Introduction

The giant river prawn (Macrobrachium rosenbergii, De Man 1879) is a tropical species highly valued for freshwater aquaculture. Characteristics such as rusticity and resistance to disease, consistent reproductive performance, and high fecundity under captivity make this species particularly relevant for global prawn farming (Cavallo et al. 2001; Pillai et al. 2022). In 2018, the global production of M. rosenbergii was 234.4 thousand tonnes (FAO 2020). Since 2007, several countries including Brazil have been recovering from negative economic impacts on prawn production and are now expanding their farming of M. rosenbergii (David et al. 2018). The progress demands continue high-quality scientific studies to support a sustainable and profitable growing industry. This includes the development of sedatives and anaesthetics to be used during prawn transportation, handling, biometric procedures, and sampling.

In recent years, the Intergovernmental Panel on Climate Change has drawn attention that different anthropogenic activities have contributed to an increase in global temperature, an escalation in the atmospheric concentration of carbonic acid, and a reduction in the pH in both terrestrial and aquatic environments (Collins et al. 2013; IPCC 2014; Abram et al. 2019). With the evidence of water acidification, it is necessary to better comprehend whether different water pHs would influence prawn production and modulate the effect of anaesthetic agents.

Different anaesthetics are used in decapods aquaculture. Many of them, however, may cause adverse effects on both animals and handlers. Studies have proposed natural alternatives to synthetic anaesthetics, for instance, menthol (Li et al. 2018a), eugenol (Jiang et al. 2020), and the essential oils of Lippia alba and Ocimum gratissimum (Becker et al. 2021). Due to their essential oils, aromatic plants are gaining popularity in aquaculture. Essential oils are natural compounds formed by a complex blend of low molecular weight, fat-soluble, and volatile substances; they are secondary metabolites extracted from the secretory glands of herbs and parts of a plant (Ingraham 2018).

The essential oil extracted from the aromatic herb Ocimum gratissimum L., Lamiaceae family (common names: clove basil, African basil, wild basil), has been used in several routine procedures in aquaculture, for example, as antimicrobial and antiparasitic (Bandeira Jr et al. 2017), sedative and anaesthetic (Souza et al. 2019; Boaventura et al. 2020, 2021; Silva et al. 2020; Becker et al. 2021; Ferreira et al. 2021), and anthelmintic (Meneses et al. 2018; Silva et al. 2020). Original from tropical Africa, India, and Southeast Asia, O. gratissimum currently has a pantropical and subtropical distribution across the world (Rojas-Sandoval 2018). The therapeutic properties of O. gratissimum essential oil are attributed to its main compounds (eugenol and 𝛽-caryophyllene). The common Brazilian chemotypes contain the major compounds eugenol and 1,8-cineole (Silva et al. 2012; Ribeiro et al. 2016; Silva et al. 2020). As O. gratissimum is currently consumed as spice and tea by humans (Di Stasi et al. 2002), O. gratissimum is expected to be accepted and approved to be used as an anaesthetic agent for fish and crustaceans used for human consumption.

Thus, our study aimed at evaluating the anaesthetic effect of O. gratissimum essential oil on juvenile giant river prawn (M. rosenbergii) exposed to different water pHs. That is, comparing the anaesthetic effect of the EO using the ideal water pH for M. rosenbergii juveniles’ production (i.e. water pH 7.0–8.5, FAO 2003) and mild acid water simulating water acidification (e.g. water pH 6.02). To the best of our knowledge, this is the first study reporting the potential use of O. gratissimum essential oil in freshwater prawns.

Materials and methods

Plant origin, essential oil extraction, and characterization

Leaves of O. gratissimum were collected from the botanical collection of the Federal University of Santa Maria (UFSM) campus Frederico Westphalen, Rio Grande do Sul, Brazil. The specimen was identified by Adelino Alvares Filho and deposited in the Biology Department Herbarium, Federal University of Santa Maria, Brazil, register number SMDB 11167. The essential oil (EO) was extracted from the leafy material by hydro-distillation using a Clevenger-type apparatus for 2 h (European Pharmacopeia 2007). In order to separate the EO from the hydrolate, both were collected and transported to the UFSM Campus Santa Maria, where the mixture was transferred to a separating funnel, being subjected to liquid-liquid partition with hexane (analytical degree). The hexane fraction was concentrated in a rotary evaporator (40 °C), obtaining the pure EO. The essential oil of Ocimum gratissimum (EO-OG) was then stored at -4° C, in an amber glass bottle, until further analysis and use. The EO-OG composition was analysed by gas chromatography-mass spectroscopy (GC-MS)–total ion chromatogram (TIC), performed with a gas chromatograph (Agilent-7890A) coupled with a mass selective detector (Agilent-5975C), using HP5-MS column and EI-MS of 70eV. Conditions were as described by Bandeira Jr et al. (2017), i.e. split inlet 1:100, program temperature 40–320 °C at 4 °C min−1, carrier gas He, flow rate of 1 mL min−1, injector (2 μL), and detector temperature 250 °C. Kováts retention index and mass spectral library were used for comparative identification of the EO-OG chemical composition (NIST 2010). Table 1 shows the major constituents of the EO-OG.

Table 1 Chemical composition of Ocimum gratissimum leaves essential oil
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Experimental design

Juvenile Macrobrachium rosenbergii (weight 0.40 ± 0.03 g; length: 2.43 ± 0.08 cm), regardless of sex, were obtained from the Shrimp Culture Laboratory, Federal University of Paraná, Palotina, Paraná, Brazil. Animals were initially kept for 30 days in a 300-L tank, nursery system, with constant aeration and continuous water flow, and fed with a commercial shrimp diet (Guabitech® Inicial PL, 40% crude protein) twice a day at 08h30 and 16h30 at equal rations. Water parameters were monitored twice a day and kept under optimal conditions for M. rosenbergii farming throughout the trial. The average ± SD observed during the trial were water temperature (20.30 ± 0.82 °C), dissolved oxygen (7.82 ± 1.34 mg L−1), total ammonia nitrogen (0.045 ± 0.035 mg N L−1), nitrite (0.028 ± 0.038 mg L−1), nitrate (4.426 ± 4.339 mg L−1), alkalinity (85.0 ± 7.29 mg CaCO3 L−1), and hardness (83.8 ± 23.2 mg CaCO3 L−1). Animals in the moult stage were not used and no moulting was observed during or after the exposure time to EO-OG. Length measurements occurred once a day with 25 postlarvae (PL) per tank. Mortality was monitored twice a day. Natural photoperiod was set at 06h/18h, light/dark cycle.

After 30 days, prawns were fasted for 24 h before the beginning of the anaesthetic trial. Subsequently, 144 prawns were randomly collected from the nursery system and distributed into glass recipients with 300 mL freshwater content, divided into 24 (3 prawns per unit, per water pH) experimental units. Prawns were then exposed to the following concentrations of EO-OG, previously diluted in ethanol analytical degree (1:10, v/v): 0 (negative control with freshwater only), 100, 200, 300, and 400 μL L−1. A sixth group of animals was exposed to the concentration of ethanol used to dilute the highest concentration of EO-OG, which corresponded to 3600 μL L−1 of ethanol analytical degree (vehicle control). In each of the experimental groups, performed in quadruplicate, three animals were used simultaneously per aquarium, totalling 12 juveniles of M. rosenbergii per concentration of EO-OG divided into four experimental units. Each shrimp was used only once. Additionally, each EO-OG concentration was tested at two different water pHs: alkaline (8.08 ± 0.10) and acidic (6.42 ± 0.15). Figure 1 illustrates the experimental design. The water pH was adjusted by adding sodium bicarbonate (NaHCO3) or hydrochloric acid (1M HCl), respectively.

Fig. 1
figure 1

Schematic experimental design of juvenile M. rosenbergii exposed to different concentrations of EO-OG in A alkaline water and B acidic water

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Sedation and anaesthesia induction and recovery

Sedation and anaesthesia induction and recovery times were evaluated as described by Coyle et al. (2005). The sedation state was characterized as a partial loss of balance and the presence of a positive reaction to touch stimulus. The anaesthesia state was attributed to the complete loss of balance and lack of reaction to touch stimulus. Finally, the recovery stage was determined when the shrimp regained balance and reached the normal, upright position at the bottom of the tank. The maximum observation time of the induction stage was 30 min. A digital stopwatch was used to record the times, expressed in seconds. After a maximum of 30 min, animals were transferred to a recovery aquarium with anaesthetic-free water. Animal survival was observed immediately after reaching the recovery time, and again after 24 h.

Statistical analysis

All data are expressed as mean ± standard error of the mean (S.E.M.). The normality and homoscedasticity of variances were confirmed with Shapiro-Wilk and Levene’s tests, respectively. Comparison of anaesthesia induction and recovery times and different EO-OG concentrations was performed using Nested-ANOVA, and comparison among different water pH was performed using two-way ANOVA, followed by Tukey post hoc test. When necessary, logarithmic transformation was used to make the data satisfy the ANOVA assumptions. Additionally, the evaluation of the anaesthetic activity was performed through exponential regression analysis (concentration × time of induction to anaesthesia). Statistical analyses were performed using Minitab 17, Sigma Plot 14.0, and Statistica 7.1 software, with a significance level of 95% (p < .05).

Results

The EO-OG induced sedation and anaesthesia in juvenile M. rosenbergii at all tested concentrations. The increase in the EO-OG concentration proportionally decreased the time required for sedation and anaesthesia, regardless of the water pH; that is, induction times for sedation and anaesthesia with EO-OG were concentration dependent. The significantly highest recovery time was observed in prawns anaesthetised with the highest EO-OG concentration (400μL L−1) when in alkaline water, and 400μL L−1 and 200 μL L−1 when prawns were kept in acidic water (Table 2). Thus, the highest tested concentration of EO-OG (400 μL L−1) resulted in the significantly fastest sedation and anaesthetic effect (2.0–2.5 min and 3.5–4.1 min, respectively). Alkaline water accelerated the EO-OG anaesthesia induction effect, with a recovery time of 16 min, in both tested water pHs. Anaesthesia recovery time did not present a direct relation with the EO-OG concentrations used, particularly in acidic water (Table 2).

Table 2 Time (seconds) to sedation, anaesthesia induction, and anaesthesia recovery in Macrobrachium rosenbergii juveniles exposed to different concentrations of Ocimum gratissimum essential oil and two different water pHs
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There was a significant difference in the anaesthetic effect of EO-OG in the three analysed parameters (i.e. times of sedation, anaesthesia induction, and anaesthesia recovery; p < .05). Our results also showed a significant interaction effect between anaesthetic concentration and water pHs (interaction effect p < .05), suggesting an influence of both factors on the anaesthetic effect of EO-OG (Table 3). Sedation induction time was significantly higher on M. rosenbergii kept in acidic water when exposed to all EO-OG tested concentrations (p < .001), except 300 μL L−1. However, anaesthesia induction and recovery times in mild alkaline and acidic water were not significantly different. Ethanol analytical degree per se (vehicle control) did not present any sedative or anaesthetic effect in the juvenile M. rosenbergii; hence, it is conceivable not to have any synergic effect with EO-OG. Prawns in the negative control group did not show any symptom related to sedation or anaesthesia. No mortality occurred from anaesthesia induction or 24 h after exposure to EO-OG in any of the tested concentrations or different water pHs studied.

Table 3 Interaction between anaesthetic concentration of Ocimum gratissimum essential oil and water pH on the sedation and anaesthesia times, and anaesthesia recovery in Macrobrachium rosenbergii juveniles
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Discussion

Our study proposes the use of the EO-OG as an effective natural anaesthetic for the prawn M. rosenbergii. The concentration of 400 μL L−1 proved to be the most appropriate to be used in routine procedures with juveniles of M. rosenbergii. This concentration showed the ideal time for sedation, anaesthesia induction, and recovery for short-duration procedures on shrimp farms, i.e. less than 5 min to induce deep anaesthesia and close to 15 min for full anaesthesia recovery (Keene et al. 1998; Tsantilas et al. 2006; Park et al. 2009). Practices and procedures such as weighting, sampling, transportation, and welfare checks can benefit from sedation and analgesia. Handling, air exposure, transportation, and disturbance cause distress in prawns and impact animal health and performance (Fotedar and Evans 2011; Kamaruding and Abdullah 2021). Therefore, the use of anaesthetics contributes to improve animal welfare and, at the same time, facilitates routine procedures (Saydmohammed and Pal 2009).

Our results demonstrated that the induction times of sedation and anaesthesia were concentration dependent, decreasing with the increase of EO-OG concentration, while the anaesthesia recovery time was independent of the previously applied anaesthetic concentration. Parodi et al. (2012) observed a similar pattern when using the essential oils of Lippia alba and Aloysia triphylla to anaesthetise the Pacific white shrimp (P. vannamei). Becker et al. (2021) also reported a similar result when anaesthetising the São Paulo shrimp (Penaeus paulensis) and P. vannamei with the essential oils of L. alba and O. gratissimum. As such, it is recommended to use the proper effective anaesthetic concentration to control sedation and anaesthesia induction time and depth.

Eugenol is the main component of EO-OG (Bandeira Jr et al. 2017). This compound is one of the most used anaesthetics for decapod crustaceans and has been reported to satisfactory induce anaesthesia in shrimps and prawns, particularly for immersion anaesthesia (de Souza Valente 2022). Eugenol blocks sodium, potassium, and calcium channels, hence decreasing the proprioception (Cowing et al. 2015). Parodi et al. (2012) reported fast, deep anaesthesia of P. vannamei postlarvae in 4 min, using 175 μL L−1 of eugenol, while P. vannamei subadults are anaesthetized in 3 min with 400 μL L−1 of eugenol. Similarly, juveniles of green tiger prawn (Penaeus semisulcatus) can be anaesthetized with 100 and 200 μL L−1 of clove oil (80% eugenol) within 5 and 2 min, respectively (Soltani et al. 2004). Anaesthesia of small grass shrimp (Penaeus sinensis) in less than 10 min can be obtained with 100–200 μL L−1 eugenol, but bigger grass shrimps need higher concentrations (300–500 μL L−1) and mortality occurs at 20–28 °C (Li et al. 2018b). Postlarvae of M. rosenbergii anaesthetized at 28 °C with 60 μL L−1 clove oil in 9.5 min and recovered in 14.5 min, but juveniles needed a much higher concentration (750 μL L−1) to anaesthetize in 10.1 min and all animals died (Vartak and Singh 2006). Saydmohammed and Pal (2009) observed that even with 800 μL L−1 eugenol, M. rosenbergii took 21 min to anaesthetize at 30 °C, while Coyle et al. (2005) noted that at 24 °C, 45 min of exposure to 300 μL L−1 eugenol was needed to anaesthetize this species. Despite the high concentrations of eugenol and long induction time reported to be necessary to anaesthetize M. rosenbergii in these cited studies, in the present research, we found that EO-OG was effective to anaesthetize M. rosenbergii in a shorter time and using a lower concentration. This dissimilarity may be attributable to the synergic effects among the different compounds of EO-OG. On the other hand, ethanol did not lead to any sedative or anaesthetic effect thus acting only as an organic solvent without any isolated or synergic effect with EO-OG. The EO-OG was also more efficient to induce anaesthesia than eugenol in P. paulensis (Becker et al. 2021) and silver catfish, Rhamdia quelen (Silva et al. 2012), but not in other fish species, due to the different composition (1,8-cineole was the main compound) (Silva et al. 2020) or species-specific responses (Boaventura et al. 2020; Ferreira et al. 2021).

The major constituents of the EO-OG used in our study were eugenol and β-caryophyllene, followed by the minor constituents copaene, E-β-ocimene, and germacrene D. Despite eugenol being the major anaesthetic component of the studied EO-OG, the other constituents are worthy of a note. The β-caryophyllene is a sesquiterpene plant volatile reported as a potent natural analgesic for mammals (Fidyt et al. 2016). The proportion of β-caryophyllene found in the essential oil of Aloysia triphylla presents a significant relationship with the time to induce anaesthesia in silver catfish (Parodi et al. 2020). Parodi et al. (2020) observed that the concentration of β-caryophyllene shows a negative relation with the anaesthetic induction; i.e. the increase of β-caryophyllene concentration on the essential oil leads to a faster induction of deep anaesthesia. Notably, the lower β-caryophyllene concentrations found by these authors (5.4 to 12.7% in 2009 and 2.4 to 53.6% in 2010) are comparable to that one identified on the present study (i.e. 5.3%). This compound is also found in other essential oils with anaesthetic effect as Lippia alba in tambaqui (dos Santos Batista et al. 2018) and shrimp (Parodi et al. 2012; Becker et al. 2021), and Syzygium aromaticum on crayfish (in a commercial formulation of clove oil; Ghanawi et al. 2019). It also occurs in essential oils such as Humulus lupulus, Ocimum campechianum, and Origanum vulgare (Tsuchiya 2017). Copaene is a sesquiterpene that can be found in essential oils like Cinnamomum zeylanicum (Plata-Rueda et al. 2018), Decaspermum parviflorum (Khanh et al. 2020), and Vernonia patula (Hoi et al. 2021). Likewise, ocimene is a plant volatile from the terpenoid class and is among the main constituents (> 1%) of several additional essential oils described with anaesthetic effects for aquatic animals and others, such as Piper divaricatum on tambaqui (Colossoma macropomum, Vilhena et al. 2019), Nectandra grandiflora on Nile tilapia (Oreochromis niloticus, Rodrigues et al. 2021), A. triphylla on Nile tilapia (Oreochromis niloticus, Teixeira et al. 2017), and Lavandula angustifolia on chronic pain mice model (Donatello et al. 2020). In like manner, germacrene D is a sesquiterpene hydrocarbon and one of the constituents of essential oils used to anaesthetize aquatic animals, including Nectandra megapotamica on fat snook (Centropomus parallelus, Tondolo et al. 2013) and Hyptis mutabilis on silver catfish (Rhamdia quelen, Silva et al. 2013).

High-quality water parameters are essential for the proper development of aquatic organisms. Parameters such as water pH, hardness, and temperature interfere with the anaesthetic potential of different agents (Gomes et al. 2011). For instance, water temperature and salinity influence the anaesthesia induction and recovery times of the green tiger prawn (Penaeus semisulcatus) exposed to clove oil (Soltani et al. 2004). Thereby, water parameters seem to interact and influence the pharmacokinetic properties of the anaesthetic agents. In the present study, water quality parameters, except pH, were similar between the experimental groups (i.e. no significant difference) thus not influencing the sedative and anaesthetic effect of EO-OG on M. rosenbergii juvenile.

Within the context of the water bodies’ acidification, we investigated whether different water pHs could affect the anaesthesia and recovery of M. rosenbergii exposed to EO-OG. Our results suggest that there is an interaction between EO-OG anaesthetic concentration and water pH, with an influence of water pH on M. rosenbergii sedation time, leading to a significantly longer sedation time in mild acidic water. While water pH did not influence anaesthesia induction and anaesthesia recovery times under the studied conditions; indeed, acidic water has been previously suggested to impact eugenol efficacy as an anaesthetic, inducing higher sedation time in silver catfish kept in acidic water (Gomes et al. 2011). The ideal range of water pH for shrimp and prawns is 7.5 to 8.5, with values out of the range of 6.0 to 9.5 being highly detrimental (Boyd et al. 2014). Mild acidic water (e.g. pH 6.4–6.8) can negatively impact M. rosenbergii survival, growth, moulting frequency, and feeding rate, for both larvae and juvenile stages (Chen and Chen 2003; Liew et al. 2022). Taken together with our results, one recommends the constant monitoring of water pH including when anaesthetising M. rosenbergii, preferring the use of neutral to mild alkaline water. Attention should be paid to situations with a potential reduction of water pH, such as during animal transportation, particularly when associated with high densities (Hong et al. 2019).

In summary, EO-OG presents sedative and anaesthetic effects on the giant river prawn, M. rosenbergii, in a concentration-depended effect. The recommended concentration by our study is 400 μL L−1 of EO-OG in mild alkaline water. This was the effective anaesthetic concentration able to induce smooth sedation, deep anaesthesia, and full recovery in adequate time for farming procedures. The EO-OG sedation effect was influenced by water pH, while anaesthesia induction and recovery times were not modulated by water pH. EO-OG at the effective concentration is suggested to be used during routine procedures, such as animal handling, transportation, biometrics, and sampling.