.00±0.00
20 mL L-1	20.00±0.00	90.00±10.00	7.00±0.00	0.20±0.05b	0.04±0.01	7.00±0.00	26.00±0.00
EOOBM
10 mL L-1	20.00±0.00	100.00±10.00	7.50±0.50	0.40±0.05b	0.02±0.01	6.50±0.50	26.00±0.00
20 mL L-1	20.00±0.00	100.00±10.00	7.00±0.00	0.40±0.05b	0.04±0.01	7.00±0.00	26.00±0.00
Tambaqui
Pre-transport	30.00±0.00	90.00±0.00	6.00±0.50	0.15±0.05b	0.01±0.01	7.00±0.00	26.50±0.00
Control	30.00±0.00	100.00±5.00	6.00±0.50	1.00±0.05a	0.02±0.01	7.00±0.00	26.50±0.00
EOOSE
10 mL L-1	30.00±0.00	90.00±0.00	6.50±0.50	0.75±0.05a	0.02±0.01	7.00±0.00	26.50±0.00
20 mL L-1	30.00±0.00	100.00±5.00	6.50±0.50	0.85±0.05a	0.02±0.01	7.00±0.00	26.50±0.00
EOOCL
10 mL L-1	30.00±0.00	90.00±0.00	6.50±0.50	0.50±0.05ab	0.01±0.01	7.00±0.00	26.50±0.00
20 mL L-1	30.00±0.00	100.00±5.00	6.50±0.50	0.75±0.05a	0.02±0.01	7.50±0.50	26.50±0.00
EOOBM
10 mL L-1	30.00±0.00	100.00±5.00	6.50±0.50	0.75±0.05a	0.02±0.01	7.00±0.00	26.50±0.00
20 mL L-1	30.00±0.00	100.00±5.00	6.50±0.50	0.75±0.05a	0.02±0.01	7.00±0.00	26.50±0.00

• Ocimum sanctum chemotype eugenol = EOOSE, Ocimum canum chemotype linalool = EOOCL, and Ocimum basilicum chemotype methyl chavicol = EOOBM). ¹ Alkalinity (Alk) and hardness are in mg L^-1 CaCO₃. Dissolved oxygen (DO) is in mg O₂ L^-1. Total ammonia (TAM) is in mg N-NH₃ L^-1. Nitrite is in mg N-NO₂ L^-1. Temperature (Temp) is in °C. n = 3 plastic bags per treatment. Different letters indicate significant differences (p < 0.05) between the treatments (Tukey test) and the pre-transport (Dunnet test). N = 6 fish per concentration (mean ± SEM).

3. Discussion

In general, EO that act as fish anesthetics depend on their main chemical constituents to act by inhibiting neurotransmitter receptors, such as gamma-aminobutyric acid (GABA), or blocking other steps of action potential transmission in neurons (e.g., voltage-gated sodium channels) (Copatti et al., 2024). This finding has already been verified using EO from Ocimum gratissimum, whose primary compound was eugenol (73.6%), which must have contributed to inhibiting GABA receptors in silver catfish (Silva et al., 2012). In the current study, eugenol was the main compound of EOOSE. However, the anesthetic does not always affect the GABAergic system, as Heldwein et al. (2014) reported for linalool in silver catfish anesthesia. In the present study, linalool was the main compound of EOOCL. In addition, we found no reports on the action of methyl chavicol (EOOBM) on the GABAergic system in fish. This demonstrates that the mechanisms of action of several EOs still need to be further investigated. Therefore, studies that evaluate the anesthetic potential of these compounds should contribute to directing research that can deepen how the central nervous system of fish is affected by EO.

To be considered a good anesthetic, the compound must provide a state of anesthesia between 1-3 min. For full anesthetic recovery, it is recommended not to exceed 10 min (Parodi et al., 2014; Copatti et al., 2024). In the present study, all three EO tested in our research promoted anesthesia in these recommended times, but at different concentrations, which were, respectively, 200, 400, and 800 µL L^-1 of EOOSE, EOOCL, and EOOBM for freshwater angelfish and 200, 300, and 400 µL L^-1 of EOOSE, EOOCL, and EOOBM for tambaqui.

In the current study, the evaluation of the anesthesia stage showed that EOOSE had a better anesthetic potential; that is, it triggered anesthesia in both fish species with a lower concentration than the other EO. Following this same criterion, the least potent anesthetic was EOOBM. In addition, as described above, although anesthesia with EOOSE was achieved with the same concentration for both fish species, for EOOCL and EOOBM, tambaqui juveniles were anesthetized at lower concentrations than freshwater angelfish juveniles. Khumpirapang et al. (2018) evaluated EO obtained from the same plants of this study for anesthesia of Koi carp (Cyprinus carpio) but with different chemical compositions. In the study of these authors, the concentrations indicated for EOOS (eugenol 48.60%), EOOC (citral 78.05%), and EOOB (methyl chavicol 78.12%) were, respectively, 100, 200, and 300 mg L^-1.

Similarly, other studies reported fish anesthesia caused by the same compounds highlighted in our research. The linalool showed anesthetic potential in tambaqui and silver catfish at concentrations of 50 (Boaventura et al., 2022) and 180 μL L^-1 (Heldwein et al., 2014), respectively. Linalool was the primary compound of EO from Lippia alba, which had sedative and anesthetic action for tambacu at concentrations of 30 and 200 μL L^-1, respectively (Sena et al., 2016). The EO from O. canum (1,8-cineole 21.00% and b-linalool 20.18%) was efficient such as sedative and anesthetic in silver catfish when added in the water, respectively, at 25 e 200-500 mg L^-1 (Silva et al., 2015). In freshwater angelfish, eugenol has shown sedation and anesthesia with 15 and 50 mg L^-1 (De Oliveira et al., 2019). For pirapitinga (Piaractus brachypomus), eugenol was indicated for anesthesia and reduction of ventilatory frequency in concentrations between 50 and 100 mg L^-1 (Ferreira et al., 2021). A concentration of 75 mg L^-1 was found by Vidal et al. (2008) for anesthesia of Nile tilapia. For tambaqui, the EO from O. basilicum (methyl chavicol 66.51% and linalool 20.90%) caused sedation at low concentrations (25 μL L^-1), and the opposite was verified for anesthesia, where the indicated concentration was 1000 μL L^-1 (Ventura et al., 2021). This same compound was effective in anesthetizing pacu (P. mesopotamicus) with 300 μL L^-1, being rapidly eliminated from the tissues (Ventura et al., 2024). Similarly, in rainbow trout, the EO from O. basilicum (methyl chavicol 72.57% and linalool 21.60%) was indicated for anesthesia when used in a concentration of 300 mg L^-1 (Yigit et al., 2022). The EO from Ocimum micranthum (methyl chavicol 58.20% and linalool 29.80%) can sedate grass carp (Ctenopharyngodon idella) and silver catfish with 25 μL L^-1, while to anesthetize these species is required 100 and 200 μL L^-1, respectively (Zeppenfeld et al., 2019).

In the present study, the concentration necessary to reach the sedation stage was similar when comparing the EO and fish species, and the use of 10 or 20 μL L^-1 can be assumed. Based on this finding, the simulated transport experiment (6 h) was performed in the current study, carried out under laboratory conditions using a low stocking density. Commonly, fish farms use high stocking densities for fish transportation, and it is suggested that future experiments be performed based on this condition. In addition, closed-system transport can increase water ammonia levels (Sena et al., 2016) and reduce the water dissolved oxygen levels (Silva et al., 2012).

The use of EO during transportation can alter the values of water quality parameters, causing a decrease in ammonia excretion and use of dissolved oxygen (Copatti et al., 2024); however, in the present study, only total ammonia was influenced by the treatments tested. For freshwater angelfish, the use of EO was able to reduce the total ammonia in the water. On the other hand, for tambaqui, this was not verified, where only the 10 mL EOOCL L^-1 treatment did not differ from the pre-transport group concerning the level of total ammonia in the water. This finding, at least for freshwater angelfish, is essential, as this nitrogenous compound is toxic to fish. Fish excrete a large part of the ammonia through the gills; however, when there is an accumulation of plasma ammonia and toxic levels are reached, electrolyte imbalance occurs, which can cause the death of the animals (Ip & Chew, 2010).

The use of sedative substances does not always interfere with water quality parameters. Studies evaluating the transport of freshwater angelfish with EO from Cymbopogon citratus and Lippia sidoides (De Oliveira et al., 2022; Castro Neto et al., 2024) and of tambacu with L. alba (Sena et al., 2016) show no differences in water total ammonia levels. However, transportation using eugenol in freshwater angelfish (De Oliveira et al., 2019) and L. alba (citral 64.66%) in tambaqui (Da Silva et al., 2018) resulted in a reduction in this parameter.

4. Conclusions

All EO tested in this study were effective in causing anesthesia in fish, whereas EOOSE was the most effective in causing rapid anesthesia using a smaller amount of compound. For transport (6 h), it is recommended to use up to 20 mL L^-1 of any of these EOs, which promote sedation without causing mortality and, for freshwater angelfish, have also shown effectiveness in reducing total ammonia levels in the water.

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Edited by

• Editor:
  Luiz Vitor Oliveira Vidal

Publication Dates

• Publication in this collection
  07 Apr 2025
• Date of issue
  2025

History

• Received
  29 Nov 2024
• Accepted
  11 Feb 2025