Efficiency of clove oil as an anesthetic in blue swimming crab Portunus segnis

ABSTRACT

Objective

Anesthetics are essential for reducing stress, facilitating handling, and preventing injury in aquatic animals. They are extensively employed in tagging and blood and hemolymph sampling. However, synthetic anesthetics can adversely affect the physiology and safety of living organisms. This study aimed to evaluate the efficacy of clove oil as a natural anesthetic in reducing handling stress in blue swimming crabs Portunus segnis by evaluating the effects of different concentrations on anesthetic induction and recovery times.

Methods

We compared three doses of clove oil (150, 300, and 450 μL/L) to assess anesthetic induction and recovery times of the crabs. Samples were collected from the northern Persian Gulf coastline (Hormozgan, Iran). A total of 45 crabs (weight [mean ± SD] = 51.24 ± 3.08 g; carapace width = 8.61 ± 0.17 cm) were exposed to the selected concentrations. After the anesthetic bath, anesthesia times (stages A1, A2, and A3) and recovery times (stages R1, R2, and R3) were individually recorded.

Results

Required optimal times for all anesthesia stages were approximately 283 ± 13, 423 ± 22, and 856 ± 61 s, whereas the times for recovery stages were 296 ± 8, 455 ± 16, and 753 ± 27 s for the three doses. Deep anesthesia was observed in all treatments, and no mortality was recorded during the experiment or the 48-h observation period. Data analyses indicated a significant difference between treated crabs’ induction and recovery times at the selected doses. The induction and recovery times were positively correlated with the weight of crabs.

Conclusions

The findings of this study demonstrate that clove oil at a concentration of 300 μL/L is an effective anesthetic for blue swimming crabs, providing a practical solution for minimizing handling stress in aquaculture. The natural origin and safety profile of clove oil make it a suitable alternative to synthetic anesthesia. Future research should explore the long-term effects on crab health and meat quality to further refine the use of clove oil in commercial settings.

INTRODUCTION

In aquaculture, managing stress during the handling and transportation of aquatic animals is crucial due to its ­significant impact on survival rates and meat quality. Stress in aquatic ­species can lead to negative outcomes, such as reduced growth, compromised immune function, and increased mortality (Akbary et al., 2016). Therefore, minimizing stress is essential not only for animal welfare but also for the economic efficiency of aquaculture operations. Additionally, the growing demand for high-quality seafood in international ­markets has ­further increased the live shipping of various species (Bondad-Reantaso et al., 2012; Willis et al., 2024).

One effective method to reduce handling stress is through anesthetic agents that temporarily immobilize animals. This facilitates easier handling while reducing the risk of injury (Taylor & Roberts, 1999). Anesthetics are widely utilized for tagging purposes, artificial reproduction practices, blood sampling procedures, and pathological analyses (Archibald et al., 2019; Cook et al., 2018; Purbosari et al., 2019; Soulsbury et al., 2020).

However, synthetic anesthetics may alter the physiological condition of aquatic animals, affecting muscle quality by causing lactic acid accumulation during transport (Jia et al., 2022). They often have harmful side effects on the nervous system of crustaceans compared to natural alternatives (da Cunha et al., 2010; Tang et al., 2012; Wycoff et al., 2018). For instance, tricaine methanesulfonate (MS-222) is a commonly used anesthetic in aquatic species due to its rapid induction and recovery times, ease of administration, and low concentrations (Archibald et al., 2019; Wagner et al., 2003). Nonetheless, it may induce adverse physiological consequences in crustaceans and can be restricted in some countries (Topic Popovic et al., 2012). Additionally, 2-phenoxyethanol is a low-toxicity anesthetic that is economical for short-term use in aquatic species (Barata et al., 2016), but it may irritate both animals and handlers. Eugenol and isoeugenol, the active components of clove oil, are considered natural anesthetics for aquatic species (Vatanparast et al., 2017) and have minimal effects on handlers and/or test animals at recommended dosages (Waterstrat & Pinkham, 2005; Zahl et al., 2012). Although carbon dioxide (CO₂) is an affordable option with low toxicity across some aquatic species, its inconsistent results limit its use among crustaceans (Parodi et al., 2012), while ionic or electrical anesthesia poses challenges regarding cost, implementation difficulties, and long-term health effects in large-scale aquaculture operations (Rotllant et al., 2023).

Recent studies have explored natural products like clove oil as alternatives due to their effectiveness and favorable safety profile (Fujimoto et al., 2018; Ghanawi et al., 2019; Premarathna et al., 2016). Clove oil is derived from the dried flower buds of Eugenia caryophyllata, which contain eugenol, recognized for its anesthetic properties (Bilbao et al., 2010; Carneiro et al., 2019). Its dark brown color and strong aromatic odor characterize it well within this context (Ross & Ross, 2009), aligning with growing demands for sustainable practices due to its natural origin and cost-effectiveness, which make it suitable for small- or large-scale operations.

Existing literature has demonstrated promising results regarding clove oil’s efficacy as an anesthetic across various crustacean species. Premarathna et al. (2016) demonstrated that clove oil allowed for a stress-free induction in blood-spotted crabs Portunus sanguinolentus at a concentration of 0.25 mL/L of seawater while effectively maintaining anesthesia over time. Jiang et al. (2020) found that clove oil concentrations ranging from 60 to 210 mg/L successfully induced rapid and deep anesthesia in giant tiger prawns Penaeus monodon, highlighting its efficiency as an anesthetic agent. Similarly, Li et al. (2018) noted effective clove oil doses of around 200 μL/L when testing grass shrimp Palaemonetes sinensis. Seol et al. (2007) reported linear decreases in induction timing correlated with increased concentrations of clove oil applied to Korean octopus Octopus minor, with a notable efficacy observed at a concentration of 200 mg/L. Soltani et al. (2004) highlighted shorter sedation times when using higher doses for green tiger prawns Penaeus semisulcatus. Ghanawi et al. (2019) found rapid induction and recovery times in Australian redclaw crayfish Cherax quadricarinatus at clove oil concentrations of 375 and 500 μL/L, with 500 μL/L proving more effective. In contrast, Zhu et al. (2023) found that clove oil achieved a postanesthesia survival rate of 100% in green mud crabs Scylla paramamosain. Furthermore, Elmas and Karadal (2022) highlighted significant reductions in induction times at clove oil concentrations of 1,000 μL/L compared to lower concentrations (200 and 350 μL/L) when applied to red swamp crayfish Procambarus clarkii under controlled temperatures. Similarly, Jensen et al. (2013) reported that clove oil induced rapid anesthesia in green rock lobsters Sagmariasus verreauxi, achieving effective immobilization at concentrations as low as 0.032%.

The blue swimming crab Portunus segnis is a species of significant commercial value, particularly within tropical waters of the Indo-Pacific Ocean (Gheshlaghi et al., 2022; Lai et al., 2010; Prangnell & Fotedar, 2006). This species is known for its high-quality meat and its demand in both domestic and international markets (Sudtongkong, 2006). However, handling these highly active crabs poses substantial challenges since they are susceptible to stress during transport, leading to injuries or increased mortality rates, which ultimately exert negative impacts on meat quality.

Despite the promising findings on the effectiveness of clove oil as an anesthetic across various crustacean species, its application in blue swimming crabs remains understudied. Given the commercial significance of this species and the importance of optimizing handling practices to minimize stress during aquaculture operations, further investigation is required. This study aims to address the research gap by evaluating the effects of various clove oil concentrations on induction and recovery times in blue swimming crabs. Specifically, we identify effective concentrations of clove oil for anesthesia in blue swimming crabs while ensuring rapid recovery, thereby contributing to improved handling practices in aquaculture. Additionally, we explore how clove oil compares to synthetic anesthetics regarding safety and we highlight future research directions concerning the long-term effects on crab health and meat quality.

METHODS

The primary objective of this experiment was to determine the efficiency of clove oil as a stress-reducing agent in blue swimming crabs by evaluating its effects on anesthesia induction and recovery times as well as behavioral responses during handling. The methodology involved different concentrations of clove oil, thereby allowing for the optimization of anesthesia and recovery times.

Study area

The Persian Gulf, situated in Western Asia between Iran and the Arabian Peninsula, is characterized by a semi-enclosed sea with shallow depths. The sampling location of this study was on the northern coast of the Persian Gulf in Bandar Abbas, situated within the Hormozgan province along the Iranian coastline of the Persian Gulf (Figure 1).

Experimental setup

The experiment was conducted in February 2023 at the University of Hormozgan fisheries laboratory in Iran. Fifty healthy specimens of live male blue swimming crabs with intact appendages and at similar stages of development (weight [mean ± SD] = 51.24 ± 3.08 g; carapace width = 8.61 ± 0.17 cm) were randomly collected from intertidal, fixed stake-net traps along a beach (study area) and transported to the laboratory in tanks with portable oxygen injection, arriving within 2 h of collection. For standardization, all crabs were selected to have comparable weights and similar carapace widths. Additionally, only male crabs were selected as experimental animals to avoid the potential effects of reproduction on the tested parameters. Morphological traits were used to determine sex.

Collected specimens were acclimated for 1 week in glass aquaria (60 × 30 × 30 cm) containing 15 L of aerated seawater with a salinity of 33 ± 2‰ under standard laboratory conditions of temperature, photoperiod (12 h light : 12 h dark; Ikhwanuddin, 2020; Romano & Zeng, 2006), and aeration. During the experiment, parameters of the aerated seawater were maintained as follows: temperature at 28 ± 1°C (Ikhwanuddin, 2020; Romano & Zeng, 2006), salinity at 33 ± 2‰ (Ikhwanuddin, 2020; Romano & Zeng, 2006), pH at 7.8 ± 0.2, and dissolved oxygen at 8.5 ± 0.7 mg/L (Table 1). The crabs were fed twice daily (0900 and 1600 hours) at 2% biomass with a 28% crude protein commercial feed (Faradaneh Company). Seventy percent of the aquarium water was replaced daily. Uneaten food, feces, and debris were siphoned daily, and checks were conducted for any mortalities, including incidences of molt death syndrome, each day (Carneiro et al., 2019). The experimental setup was placed in a climate-controlled room to minimize environmental variation. Crabs are highly territorial and can exhibit aggression. To mitigate aggressive behavior, every crab should have its own space to be alone. A half-round PVC pipe was used to cover a portion of each pie-shaped tank section (Supriyono et al., 2017). The PVC pipe served as a place for crabs to hide when feeding and provided a space for molting, as crabs are most susceptible to attacks during the molt. Each tank was fitted with an air stone within the center of the tank to facilitate water circulation and oxygenation.

Preparation of anesthetics

Clove oil was diluted in low, medium, and high doses to create stock solutions, which were then added to the seawater in each treatment aquarium to achieve the desired concentrations (150, 300, and 450 μL/L). Because previous studies on crustaceans used clove oil doses based on units of microliters per liter, this scale was used to compare the results. For immersion tests, Nourhan brand clove oil (85–95% eugenol; Moohaya Company) was dissolved in 1:10 ethanol as described by previous authors (Barata et al., 2016; Cooke et al., 2004; Neiffer & Stamper, 2009) and was stored in brown stock solution vials until use to ensure uniformity (Carneiro et al., 2019).

Experimental design

After acclimatization to the laboratory and experimental setup for 1 week, a total of 45 crabs were randomly anesthetized with 150-, 300-, and 450-μL/L clove oil treatments, with five replicates per treatment. Specific anesthetic concentrations (stock solutions) were prepared in a tank containing 10 L of aerated seawater. A 60- × 30- × 30-cm glass tank with 3 L of seawater was used for the induction process. Porous air stones linked to a silicone hose via a 9-W electromagnetic air pump (co-318; HAILEA, China) supplied continuous aeration.

Mean values for the water quality parameters were within the range considered ideal for blue swimming crabs (Romano & Zeng, 2006). Twenty-four hours before the experiment started, feeding was stopped. The crabs were individually placed in aquariums containing the three doses of clove oil. The time required to reach the desired anesthesia stage was recorded based on behavioral responses. Subsequently, a chronometer was used to record induction times as described by Yoshikawa et al. (1988). Due to the lack of studies on crustaceans, especially crabs, information on the anesthesia and recovery stages of fish was used. The anesthesia phases illustrated by Iwama et al. (1989) were adopted for this study, consisting of three induction stages (A1, A2, and A3) and three recovery stages (R1, R2, and R3). Every crab was maintained in its tank until the complete loss of equilibrium, or deep anesthesia, was noticed (stage A3). The timer was stopped, and the anesthetic time was noted using the methodology of Gardner (1997). The protocol presented in Table 2 was used to determine the various anesthesia induction and recovery phases. The recovery times, defined as the duration required for each crab to return to normal swimming behavior, were assessed after the induction period. Each crab’s recovery time was monitored, recorded, and documented separately for each concentration until it reached stage R3. After achieving deep anesthesia, the crabs were transferred from their tanks to a 100-L tank with constant aeration and monitored for 24 h to assess their survival and mortality rates. Notably, deep anesthesia was not observed at two lower concentrations of clove oil (75 and 100 µL/L), with the 75-µL/L concentration being ineffective.

Statistical analyses

Normality of data and homogeneity of variance were examined using one-sample Kolmogorov–Smirnov and Levene’s tests. One-way ANOVA and Tukey’s multiple comparison tests were used to compare induction times (stages A1, A2, and A3) and recovery times (stages R1, R2, and R3) among the three doses of clove oil (α = 0.05). Pearson’s correlation was used to test the relationship between crab weight and the induction and recovery times. All analyses were conducted at a significance level of 0.05 using IBM SPSS Statistics version 20.0, ensuring the transparency and replicability of this study.

RESULTS

No crab mortality was observed at any clove oil concentration during the experimental exposures or during the 24-h postexposure recovery period. There was a significant difference in the times of anesthetic induction for the 150-μL/L concentration compared to the other concentrations (P < 0.001). Anesthetic induction and recovery times for all stages are presented in Figures 2 and 3.

With an increase to 300- and 450-μL/L concentrations, the times for stage A1 decreased (P < 0.001), but there was no significant difference between these dosages. The time to reach stage A1 was significantly (P < 0.001) longer for the 150-μL/L dose (Figure 2). The same result was observed for stages A2 and A3, and the anesthesia rate required to reach these stages significantly increased at the 300- and 450-μL/L concentrations. Moreover, the time required to reach stage A2 at the 450-μL/L concentration (421 s) was significantly shorter than those for the other concentrations. For stage A3, the period required to attain deep anesthetic induction at the 150-μL/L dosage (1,058 s) was significantly longer compared to the higher dosages. There was no difference for the 300- and 450-μL/L concentrations to reach stage A3. However, at doses of 300 and 450 μL/L, the duration to reach stage A2 was shown to be ­considerably (P < 0.001) shorter than the duration at the 150-μL/L dosage. Blue swimming crabs that were exposed to the 150-μL/L dose took the longest time to reach stage A2 (Figure 2). Significant differences (P < 0.001) were found between the highest clove oil concentrations (300 and 450 μL/L) and the lowest concentration (150 μL/L) in the time taken by crabs to reach stage A3 (Figure 2).

The amount of time crabs needed to recover after anesthesia induction at the different doses differed significantly (Figure 3). Crabs that were exposed to 300- and 450-μL/L doses had longer recovery times (stage R3; P < 0.004) than crabs that were exposed to 150 μL/L. The recovery durations for the 300- and 450-μL/L concentrations did not differ significantly (Figure 3). The shortest recovery time (stage R1; mean ± SE = 240 ± 14 s) was observed in crabs that were exposed to 150 μL/L. The maximum recovery times (stage R3) were observed at the 300- and 450-μL/L doses, with averages of 753 ± 27 and 753 ± 29 s, respectively.

Results of Pearson’s correlations examining the relationship between crab weight and anesthesia duration and recovery are presented in Table 3. Anesthesia and recovery times across the range of blue swimming crab sizes (from smaller to larger individuals) appeared to trend upward at the 300- and 450-μL/L concentrations. The significance level for all concentrations except 150 μL/L in stage A1 was less than 0.05. This relationship was strong for stages A2 and A3 and moderate for stage A1. The relationship’s total intensity was 13.98% for stage A1, 30.36% for stage A2, and 23.52% for stage A3. Pearson’s correlation showed that crab weight was correlated with the time of anesthesia. The correlation between crab weight and recovery time at all concentrations was confirmed as less than 1%, but the correlation at 150 μL/L was confirmed as less than 5% only for stage R1. Therefore, the correlation coefficients were significant for all concentrations. The intensity of the relationship was 38.13% for stage R1, 66.58% for stage R2, and 83.72% for stage R3.

DISCUSSION

Using anesthetics in aquaculture, particularly for handling and transporting crustaceans like the blue swimming crab, is critical for minimizing stress and ensuring the welfare of the animals (Berlinsky et al., 2016; Öğretmen et al., 2014). This study assessed the efficacy of clove oil as a natural anesthetic at various concentrations, providing valuable insights into effective levels for inducing anesthesia and facilitating recovery.

Clove oil demonstrated effectiveness at concentrations of 300 and 450 µL/L at a constant temperature of 29 ± 1°C, achieving anesthetic induction (stage A1) in less than 4.5 min, compared to approximately 6 min with a concentration of 150 µL/L (Table 3). These findings highlight the significance of induction speed in reducing exposure to stressful handling conditions (Carneiro et al., 2019; Gullian & Villanueva, 2009; Pawar et al., 2011). Notably, higher clove oil concentrations led to quicker induction times while maintaining acceptable recovery durations without significant mortality or prolonged adverse effects during the recovery phases.

Although previous research has demonstrated clove oil’s effectiveness across various crustacean species, including the blood-spotted crab, giant tiger prawn, and several shrimp species (e.g., grass shrimp), unique physiological responses exhibited by crabs necessitate tailored investigations (Jiang et al., 2020; Li et al., 2018; Premarathna et al., 2016). Our results align with earlier observations indicating that elevated doses result in reduced sedation periods among crustaceans (Ghanawi et al., 2019; Soltani et al., 2004). Our observations suggest an effective concentration of around 300 μL/L for blue swimming crab applications, consistent with findings from studies on green mud crabs, which achieved high survival rates after anesthesia usage (Zhu et al., 2023).

Importantly, there was no significant difference between recovery times at concentrations above 300 µL/L (P > 0.05), suggesting no added benefit from increasing the dose past this point, in agreement with other studies showing that higher clove oil concentrations do not always improve anesthesia results but can lead to longer recovery times.

Synthetic anesthetics are associated with physiological disturbances such as neurotoxicity (Wycoff et al., 2018). Therefore, their use should always be approached cautiously (Fotedar & Evans, 2011; Hajek et al., 2009; Parodi et al., 2012). Conversely, clove oil is considered safer due to its lower incidence of side effects (Ghanawi et al., 2019). Its lower cost compared to synthetic options further enhances its appeal in commercial settings while improving aquatic animal welfare (Berlinsky et al., 2016; Öğretmen et al., 2014).

However, it should be noted that anesthesia with clove oil can result in slower recovery rates due to factors such as chemical receptor specificity (Souza et al., 2018). Additionally, intravascular administration may cause autotomy or mortality associated with limb autoionization, which occurs in an attempt to block the extreme loss of hemolymph, a concern that was highlighted by previous research (Minter et al., 2013; Souza et al., 2018).

Stressful transportation conditions often lead to significant losses in both welfare and economic value. Based on Shaluei et al. (2012), the sedation stage (i.e., stage A1) allows for transportation without losing equilibrium, suggesting that a concentration of 150 µL/L at a water temperature of 29 ± 1°C could enhance safety during anesthesia and recovery procedures in routine management tasks (Table 3).

This study provides valuable insights into clove oil’s effectiveness as an anesthetic for blue swimming crabs, but several limitations must be acknowledged regarding generalizability across crustacean species due to unique physiological responses and environmental influences affecting anesthesia efficacy. Most existing studies center on adult crabs; thus, responses from juveniles, larvae, or reproductive individuals remain unclear. Understanding how these life stages react to clove oil is essential for successful culture practices within portunid species. Future studies should (1) validate these results under commercial conditions to ensure practical applicability across diverse aquaculture environments and (2) investigate the long-term effects of clove oil exposure on crab health and behavior. Moreover, ongoing research could explore the combination of clove oil with other stress-reducing methods, such as optimized handling protocols or supplemental oxygen during transportation, and could assess whether varying concentrations or repeated exposures influence the flavor profile of crab meat. Integrating clove oil with best practices could further enhance crustacean welfare and productivity in aquaculture processes while ensuring that its use does not compromise the sensory qualities of the final product.

Conclusions

The findings of this study confirm that clove oil at a concentration of 300 µL/L is a practical anesthetic for blue swimming crabs. This concentration enables rapid induction of anesthesia with manageable recovery times, making it suitable for various applications in aquaculture. Clove oil is also a natural and cost-effective alternative to synthetic anesthetics. Its use aligns with the increasing demand for sustainable aquaculture practices and supports eco-friendly procedures within the industry. Clove oil presents a promising solution for enhancing welfare during handling and transportation in crab aquaculture.

DATA AVAILABILITY

The data sets generated for this study are available upon request from the corresponding author.

ETHICS STATEMENT

All authors declare that the use of animals for this research complies with the requirements of the ethical principles and the national norms and standards for conducting medical research in Iran on the use of animals for experimentation; therefore, we faithfully comply with the Code of Practice for Housing and Care of Animals Used in Scientific Procedures. The project has been approved by the Biomedical and Animal Research Ethics Committee and evaluated by the Research Ethics Committees of Hormozgan University of Medical Sciences, Iran (Approval ID IR.HUMS.REC.1401.036).

FUNDING

None declared.

ACKNOWLEDGMENTS

We are thankful to the Department of Marine Science and Technology, Hormozgan University, for the support. Author contributions are as follows: Pegah Gheshlaghi wrote the original draft and contributed conceptualization, methodology, resources, formal analysis, investigation, visualization, data curation, and validation; Ehsan Kamrani contributed investigation, visualization, supervision, and writing (review and editing); and Moslem Daliri contributed writing (review and editing), investigation, data curation, and supervision.

REFERENCES

Author notes