Rosmarinus officinalis essential oil components promote anesthesia in Colossoma macropomum (Cuvier, 1818) through behavioral and cardiorespiratory modulation

Rosmarinus officinalis essential oil components promote anesthesia in Colossoma macropomum (Cuvier, 1818) through behavioral and cardiorespiratory modulation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Rosmarinus officinalis essential oil components promote anesthesia in Colossoma macropomum (Cuvier, 1818) through behavioral and cardiorespiratory modulation Lorena Cristina Nunes Almeida, Priscille Fidelis Pacheco Hartcopff, and 12 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7964102/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Anesthetic management plays a crucial role in maintaining the welfare and physiological stability of fish during routine aquaculture and research procedures. The essential oil of Rosmarinus officinalis (ROEO) has emerged as a promising natural anesthetic, yet its cardiophysiological effects in Amazonian species remain underexplored. This study assessed the behavioral, electrocardiographic, and respiratory responses of Colossoma macropomum (tambaqui) exposed to ROEO, aiming to define safe and effective concentrations for use as a plant-derived anesthetic in aquaculture. Juvenile tambaqui were exposed to immersion baths containing 100–200 µL L⁻¹ of ROEO for 5 min. Behavioral endpoints (latency to loss and recovery of postural reflex), electrocardiographic parameters (heart rate, QRS amplitude, R–R, P–Q, and Q–T intervals), and opercular frequency were monitored. The oil composition was determined by gas chromatography. Statistical analyses were performed using one-way ANOVA followed by Tukey’s test ( p < 0.05). ROEO produced a clear dose-dependent anesthetic response, characterized by faster induction and prolonged recovery times at higher concentrations. Cardiac monitoring revealed reversible bradycardia (up to − 56%), elongation of R–R and Q–T intervals, and a slight reduction in QRS amplitude without arrhythmias. Opercular frequency decreased by up to 38% at 200 µL L⁻¹, indicating moderate respiratory depression. All effects were reversible within the recovery period. Eucalyptol (47.5%), camphor (19.3%), and α-pinene (12.2%) were the main phytoconstituents identified. Rosmarinus officinalis essential oil provides a safe, reversible, and concentration-dependent anesthetic effect in C. macropomum , maintaining sinus rhythm and physiological recovery. These results highlight ROEO as a biodegradable, low-toxicity, and effective alternative to synthetic anesthetics, reinforcing its applicability for sustainable and welfare-oriented aquaculture practices. Rosmarinus officinalis Colossoma macropomum fish anestesia electrocardiography aquaculture welfare natural anesthetics eucalyptol Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1. Introduction The use of anesthesia in aquaculture and scientific research involving fish plays a fundamental role in maintaining the health and welfare of these animals. Handling procedures, both for productive and experimental purposes, often involve invasive techniques that can cause stress and physical harm to fish (Aktop et al., 2019 ; Brønstad, 2022 ; Barbas et al., 2021 ; Kheawfu, 2021; Reis et al., 2024 ). In this context, anesthetic use is essential to minimize such impacts and ensure more humane practices, providing safety for both animals and operators or consumers, while reflecting a commitment to ethical principles in animal management (Aydın and Barbas, 2020 ; Rairat et al., 2021 ; Schroeder et al., 2021 ; Mocho, 2024 ). Synthetic anesthetics, such as MS-222 (tricaine methanesulfonate) and Benzocaine, are widely used in aquaculture. However, their use has been associated with undesirable side effects, including respiratory and cardiac depression, as well as the potential accumulation of residues in fish tissues, compromising aquaculture product quality and causing adverse effects on animal health and the environment (Taheri Mirghaed et al., 2018 b; Can and Sümer, 2019 ; Neiffer, 2021 ). Consequently, there has been growing interest in safer and more sustainable alternatives, such as plant-derived natural anesthetics, which offer a more favorable profile in terms of toxicity, biodegradability, and economic accessibility (Bianchini et al., 2017 ; Seyidoglu and Yagcilar, 2020 ; de Araújo et al., 2021 ; Rucinque, 2021; Elmas and Karadal, 2022 ; Zeng, 2022). Among the promising natural anesthetics is the essential oil of Rosmarinus officinalis , a perennial plant from the Lamiaceae family widely recognized for its diverse pharmacological properties, including antibacterial, antidiabetic, anti-inflammatory, antitumor, and antioxidant actions (Andrade et al., 2018 ; Al Zuhairi, 2020; Zhong et al., 2021; Al-Mahariki et al., 2022; Li Pomi et al., 2023 ; Sharma, 2024). Among its main components, 1,8-cineole (eucalyptol) stands out in experimental studies due to its ability to act on the central nervous system, activating transient cation channels that promote desensitization and result in anesthetic effects (Caceres, 2017; Khumpirapang et al., 2018 ; Sadeh et al., 2019 ; Cai et al., 2021 ; Dhouibi, Flamini & Bouaziz, 2023 ). In addition to its anesthetic properties, 1,8-cineole also influences autonomic cardiovascular regulation, demonstrating effects such as bradycardia and reduced blood pressure, underscoring its therapeutic potential (Lahlou et al., 2002 ; Moon, 2014; Wang, 2021). In fish experiments, 1,8-cineole has proven to be an effective anesthetic, with induction and recovery times directly dependent on the concentration used. In common carp ( Cyprinus carpio ), results indicate that higher concentrations reduce induction time, highlighting the potential of 1,8-cineole as a promising natural anesthetic for use in aquaculture (Hoseini et al., 2020 ; Mirghaed et al., 2018 ; Mazandarani & Hoseini, 2017 ). Furthermore, studies in rats have shown that intravenous administration of 1,8-cineole significantly reduces mean arterial pressure and heart rate in a dose-dependent manner, also indicating its effect on the cardiovascular system (Lahlou et al., 2002 ). Despite these advances, there remains a research gap concerning the cardiovascular effects of 1,8-cineole in different fish species, which justifies the need to expand its evaluation. The present study aims to investigate the anesthetic effect of Rosmarinus officinalis essential oil in Colossoma macropomum (tambaqui), one of the most widely farmed species in Brazilian aquaculture. It was sought to determine optimal dose-response relationships for different concentrations of the oil and to assess cardiovascular safety and behavioral effects in fish during the induction and recovery phases of anesthesia. This study aims to provide a safer and more sustainable alternative to synthetic anesthetics, contributing to animal welfare and promoting more ethical and sustainable practices in aquaculture. 2. Materials and Methods 2.1. Experimental Animals The subjects used were Colossoma macropomum tambaqui (n = 108), housed in aquariums in the Experimental Animal Facility of the Laboratory of Pharmacology and Toxicology of Natural Products at the Federal University of Pará (ICB/UFPA). The environment was maintained at a controlled temperature (25–27°C) with a 12 h light: 12 h dark photoperiod. The fish were fed commercial feed (32% protein) twice daily until satiation. Simultaneously, uneaten food and feces were removed by siphoning, and the water was partially renewed (approximately 40% of tank volume) with water from the same source. During the acclimatization period (20 days), water quality variables such as water temperature (°C) (26.5°C) and pH (pH = 7.5) were monitored. The project was previously approved by CEUA-UFPA under registration 5846030624 (ID 002640). 2.2. Organoleptic Properties and Chromatographic Analysis Rosemary essential oil ( Rosmarinus officinalis ) – ROEO - was acquired from Harmonie Aromatherapy (Florianópolis, SC, Brazil, CNPJ: 11.938.821/0001–90). The oil was extracted by steam distillation, and analysis was conducted by Gas Chromatography using an AGILENT 7820A Gas Chromatograph under the following conditions: Column: RXi-5MS 30 m x 0.25 mm x 0.25 µm (Restek). Temperature program: Column at 50°C (0 min), increasing at 3°C/min to 200°C. Injector: 200°C, split ratio: 1/50. FID detector: 220°C. Injection volume: 1 µL (1% in ethyl acetate). The phytoconstituents of the oil were identified as follows: Eucalyptol (47.5%), Camphor (19.3%), α-pinene (12.2%), β-pinene (7.8%), and β-caryophyllene (4.6%). Table 1 here Table 1 Chemical composition Rosmarinus Officinalis essential oil Retention index Identification Percentage (%) Peak 913 Alpha-pinene 12.2 1 925 Camphene 1.4 2 950 Beta-pinene 7.8 3 963 Myrcene 0.5 4 995 Para-cymene 0.7 5 1000 Limonene 2.3 6 1003 Eucalyptol 47.5 7 1031 Gamma terpinene 0.1 8 1073 Linalool 1.2 9 1121 Camphor 19.3 10 1148 Borneol 0.1 11 1159 Terpinen-4-ol 0.3 12 1174 Alpha terpineol 1.0 13 1281 Bornyl acetate 0.2 14 1425 Beta-caryophyllene 4.6 15 1461 Humolene 0.2 16 Others 0.2 Source: Prepared by the authors, 2024 Figure 1 here 2.3. Experimental Design 2.3.1. Experiment with Rosmarinus officinalis Essential Oil (ROEO) Juvenile tambaqui (26.18 ± 3.5g) were randomly assigned to the following treatments: a) Control; b) vehicle group (fish subjected to immersion bath with 2 ml of 70% alcohol diluted in 1 liter of aquarium water); c) fish treated with ROEO 100 µL L⁻¹; d) 125 µL L⁻¹; e) 150 µL L⁻¹; f) 175 µL L⁻¹; and g) 200 µL L⁻¹. All fish underwent anesthetic induction with a contact period of 5 minutes, followed by observation during recovery for 5 minutes in water without ROEO. For each recording, n = 9 per treatment was used (immersion bath with ROEO and recovery post-immersion bath), totaling 108 animals. 2.3.2. Experiment 1 - Behavioral Analysis of Anesthetic Induction and Recovery Considering the exposure time for the following ROEO treatments: a) 100 µL L⁻¹, b) 125 µL L⁻¹, c) 150 µL L⁻¹, d) 175 µL L⁻¹, and e) 200 µL L⁻¹ ( n = 9 per treatment), the latency to the behavior of postural reflex loss (characterized by lateral recumbency) was evaluated. Following removal from ROEO contact, the latency for recovery of postural reflex was recorded. 2.3.3. Experiment 2 - Electrocardiogram (ECG) Analysis For cardiac function analysis and monitoring, groups were divided as follows: a) Control group; b) vehicle group; c) treated group with ROEO 100 µL L⁻¹; d) 125 µL L⁻¹; e) 150 µL L⁻¹; f) 175 µL L⁻¹; and g) 200 µL L⁻¹ (a total of 63 animals). Electrodes were made from 0.3 mm diameter, 10 mm long 925 silver electrodes, and subsequently isolated with liquid insulation. Electrodes were non-conjugated. The reference electrode positioning followed the cardiac vector indication (negative pole at the cardiac base and positive pole at the apex). The reference electrode was fixed to the ventral portion of the left opercular opening 0.2 mm past the opercular cavity. The recording electrode was inserted 2.0 mm from the right opercular opening, capturing the signal on lead D1. The electrodes were connected to a high-impedance amplifier (Grass Technologies, Model P511) for ECG recording, enabling analysis of heart rate (bpm), QRS complex amplitude (mV), QRS complex duration (ms), and R-R (ms), P-Q (ms), and Q-T intervals (ms). 2.3.4. Experiment 3 - Opercular Activity Recording For opercular activity analysis, 925 silver electrodes were made with a 0.5 mm diameter and 15 mm length, conjugated at a 7 mm distance and isolated with liquid insulation. The electrodes were placed at the center of the right opercular opening to record opercular beating, assessing the frequency (beats per minute) and power of opercular beating (mV²/Hz). 2.4. Recording and Analysis of Data The electrodes were connected to a digital data acquisition system via a differential high-impedance amplifier (Grass Technologies, Model P511), set with 0.3 to 300 Hz filtering, 2000X amplification, and monitored by an oscilloscope (ProteK, Model 6510). Recordings were continuously digitized at a 1 KHz rate on a computer equipped with a data acquisition board (National Instruments, Austin, TX), and stored on a hard drive for further processing via specialized software (LabVIEW express). The acquired signals were analyzed using a tool developed in Python version 2.7. Numpy and Scipy libraries were used for mathematical processing, while Matplotlib was used for graph plotting. The graphical interface was created with the PyQt4 library (Da Paz, 2024). 2.5. Statistical Analysis After confirming normality and homogeneity of variances with the Kolmogorov-Smirnov and Levene tests, respectively, comparisons of mean power values were made via one-way ANOVA, followed by Tukey’s test. GraphPad Prism® 8 software was used for the analyses, with p < 0.05, *p < 0.01, and **p < 0.001 considered statistically significant in all cases. 3. Results The behavioral analysis showed that ROEO caused a concentration-dependent loss of postural reflex, with higher doses resulting in shorter latency for reflex loss onset. Fish exposed to 100 µL L⁻¹ exhibited an average postural reflex loss at 214.1 ± 16.66 s, which was similar to the group treated with 125 µL L⁻¹ (p = 0.060). However, these were longer than in the other groups. The group treated with 150 µL L⁻¹ (151.4 ± 18.76 s), 175 µL L⁻¹ (131.2 ± 8.89 s), and 200 µL L⁻¹ (110.9 ± 13.57 s) were significantly different (Fig. 2 A). The recovery of postural reflex in the group treated with 100 µL L⁻¹ ROEO occurred in 87.0 ± 11.75 s and was shorter than in the other groups: 125 µL L⁻¹ (124.6 ± 12.38 s), 150 µL L⁻¹ (154.7 ± 15.80 s), 175 µL L⁻¹ (172.2 ± 11.01 s), and 200 µL L⁻¹ (208.1 ± 16.13 s). All groups showed recovery time dependent on the concentration used, with higher concentrations resulting in longer recovery times, indicating a slower reversibility of the effect for groups that received higher concentrations (Fig. 2 B). Figure 2 (A and B) here The normal electrocardiogram of the tambaqui exhibited a sinus rhythm with an average frequency of 86.89 ± 2.47 bpm. The P wave, QRS complex, and T wave can be identified (Figures A, B, and C). The intervals between R-R deflections, as well as the P-Q and Q-T intervals, remained regular considering the normal cardiac parameters. Thus, the interference of increasing doses of ROEO was measured. For the control group, the heart rhythm was sinusoidal during the 5-minute recording (Fig. 3 ). Figure 3 (A, B and C) here The cardiac activity in the control group showed a mean frequency of 86.89 ± 2.47 bpm, which was similar to the vehicle group (p = 0.999), with a sinus rhythm and all cardiac deflections present on the electrocardiogram (Figs. 3 and 3 A). In the 30-second amplification, all graph elements were visible: P wave, QRS complex, and T wave in a 5-second amplification, allowing for the evaluation of intervals during the immersion bath in ROEO and their recovery. This enabled the identification of atrial activity represented by P waves, ventricular activity by QRS complexes, and ventricular repolarization represented by T waves (Figs. 4 A, B, C, D, E, and F). During immersion treatment with 100 µL L⁻¹, the fish showed a 40.66% decrease in heart rate compared to the control group (Fig. 4 B). The group treated with 125 µL L⁻¹ of ROEO exhibited a 45.78% decrease (Fig. 4 C), while the group treated with 150 µL L⁻¹ showed a 55.49% reduction (Fig. 4 D). The 175 µL L⁻¹ group decreased by 56.51% (Fig. 4 E), and the group treated with 200 µL L⁻¹ exhibited a 56.77% reduction (Fig. 4 F). The treated fish presented bradycardia while maintaining sinus rhythm (Figs. 4 B, C, D, E, and F). Figure 4 (A, B, C, D, E and F) here The heart rate was significantly affected by the increasing concentrations of ROEO. The control group had an average heart rate of 86.89 ± 2.47 bpm, and the vehicle group showed 86.67 ± 3.46 bpm, both being similar (p = 0.999). However, these values were higher than those in the treated groups. The group treated with 100 µL L⁻¹ had an average heart rate of 51.56 ± 2.60 bpm, which was higher than the other treated groups. The group treated with 125 µL L⁻¹ had an average of 47.11 ± 1.76 bpm, which was also higher than the groups treated with the higher doses. The group treated with 150 µL L⁻¹ had an average of 38.67 ± 2.44 bpm, which was like the groups treated with 175 µL L⁻¹ and 200 µL L⁻¹ (p = 0.966) (Fig. 5 A). The average amplitude of the QRS complex for the control group was 1.168 ± 0.27 mV, and for the vehicle group, it was 1.15 ± 0.27 mV, which were similar to the groups treated with 100 µL L⁻¹, 125 µL L⁻¹, and 150 µL L⁻¹, as well as 175 µL L⁻¹ (p = 0.747). The control and vehicle groups had greater amplitudes than the group treated with 200 µL ·L⁻¹, which measured 0.713 ± 0.134 mV (Fig. 5 B). The average RR interval for the control group was 696.7 ± 19.33 ms, which was similar to the vehicle group (p = 0.999), and was shorter compared to the other treated groups. The group treated with 100 µL L⁻¹ had an average RR interval of 1166 ± 58.31 ms, like the group treated with 125 µL L⁻¹ (p = 0.1106) and was shorter than the other treated groups. The group treated with 150 µL L⁻¹ had an RR interval of 1545 ± 91.82 ms, which was similar to the groups treated with 175 µL L⁻¹ and 200 µL L⁻¹ (p = 0.2725) (Fig. 5 C). The average PQ interval for the control group was 115.1 ± 6.27 ms and showed no difference from the vehicle group (p = 0.999), as well as from the groups treated with 100 µL L⁻¹ and 125 µL L⁻¹ (p = 0.1060). The group treated with 150 µL L⁻¹ had an average of 134.8 ± 8.21 ms, and the group treated with 175 µL L⁻¹ was similar (p = 0.6530). The group treated with 175 µL L⁻¹ was also like the group treated with 200 µL L⁻¹ (p = 0.2505) (Fig. 5 D). The average duration of the QRS complex for the control group during induction was 27.22 ± 2.72 ms, which was similar to the vehicle group (p = 0.999) but shorter than the other groups. The groups treated with 100 µL L⁻¹ (38.11 ± 4.06 ms) were like the group treated with 125 µL L⁻¹ (p = 0.9999). The group treated with 150 µL L⁻¹ (44.33 ± 3.87 ms) was like the group treated with 175 µL L⁻¹ (p = 0.6513). The group treated with 200 µL L⁻¹ (51.67 ± 6.083 ms) was greater than the other groups but was like the group treated with 175 µL L⁻¹ (p = 0.2251) (Fig. 5 E). For the control group, the average QT interval during induction was 290.3 ± 10.51 ms, which was similar to the vehicle group (p = 0.9967) and shorter than the other groups. The group treated with 100 µL L⁻¹ had an average QT interval of 375.7 ± 13.59 ms, and the 125 µL L⁻¹ group was not significantly different (p = 0.999). The group treated with 150 µL L⁻¹ (435.7 ± 18.91 ms) was like the group treated with 175 µL L⁻¹ (p = 0.9525). The group treated with 200 µL L⁻¹ (474.1 ± 16.44 ms) was greater than the other groups (Fig. 5 F). Figure 5 (A, B, C, D, E and F) here During the recovery period following exposure to the ROEO concentrations of 100 µL L⁻¹, 125 µL L⁻¹, 150 µL L⁻¹, 175 µL L⁻¹, and 200 µL L⁻¹, a reversibility of the electrocardiographic alterations was observed (Figs. 6 A, B, C, D, and E), occurring more slowly in the groups treated with higher concentrations. The cardiac rhythm remained sinusoidal, with a gradual increase in heart rate approaching levels observed in the control group. For the group treated with 100 µL L⁻¹ of ROEO, the heart rate recovered 99.48% compared to the control (Fig. 6 A). The recovery percentages for the other groups were as follows: 125 µL L⁻¹ (96.16%) (Fig. 6 B), 150 µL L⁻¹ (74.68%) (Fig. 6 C), 175 µL L⁻¹ (68.54%) (Fig. 6 D), and 200 µL L⁻¹ (66.49%) (Fig. 6 E). During the recovery period, no arrhythmias were observed, and the reversal of ROEO effects on the heart was gradual and concentration dependent. Figure 6 (A, B, C, D and E) here The amplitude of the QRS complex during recovery for the control group was 1.168 ± 0.27 mV, which was similar to the other groups (F (6, 56) = 0.375, p = 0.891) (Fig. 7 B). The average RR interval during recovery for the control group was 686.7 ± 19.33 ms, showing no statistical difference compared to the vehicle, 100 µL L⁻¹, and 125 µL L⁻¹ groups (p = 0.7572); however, it was shorter than the other groups. The groups treated with higher concentrations, 150 µL L⁻¹ (926.1 ± 43.80 ms), were shorter than the 175 µL L⁻¹ (1009 ± 57.90 ms) and 200 µL L⁻¹ (1043 ± 85.05 ms) groups. The 175 µL L⁻¹ and 200 µL L⁻¹ groups were similar (p = 0.5171) (Fig. 7 C). The PQ interval during recovery for the control group was 115.1 ± 6.27 ms, which was like the vehicle, 100 µL L⁻¹, 125 µL L⁻¹, and 150 µL L⁻¹ groups (p = 0.1260). The group treated with 175 µL L⁻¹ (125.3 ± 4.062 ms) was like the group treated with 200 µL L⁻¹ (p = 0.9543) but was longer than the other groups (Fig. 7 D). The duration of the QRS complex during recovery for the control group averaged (27.22 ± 2.72 ms) and was like the other groups (F (6, 56) = 2.88; p = 0.0161) (Fig. 7 E). During recovery, the QT interval for the control was 290.3 ± 10.51 ms, which was like the vehicle, 100 µL L⁻¹, 125 µL L⁻¹, and 150 µL L⁻¹ groups (p = 0.1525). The groups treated with 150 µL L⁻¹ (311.8 ± 14.06 ms) were like the 175 µL L⁻¹ group (p = 0.1142). The group treated with 200 µL L⁻¹ (354.9 ± 22.27 ms) was like the group treated with 175 µL L⁻¹ (p = 0.1894) (Fig. 7 F). Figure 7 (A, B, C, D, E and F) here During the treatment with ROEO at concentrations of 100 µL L⁻¹, 125 µL L⁻¹, 150 µL L⁻¹, 175 µL L⁻¹, and 200 µL L⁻¹, the frequency of opercular movement decreased with higher concentrations (Fig. 8 A, B, C, D, E, F, and G). The control group exhibited a mean of 98.67 ± 6.92 movements per minute (mpm), which was similar to the vehicle group (p = 0.9282) but higher than the other treatment groups. The group treated with 100 µL L⁻¹ showed a reduction in opercular beats of 22.52%. The group treated with 125 µL L⁻¹ exhibited a decrease of 21.17%, while the group treated with 150 µL L⁻¹ showed a reduction of 31.08%. The group treated with 175 µL L⁻¹ reduced opercular activity by 32.43%, and the group treated with 200 µL L⁻¹ demonstrated a decrease of 37.95%. The group treated with 100 µL L⁻¹ (76.44 ± 3.97 mpm) was like the group treated with 125 µL L⁻¹ (p = 0.9980), but higher than the other treatment groups. The group treated with 150 µL L⁻¹ (68.00 ± 4.123 mpm) was like the groups treated with 175 µL L⁻¹ and 200 µL L⁻¹ (p = 0.0995) (Fig. 8 H). The decrease in power in the opercular movement records was observed during the induction of anesthesia. The control group showed a mean power of 0.3165 ± 0.0609 mV²/Hz, which was similar to the vehicle group (p = 0.134) but greater than that of the other treatment groups. The group treated with 100 µL L⁻¹ showed a mean of 0.189 ± 0.025 mV²/Hz, which was like the group treated with 125 µL L⁻¹ (p = 0.5722). The group treated with 125 µL L⁻¹ (0.155 ± 0.0301 mV²/Hz) was like the groups treated with 150 µL L⁻¹ and 175 µL L⁻¹ (p = 0.1882). The group treated with 200 µL L⁻¹ (0.04063 ± 0.0087 mV²/Hz) was lower than the other groups (Fig. 8 I). Figure 8 (A, B, C, D, E, F, G, H and I) here During the recovery period from anesthesia, opercular movement showed recovery (Fig. 9 A, B, C, D, and E). The control group had a mean of 98.67 ± 6.92 mpm, which was like the vehicle group, the group treated with 100 µL L⁻¹, the group treated with 125 µL L⁻¹, and the group treated with 150 µL L⁻¹ (p = 0.1338), but higher than the other treated groups. The group treated with 175 µL L⁻¹ (81.67 ± 5.52 mpm) was like the group treated with 200 µL L⁻¹ (p = 0.2669) (Fig. 9 F). An increase in power in the opercular movement recordings was observed during recovery from anesthesia. The control group had a mean power of 0.3165 ± 0.0609 mV²/Hz, which was like the recovery groups of the vehicle, treated with 100 µL L⁻¹, 125 µL L⁻¹, and 150 µL L⁻¹ (p = 0.5085). The group treated with 125 µL L⁻¹ was like the group treated with 200 µL L⁻¹ (p = 0.0549). The group treated with 200 µL L⁻¹ was like the groups treated with 150 µL L⁻¹ and 175 µL L⁻¹ (p = 0.0603) (Fig. 9 G). Figure 8 (A, B, C, D, E, F and G) here 4. Discussion The results of this study demonstrate that Rosmarinus officinalis essential oil (ROEO) exerts a concentration-dependent anesthetic effect on Colossoma macropomum (tambaqui). The group treated with 200 µL L⁻¹ of essential oil showed a significantly faster loss of postural reflex compared to groups with lower concentrations, such as 100 µL L⁻¹, indicating a faster anesthetic action at higher concentrations. These findings align with the study by Bianchini et al. ( 2017 ), which evaluated the anesthetic effects of essential oil compounds like carvacrol and thymol and observed a clear relationship between concentration and anesthetic response, with significantly shorter induction times as concentrations increased across all induction stages. Uehara et al. (2018), in a comparative study of synthetic and natural anesthetic agents, highlighted that clove oil exhibited effective anesthetic action across all tested concentrations, with dose-dependent effects on both anesthetic induction and recovery time. Similarly, the lower concentrations of R. officinalis essential oil evaluated in this experiment, such as 100 µL L⁻¹ and 150 µL L⁻¹, required a longer latency time for anesthetic action than the other concentrations. Meanwhile, postural reflex recovery time was longer in groups treated with higher doses, demonstrating the dose-dependent and gradual reversibility of the anesthetic effect - a characteristic often described in the literature for natural anesthetics (Hoseini et al., 2019). These observed effects may be attributed to the composition of ROEO, particularly its main component, 1,8-cineole, whose action on the central nervous system is well-documented in the literature. High-performance gas chromatography analysis revealed that 1,8-cineole comprises 47.5% of the oil composition, followed by camphor (19.3%) and α-pinene (12.2%). These terpenoid compounds are known for their biological properties, including antioxidant, anti-inflammatory activities, and synergistic effects in various applications. The anesthetic effect of R. officinalis is closely linked to 1,8-cineole, which modulates GABA-A receptors in the central nervous system and blocks voltage-dependent sodium channels in peripheral nerves, resulting in reduced neuronal excitability (Dougnon & Ito, 2020 ; Ferreira et al., 2015; Uusi-Oukari & Korpi, 2010). Studies such as Khumpirapang et al. ( 2018 ) have demonstrated the potent depressant effect of 1,8-cineole on the central nervous system, explaining the rapid anesthetic induction observed at higher ROEO concentrations. Additionally, 1,8-cineole significantly impacts the cardiovascular system, inducing bradycardia and modulating cardiac electrical activity. In this study, prolonged R-R and QT electrocardiographic intervals were observed, similar to effects reported in fish treated with other essential oils containing 1,8-cineole or camphor (Mazandarani & Hoseini, 2017 ). The reduction in QRS complex amplitude at higher doses (200 µL L⁻¹) may be associated with ventricular depression caused by R. officinalis , indicating a direct effect on heart contractility. Studies with essential oils, such as eucalyptus oil, have also reported reduced QRS complex amplitude, suggesting that these compounds may modulate cardiac electrical activity, affecting myocardial cell function (Taheri Mirghaed et al., 2018 ). The extension of the R-R interval observed at higher ROEO doses suggests modulation of the interval between heartbeats, consistent with induced bradycardia. These findings indicate that ROEO affects the cardiac cycle in a concentration-dependent manner, similar to findings in studies with clove oil and lemongrass (Bianchini et al., 2017 ). Furthermore, the increase in the P-Q interval and prolonged QRS complex duration at higher ROEO doses indicate delayed atrioventricular conduction, possibly related to the action of 1,8-cineole on calcium channels and the cardiac conduction system (Aktop et al., 2019 ). This effect is observed in other natural anesthetics and should be considered a critical parameter to determine safe doses, avoiding cardiac blocks or prolonged alterations in fish cardiac electrical conduction. Prolongation of the QT interval at higher ROEO doses suggests increased ventricular repolarization time, which may indicate a risk of arrhythmias if used at inappropriate concentrations. This finding is relevant because anesthetics that prolong the QT interval are often associated with a higher risk of arrhythmias (Schroeder et al., 2021 ). However, the reversibility of the effect after recovery, without arrhythmias, demonstrates that ROEO is safe within the tested dose range. The electrocardiographic data show that, for all concentrations of R. officinalis , there was a significant dose-dependent reduction in heart rate, but sinus rhythm was maintained, indicating that R. officinalis did not compromise the normal electrical function of the heart. This suggests that ROEO can be considered a safe anesthetic for use in fish, with a cardioprotective effect and no arrhythmias. These results support the findings of Krasteva, Yankova, & Ivanova ( 2021 ), who evaluated R. officinalis oil in Cyprinus carpio L. and found that, in addition to dose-dependent anesthetic induction, all oil concentrations were safe for fish transport for up to 4 hours without causing harm to the animals. The complete reversibility of the electrocardiographic changes observed during recovery with ROEO reinforces its safety profile for aquaculture, with sinus rhythm being progressively restored. This is especially advantageous, as natural anesthetics, like ROEO, often exhibit slower recovery due to their prolonged bioavailability (Can & Sümer, 2019 ). The near-total recovery of heart rate at lower concentrations also suggests that moderate ROEO doses can induce effective anesthesia without compromising long-term cardiovascular function. In contrast, MS-222 causes an abnormal respiratory response in fish, with rapid and irregular gill movements, indicating respiratory depression and stress. This abrupt behavior contrasts with essential oils, which tend to promote a smoother, more controlled anesthetic induction, reducing stress during the process and highlighting the safety of essential oils as natural anesthetics in aquaculture. This study demonstrated that Rosmarinus officinalis essential oil exerts a dose-dependent anesthetic effect on Colossoma macropomum . The findings also indicated that the main component of ROEO, 1,8-cineole, plays a key role in modulating the central nervous system and regulating cardiac activity. Electrocardiographic data revealed changes such as bradycardia and prolongation of the R-R and QT intervals, effects that were gradually reversed during recovery, reinforcing the safety of R. officinalis as a natural anesthetic. R. officinalis provided a gentler anesthetic induction with low impacts on the respiratory function of the fish, providing a more controlled anesthetic transition. These findings indicate that R. officinalis, in addition to being effective, is a safer and more sustainable alternative for fish management in aquaculture, offering an option that minimizes the side effects associated with synthetic anesthetics. Given the demonstrated efficacy and safety of R. officinalis, future studies may focus on assessing its impact on other fish species and examining its use under long-term conditions, such as for prolonged fish transport and handling. Research on the elimination of R. officinalis compounds and potential bioaccumulation may also provide important insights to ensure the safety of continued use. Furthermore, further studies on optimizing R. officinalis concentrations may help establish clear guidelines for its routine use in aquaculture, consolidating its potential as a natural anesthetic. Declarations Acknowledgments The authors thank the Laboratory of Pharmacology and Toxicology of Natural Products and the postgraduate program at the Federal University of Pará for the support given in carrying out the experiments in the article. Clinical trial number The study was experimental with fish and does not involve clinical trials on humans or companion animals, therefore is not applicable. Consent to participate No human participants were involved; the study was conducted under institutional ethics approval (CEUA/UFPA), which already covers the consent requirement, therefore it is not applicable. Consent publish All authors have read and approved the final version of the manuscript and consent to its publication. Ethical approval and ARRIVE statement All experimental procedures were conducted in accordance with national and institutional guidelines for animal care and use, following the principles of the Brazilian legislation (Law No. 11.794/2008) and the recommendations of the National Council for Animal Experimentation Control (CONCEA). The study protocol was approved by the Ethics Committee on Animal Use of the Federal University of Pará (CEUA-UFPA, protocol No. 5846030624/2024). Experimental design, randomization, and sample size were defined a priori to minimize the number of animals used and ensure reproducibility, in compliance with the ARRIVE 2.0 guidelines (Percie du Sert et al., 2020). Each animal constituted an independent experimental unit, and observers responsible for behavioral and electrophysiological recordings were blinded to treatment groups during data collection and analysis. Ethical approval and animal welfare statement All experimental procedures involving fish were conducted in strict accordance with the ethical principles and guidelines for animal experimentation established by the Brazilian National Council for the Control of Animal Experimentation (CONCEA; Law No. 11.794/2008). The experimental protocol was reviewed and approved by the Ethics Committee on Animal Use of the Federal University of Pará (CEUA-UFPA, protocol No. 5846030624/2024). Efforts were made to minimize the number of animals used and to reduce their suffering. Anesthetic and handling procedures were designed to ensure fish welfare throughout the experimental period, following the ARRIVE 2.0 guidelines (Percie du Sert et al., 2020). DAS statement request All data generated or analyzed during this study are included in this published article. No supplementary files are available. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Funding This work was supported by the Federal University of Pará (UFPA), through the Laboratory of Pharmacology and Toxicology of Natural Products, and by internal research grants from the Graduate Program in Biological Sciences (ICB-UFPA). No external funding agencies influenced the study design, data collection, or interpretation. CRediT authorship contribution statement Conceived and designed the experiments: Lorena Cristina Nunes de Almeida , Priscille Fidelis Pacheco Hartcopff and Moisés Hamoy . Performed the experiments: Daniella Bastos de Araújo , Axell Lins, Taissa Viana Damasceno, Luciana Esquerdo Cerqueira, Thaysa de Sousa Reis and Moisés Hamoy . Writing-original draft and editing: Axell Lins, Maria Klara Otake Hamoy, Marcelo Victor dos Santos Brito, Luana Vasconcelos de Souza, Sarah Farias Câmara, Luciana Eiro Quirino and Nilton Akio Muto . Financial support and administrative support: Moisés Hamoy . All authors have read and agreed to the published version of the manuscript. Data availability statement The datasets generated and analyzed during the current study (raw ECG traces, opercular activity records, and chromatographic profiles) are available from the corresponding author upon reasonable request. Processed data and scripts for signal analysis (Python 2.7, NumPy, SciPy, and Matplotlib) are stored in the institutional repository of the Federal University of Pará and can be provided for review or replication purposes. References Aktop Y, Aydın B, Çağatay İT (2019) Usability of coriander oil ( Coriandrum sativum ) as a herbal anesthetic on fish. Turkish Journal of Agriculture – Food Science and Technology 7(3): 23–26. https://doi.org/10.24925/turjaf.v7isp3.23-26.3128 Akrout A, Mighri H, Krid M, Thabet F, Turki H, El-Jani H, Neffati M (2012) Chemical composition and antioxidant activity of aqueous extracts of some wild medicinal plants in southern Tunisia. International Journal of Life Sciences and Medical Sciences 2: 1–4. Al-Maharik N, Jaradat N, Hawash M, Al-Lahham S, Qadi M, Shoman I, Issa L (2022) Chemical composition, antioxidant, antimicrobial and anti-proliferative activities of Rosmarinus officinalis essential oils from five sites in Palestine. Separations 9(11): 339. https://doi.org/10.3390/separations9110339 Al Zuhairi JJMJ, Kashi FJ, Rahimi-Moghaddam A, Yazdani M (2020) Antioxidant, cytotoxic and antibacterial activity of Rosmarinus officinalis L. essential oil against bacteria isolated from urinary tract infection. European Journal of Integrative Medicine 38: 101192. https://doi.org/10.1016/j.eujim.2020.101192 Andrade JM, Faustino C, Garcia C, Ladeiras D, Reis CP, Rijo P (2018) Rosmarinus officinalis L.: an update review of its phytochemistry and biological activity. Future Science OA 4(4): FSO283. https://doi.org/10.4155/fsoa-2017-0124 Aydın B, Barbas LAL (2020) Sedative and anesthetic properties of essential oils and their active compounds in fish: a review. Aquaculture 520: 734999. https://doi.org/10.1016/j.aquaculture.2020.734999 Barbas LAL, Torres MF, Costa BMP, Feitosa MJM, Maltez LC, Amado LL, Hamoy M (2021) Eugenol induces body immobilization yet evokes increased neuronal excitability in fish during short-term baths. Aquatic Toxicology 231: 105734. https://doi.org/10.1016/j.aquatox.2020.105734 Bianchini AE, Garlet QI, Da Cunha JA, Bandeira Junior G, Brusque ICM, Salbego J, Heinzmann BM, Baldisserotto B (2017) Monoterpenoids (thymol, carvacrol and S-(+)-linalool) with anesthetic activity in silver catfish ( Rhamdia quelen ): evaluation of acetylcholinesterase and GABAergic activity. Brazilian Journal of Medical and Biological Research 50(1): 1–8. https://doi.org/10.1590/1414-431x20176346 Brønstad A (2022) Good anesthesia practice for fish and other aquatics. Biology 11(9): 1355. https://doi.org/10.3390/biology11091355 Caceres AI, Liu B, Jabba SV, Achanta S, Morris JB, Jordt SE (2017) TRPM8 channels mediate the anti-inflammatory effects of eucalyptol. British Journal of Pharmacology 174(9): 867–879. https://doi.org/10.1111/bph.13760 Cai ZM, Peng JQ, Chen Y, Tao L, Zhang YY, Fu LY, Shen XC (2021) 1,8-Cineole: a review of source, biological activities, and applications. Journal of Asian Natural Products Research 23(10): 938–954. https://doi.org/10.1080/10286020.2020.1839432 Can E, Sümer E (2019) Anesthetic and sedative efficacy of peppermint ( Mentha piperita ) and lavender ( Lavandula angustifolia ) essential oils in blue dolphin cichlid ( Cyrtocara moorii ). Turkish Journal of Veterinary and Animal Sciences 43: 334–341. https://doi.org/10.3906/vet-1809-22 Dhouibi I, Flamini G, Bouaziz M (2023) Comparative study on the essential oils extracted from Tunisian rosemary and myrtle: chemical profiles, quality, and antimicrobial activities. ACS Omega 8: 6431–6438. https://doi.org/10.1021/acsomega.2c06713 de Araújo ERL, Silva JDS, Lopes LM, Torres MF, Costa BMA, Amarante CB, Sampaio LA (2021) Geraniol and citronellol as alternative and safe phytoconstituents to induce immobilization and facilitate handling of fish. Aquaculture 537: 736517. https://doi.org/10.1016/j.aquaculture.2021.736517 Dougnon G, Ito M (2020) Inhalation administration of the bicyclic ethers 1,8- and 1,4-cineole prevent anxiety and depressive-like behaviours in mice. Molecules 25: 8184. https://doi.org/10.3390/molecules25081884 Elmas E, Karadal O (2022) Anesthetic and sedative effects of plant-derived essential oils on red swamp crayfish ( Procambarus clarkii ) at different concentrations and temperatures. Nauplius 30: e2022035. https://doi.org/10.1590/2358-2936e2022035 Gonzalez-Minero F, Bravo-Diaz L, Ayala-Gomez A (2020) Rosmarinus officinalis L. (rosemary): an ancient plant with uses in personal healthcare and cosmetics. Plants 7(1): 1–17. https://doi.org/10.3390/cosmetics7040077 Hoseini SM et al. (2020) Antioxidant and immune responses of common carp ( Cyprinus carpio ) anesthetized by cineole: effects of anesthetic concentration. Aquaculture 520: 734680. https://doi.org/10.1016/j.aquaculture.2019.734680 Kheawfu K, Pikulkaew S, Rades T, Müllertz A, von Gersdorff Jørgensen L, Okonogi S (2021) Design and optimization of self-nanoemulsifying drug delivery systems of clove oil for efficacy enhancement in fish anesthesia. Journal of Drug Delivery Science and Technology 61: 102241. https://doi.org/10.1016/j.jddst.2020.102241 Kizak V, Can E, Danabaş D, Can ŞS (2018a) Evaluation of anesthetic potential of rosewood ( Aniba rosaeodora ) oil as a new anesthetic agent for goldfish ( Carassius auratus ). Aquaculture 493: 296–301. https://doi.org/10.1016/j.aquaculture.2018.05 Kizak V, Guner Y, Kayim M, Can E (2013b) The effects of clove oil on adult males and females of rainbow trout ( Oncorhynchus mykiss ) and brown trout ( Salmo trutta fario ). Journal of Food, Agriculture & Environment 11: 1542–1545. Khumpirapang N et al. (2018) In vivo anesthetic effect and mechanism of action of active compounds from Alpinia galanga oil on Cyprinus carpio . Aquaculture 496: 1–7. https://doi.org/10.1016/j.aquaculture.2018.07.017 Kondo S, El Omri A, Han J, Isoda H (2015) Antidepressant-like effects of rosmarinic acid through mitogen-activated protein kinase phosphatase-1 and brain-derived neurotrophic factor modulation. Journal of Functional Foods 14: 758–766. https://doi.org/10.1016/j.jff.2015.03.001 Krasteva V, Yankova M, Ivanova A (2021) Anesthetic effect of rosemary essential oil ( Rosmarinus officinalis L.) and its use for transport of common carp ( Cyprinus carpio L.) stocking material. Institute of Fisheries and Aquaculture Reports , Plovdiv, Bulgaria. Lahlou S et al. (2002) Cardiovascular effects of 1,8-cineole, a terpenoid oxide present in many plant essential oils, in normotensive rats. Canadian Journal of Physiology and Pharmacology 80(12): 1125–1131. https://doi.org/10.1139/Y02-142 Li Pomi F, Papa V, Borgia F, Vaccaro M, Allegra A, Cicero N, Gangemi S (2023) Rosmarinus officinalis and skin: antioxidant activity and possible therapeutical role in cutaneous diseases. Antioxidants 12(3): 680. https://doi.org/10.3390/antiox12030680 Mazandarani M, Hoseini SM (2017) Menthol and 1,8-cineole as new anaesthetics in common carp ( Cyprinus carpio ). Aquaculture Research 48: 3041–3051. https://doi.org/10.1111/are.13136 Mirghaed A et al. (2018) Anaesthetic efficacy and biochemical effects of 1,8-cineole in rainbow trout ( Oncorhynchus mykiss ). Aquaculture Research 49: 2156–2165. https://doi.org/10.1111/are.13671 Mocho JP (2024) Anaesthesia, analgesia and zebrafish: a practical guide to husbandry, welfare and research methodology. In: Zebrafish: A Practical Guide to Husbandry, Welfare and Research Methodology , p. 427. Moon HK, Kang P, Lee HS, Min SS, Seol GH (2014) Effects of 1,8-cineole on hypertension induced by chronic exposure to nicotine in rats. Journal of Pharmacy and Pharmacology 66(5): 688–693. https://doi.org/10.1111/jphp.12195 Neiffer DL (2021) Anesthesia and analgesia. In: Clinical Guide to Fish Medicine . Wiley-Blackwell, pp. 198–212. https://doi.org/10.1002/9781119259886.ch10 Percie du Sert N et al. (2020) The ARRIVE guidelines 2.0: Updated guidelines for reporting animal research. PLOS Biology 18(7): e3000410. https://doi.org/10.1371/journal.pbio.3000410 Purbosari N, Warsiki E, Syamsu K, Santoso J (2019) Natural versus synthetic anesthetics for transport of live fish: a review. Aquaculture and Fisheries Studies 4: 129–133. https://doi.org/10.1016/j.aaf.2019.03.002 Rairat T, Chi Y, Hsieh CY, Liu YK, Chuchird N, Chou CC (2021) Determination of optimal doses and minimum effective concentrations of tricaine methanesulfonate, 2-phenoxyethanol and eugenol for laboratory management in Nile tilapia ( Oreochromis niloticus ). Animals 11: 1521. https://doi.org/10.3390/ani11061521 Reis TDS, Araújo DBD, Paz CAD, Santos RG, Barbosa ADS, Souza LVD, Hamoy M (2024) Etomidate as an anesthetic in Colossoma macropomum : behavioral and electrophysiological data complement each other as a tool to assess anesthetic safety. PLOS ONE 19(8): e0305093. https://doi.org/10.1371/journal.pone.0305093 Roohi Z, Imanpoor MR (2015) The efficacy of spearmint and methyl salicylate oils as new anesthetics and their effect on glucose levels in common carp ( Cyprinus carpio ). Aquaculture 439: 1–6. https://doi.org/10.1016/j.aquaculture.2014.12.019 Rucinque DS, Ferreira PF, Leme PRP, Lapa-Guimaraes J, Viegas EMM (2021) Ocimum americanum and Lippia alba essential oils as anaesthetics for Nile tilapia: induction, recovery and sensory analysis of fillets. Aquaculture 531: 735902. https://doi.org/10.1016/j.aquaculture.2020.735902 Sadeh D et al. (2019) Interactive effects of genotype, seasonality and extraction method on chemical compositions and yield of rosemary essential oil. Industrial Crops and Products 138: 111240. https://doi.org/10.1016/j.indcrop.2019.05.068 Schroeder P, Lloyd R, McKimm R, Metselaar M, Navarro J, O’Farrell M, Readman GD, Speilberg L, Mocho JP (2021) Anaesthesia of laboratory, aquaculture and ornamental fish: proceedings of the first LASA-FVS symposium. Laboratory Animals 55(4): 317–328. https://doi.org/10.1177/0023677221998403 Seyidoglu N, Yagcilar C (2020) The anesthetic role of some herbal oils for zebrafish. Erciyes Üniversitesi Veteriner Fakültesi Dergisi 17(3): 209–214. https://doi.org/10.32707/ercivet.828319 Sharma AD, Kaur I, Kaur J, Chauhan A (2024) Chemical profiling and in-vitro antioxidant, anti-diabetic, anti-inflammatory, anti-bacterial and anti-fungal activities of essential oil from Rosmarinus officinalis L. Notulae Scientia Biologicae 16(1): 11756. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7964102","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":546280395,"identity":"65f26661-7bb1-42ab-952c-204d74c78d31","order_by":0,"name":"Lorena Cristina Nunes Almeida","email":"","orcid":"","institution":"Federal University of Pará","correspondingAuthor":false,"prefix":"","firstName":"Lorena","middleName":"Cristina Nunes","lastName":"Almeida","suffix":""},{"id":546280397,"identity":"e09c9abe-d30f-4d7f-ba05-4d4d382105a4","order_by":1,"name":"Priscille Fidelis Pacheco Hartcopff","email":"","orcid":"","institution":"Federal University of Pará","correspondingAuthor":false,"prefix":"","firstName":"Priscille","middleName":"Fidelis Pacheco","lastName":"Hartcopff","suffix":""},{"id":546280399,"identity":"950990a2-8564-470b-8ef4-b419fb6d590d","order_by":2,"name":"Daniella Bastos de Araújo","email":"","orcid":"","institution":"Federal University of Pará","correspondingAuthor":false,"prefix":"","firstName":"Daniella","middleName":"Bastos","lastName":"de Araújo","suffix":""},{"id":546280400,"identity":"dd1a91ae-d491-44d9-a848-241912d5727d","order_by":3,"name":"Axell Lins","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4UlEQVRIiWNgGAWjYHACxgMPGBh4+JmZDwA5EjJE6TmQwGDAI9nelgDSwkO0FgaDM2cMQBzCWvhnJB8Aavkjw3Aj5/OrGzUWPAzsh49uwKdF4syxBLDDGGfkbrPOOQZ0GE9a2g281hzvMQBrYZbI3WacwwbUIsFjhleL/GH+D2AtbBI5z4xz/hGhxeB4DyTEeHjOMD/ObSNCi+GZY0CHGRjzSLC3mTHn9knwsBHyi9yN5IcPPlTI2dsfZn78OedbnRw/++Fj+L0PcR6YZJMAk4SVIwDzB1JUj4JRMApGwcgBAOHwRcMGOts+AAAAAElFTkSuQmCC","orcid":"","institution":"Federal University of Pará","correspondingAuthor":true,"prefix":"","firstName":"Axell","middleName":"","lastName":"Lins","suffix":""},{"id":546280401,"identity":"f9625417-d575-4cbe-9466-e71f6c3a95fe","order_by":4,"name":"Taissa Viana Damasceno","email":"","orcid":"","institution":"Federal University of Pará","correspondingAuthor":false,"prefix":"","firstName":"Taissa","middleName":"Viana","lastName":"Damasceno","suffix":""},{"id":546280402,"identity":"d08c4af7-b7ac-42e8-8721-8f7a445be914","order_by":5,"name":"Luciana Esquerdo Cerqueira","email":"","orcid":"","institution":"Federal University of Pará","correspondingAuthor":false,"prefix":"","firstName":"Luciana","middleName":"Esquerdo","lastName":"Cerqueira","suffix":""},{"id":546280403,"identity":"f83d2ef1-e061-4975-a38a-d5cadf8f504a","order_by":6,"name":"Thaysa de Sousa Reis","email":"","orcid":"","institution":"Federal University of Pará","correspondingAuthor":false,"prefix":"","firstName":"Thaysa","middleName":"de Sousa","lastName":"Reis","suffix":""},{"id":546280404,"identity":"b9a112bc-99a7-4d5e-85c9-77d40c73f664","order_by":7,"name":"Maria Klara Otake Hamoy","email":"","orcid":"","institution":"Federal University of Pará","correspondingAuthor":false,"prefix":"","firstName":"Maria","middleName":"Klara Otake","lastName":"Hamoy","suffix":""},{"id":546280405,"identity":"13038041-78cd-4251-ab01-691ae2ce14fb","order_by":8,"name":"Marcelo Victor dos Santos Brito","email":"","orcid":"","institution":"Federal University of Pará","correspondingAuthor":false,"prefix":"","firstName":"Marcelo","middleName":"Victor dos Santos","lastName":"Brito","suffix":""},{"id":546280406,"identity":"cc2b3ef5-3bfa-4904-bcdf-e98f4b2a66ef","order_by":9,"name":"Luana Vasconcelos Souza","email":"","orcid":"","institution":"Federal University of Pará","correspondingAuthor":false,"prefix":"","firstName":"Luana","middleName":"Vasconcelos","lastName":"Souza","suffix":""},{"id":546280407,"identity":"d2cf543c-8309-42fa-984e-234dc92594a1","order_by":10,"name":"Sarah Farias Câmara","email":"","orcid":"","institution":"Federal University of Pará","correspondingAuthor":false,"prefix":"","firstName":"Sarah","middleName":"Farias","lastName":"Câmara","suffix":""},{"id":546280408,"identity":"25e7396c-fd7a-466f-ab73-42e909f820eb","order_by":11,"name":"Luciana Eiro Quirino","email":"","orcid":"","institution":"Federal University of Pará","correspondingAuthor":false,"prefix":"","firstName":"Luciana","middleName":"Eiro","lastName":"Quirino","suffix":""},{"id":546280409,"identity":"ee62283d-3d78-4160-ada2-84a925e659c0","order_by":12,"name":"Nilton Akio Muto","email":"","orcid":"","institution":"Center for the Valorization of Bioactive Compounds from the Amazon. 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07:41:12","extension":"png","order_by":73,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":18531,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-7964102/v1/6fa1e7ebde0fd6c8fd20516b.png"},{"id":96252715,"identity":"391de31d-4ee5-44ce-8107-d98f14f364db","added_by":"auto","created_at":"2025-11-19 07:41:22","extension":"png","order_by":74,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":65606,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-7964102/v1/c45bea2ab2759c0cce169681.png"},{"id":96252375,"identity":"7c7cb8bd-b544-4a5c-80a6-5d20eed5b05d","added_by":"auto","created_at":"2025-11-19 07:40:51","extension":"png","order_by":75,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":33792,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-7964102/v1/3023452a9c3d436597623b42.png"},{"id":96193533,"identity":"448dde7d-103f-4503-b1ca-6e4582211ced","added_by":"auto","created_at":"2025-11-18 15:00:32","extension":"xml","order_by":76,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":151728,"visible":true,"origin":"","legend":"","description":"","filename":"97ef3ac6eeef4a039de36732c9a0289e1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7964102/v1/a125675f3ae0a157a40f2f25.xml"},{"id":96193554,"identity":"abd6d356-cde5-4206-8a6c-5e59a2c2bb4f","added_by":"auto","created_at":"2025-11-18 15:00:34","extension":"html","order_by":77,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":170207,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7964102/v1/c24491ea406f353f245dc2dc.html"},{"id":96193503,"identity":"ef2de8e0-7144-46f0-a303-4f6c911c1b03","added_by":"auto","created_at":"2025-11-18 15:00:31","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":211040,"visible":true,"origin":"","legend":"\u003cp\u003eChromatogram of the \u003cem\u003eRosmarinus officinalis\u003c/em\u003e essential oil sample from the Chromatography Laboratory, Department of Chemistry – Federal University of Minas Gerais, Belo Horizonte, Brazil.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7964102/v1/b7e694d213586397584f71c6.png"},{"id":96193486,"identity":"3dea18c2-f23d-4465-9c4d-1c16847de5bb","added_by":"auto","created_at":"2025-11-18 15:00:30","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":226173,"visible":true,"origin":"","legend":"\u003cp\u003eAverage latencies (s) for loss of postural reflex during immersion baths with different ROEO treatments (A). Recovery of postural reflex after contact with different concentrations of ROEO (B). (ANOVA followed by Tukey's test; *\u003cem\u003ep\u0026lt;\u003c/em\u003e0.05, **p\u0026lt;0.01, and ***p\u0026lt;0.001).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7964102/v1/a88352e698fb4ceec26762e6.png"},{"id":96193487,"identity":"355ed653-53a8-4bc0-a699-7a1210282996","added_by":"auto","created_at":"2025-11-18 15:00:30","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":868412,"visible":true,"origin":"","legend":"\u003cp\u003eNormal electrocardiographic recording (ECG) of the tambaqui, \u003cem\u003eColossoma macropomum\u003c/em\u003e (A); enlarged electrocardiogram during the final 30 seconds of recording (270-300 s) (B); amplification of the last 5 seconds of the recording, demonstrating the morphological elements: P wave, QRS complex, and T wave, as well as analyzed elements: heart rate (bpm), P-Q interval, R-R interval (s), duration of QRS (s), Q-T interval (s) (C).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7964102/v1/801fa22d6f965a85d8e3fe52.png"},{"id":96193490,"identity":"e7d752d2-5660-4859-b84b-bcd71243c531","added_by":"auto","created_at":"2025-11-18 15:00:30","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":963900,"visible":true,"origin":"","legend":"\u003cp\u003eElectrocardiographic recordings demonstrating cardiac activity in juvenile \u003cem\u003eColossoma macropomum\u003c/em\u003e (left), amplification of the last 30 seconds of the 5-minute recording (270 to 300 s) (center), showing the evaluated graph elements, including the P wave, QRS complex, and T wave (amplification of 5 s) (right). Cardiac activity in juvenile \u003cem\u003eColossoma macropomum\u003c/em\u003e during immersion with the vehicle, with a description of the cardiac graph elements (A); groups treated with immersion at different concentrations of ROEO, along with their amplification and identification of cardiac deflections: 100 µL L⁻¹ (B), 125 µL L⁻¹ (C), 150 µL L⁻¹ (D), 175 µL L⁻¹ (E), and 200 µL L⁻¹ (F).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7964102/v1/d98398a5a26f9f5fa829bb61.png"},{"id":96193512,"identity":"0d07c87e-67c5-4b9f-b54d-ee3ed4d053b7","added_by":"auto","created_at":"2025-11-18 15:00:31","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":449741,"visible":true,"origin":"","legend":"\u003cp\u003eAverage values of heart rate in beats per minute (bpm) (A), average amplitude values of the QRS complex (mV) (B), average RR interval values (ms) (C), average PQ interval (ms) (D), duration of the QRS complex (ms) (E), and QT interval (ms) (F) during exposure to ROEO at concentrations of 100 µL L⁻¹, 125 µL L⁻¹, 150 µL L⁻¹, 175 µL L⁻¹, and 200 µL L⁻¹ (ANOVA followed by Tukey's test; *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001; n=9).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7964102/v1/75ef175fd6acd0a6acf31535.png"},{"id":96193478,"identity":"7532d96a-c31e-4736-a5db-bbad2a2a2fea","added_by":"auto","created_at":"2025-11-18 15:00:28","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":828548,"visible":true,"origin":"","legend":"\u003cp\u003eCardiac activity in juvenile \u003cem\u003eColossoma macropomum\u003c/em\u003e during recovery following immersion baths with different concentrations of ROEO (left). Amplification of the recording during the last 30 seconds (270-300s) for identification of cardiac deflections (center) during the recovery period after immersion with the following concentrations of ROEO (5s amplification) (right): 100 µL L⁻¹ (A), 125 µL L⁻¹ (B), 150 µL L⁻¹ (C), 175 µL L⁻¹ (D), and 200 µL L⁻¹ (E).\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7964102/v1/5163074393b6969f21e0b86d.png"},{"id":96193505,"identity":"59e80795-0867-4960-bfce-8020208783a4","added_by":"auto","created_at":"2025-11-18 15:00:31","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":347003,"visible":true,"origin":"","legend":"\u003cp\u003eMean values of cardiac parameters during recovery following exposure to different concentrations of ROEO in immersion baths at 100 µL L⁻¹, 125 µL L⁻¹, 150 µL L⁻¹, 175 µL L⁻¹, and 200 µL L⁻¹. Mean values of heart rate (bpm) (A), amplitude of the QRS complex (mV) (B), RR interval (ms) (C), PQ intervals (ms) (D), duration of the QRS complex (ms) (E), and QT interval (ms) (F). (ANOVA followed by Tukey's test; *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001; n = 9).\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-7964102/v1/30dc033c70647eca71ca5638.png"},{"id":96193516,"identity":"a2f024d0-d27a-450b-830e-0e54f2f14dfd","added_by":"auto","created_at":"2025-11-18 15:00:31","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":3654891,"visible":true,"origin":"","legend":"\u003cp\u003eRecord of opercular activity in juveniles of \u003cem\u003eColossoma macropomum\u003c/em\u003eduring immersion in different concentrations of ROEO (left). Amplification of the record over the last 10 seconds (290-300 s) (center); spectrogram of energy distribution during immersion at different concentrations of ROEO (right). Observations were made for the following groups: Control group (A); Vehicle group (B); Group treated with 100 µL L⁻¹ (C); Group treated with 125 µL L⁻¹ (D); Group treated with 150 µL L⁻¹ (E); Group treated with 175 µL L⁻¹ (F); and Group treated with 200 µL L⁻¹ (G). It presents the mean values of opercular movement per minute (mpm) (H) and the mean values of opercular movement power (mV²/Hz) (I). (ANOVA followed by Tukey’s test; *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001; n=9).\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-7964102/v1/8b8b2bb3ed4ccb9bb8a6c3bf.png"},{"id":96193479,"identity":"ecbf0f36-6e0a-4037-8087-b18624a72ed3","added_by":"auto","created_at":"2025-11-18 15:00:29","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":2403222,"visible":true,"origin":"","legend":"\u003cp\u003eOpercular movement recording in juvenile \u003cem\u003eColossoma macropomum\u003c/em\u003eduring recovery after immersion baths with different concentrations of ROEO (left). Amplification of the recording in the last 10 seconds (290-300s) to identify the movements (center), and a spectrogram indicating the energy level during the animals’ recovery (right). Data were obtained after immersion baths with the following concentrations: 100 µL L⁻¹ (A), 125 µL L⁻¹ (B), 150 µL L⁻¹ (C), 175 µL L⁻¹ (D), and 200 µL L⁻¹ (E). Mean values of opercular movements per minute (F); mean power values of opercular movement (mV²/Hz) (G). (ANOVA followed by Tukey's test; *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001; n=9).\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-7964102/v1/2ca42b953c1f2fdbd8283a90.png"},{"id":97328592,"identity":"aaae4756-12d4-4517-8080-18e8fcd30f8e","added_by":"auto","created_at":"2025-12-03 08:55:22","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":13771063,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7964102/v1/27e455c1-b7dd-4de4-8ca9-fb92ab6e09f7.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Rosmarinus officinalis essential oil components promote anesthesia in Colossoma macropomum (Cuvier, 1818) through behavioral and cardiorespiratory modulation","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe use of anesthesia in aquaculture and scientific research involving fish plays a fundamental role in maintaining the health and welfare of these animals. Handling procedures, both for productive and experimental purposes, often involve invasive techniques that can cause stress and physical harm to fish (Aktop et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Br\u0026oslash;nstad, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Barbas et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Kheawfu, 2021; Reis et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). In this context, anesthetic use is essential to minimize such impacts and ensure more humane practices, providing safety for both animals and operators or consumers, while reflecting a commitment to ethical principles in animal management (Aydın and Barbas, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Rairat et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Schroeder et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Mocho, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eSynthetic anesthetics, such as MS-222 (tricaine methanesulfonate) and Benzocaine, are widely used in aquaculture. However, their use has been associated with undesirable side effects, including respiratory and cardiac depression, as well as the potential accumulation of residues in fish tissues, compromising aquaculture product quality and causing adverse effects on animal health and the environment (Taheri Mirghaed et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2018\u003c/span\u003eb; Can and S\u0026uuml;mer, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Neiffer, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Consequently, there has been growing interest in safer and more sustainable alternatives, such as plant-derived natural anesthetics, which offer a more favorable profile in terms of toxicity, biodegradability, and economic accessibility (Bianchini et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Seyidoglu and Yagcilar, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; de Ara\u0026uacute;jo et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Rucinque, 2021; Elmas and Karadal, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Zeng, 2022).\u003c/p\u003e\u003cp\u003eAmong the promising natural anesthetics is the essential oil of \u003cem\u003eRosmarinus officinalis\u003c/em\u003e, a perennial plant from the Lamiaceae family widely recognized for its diverse pharmacological properties, including antibacterial, antidiabetic, anti-inflammatory, antitumor, and antioxidant actions (Andrade et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Al Zuhairi, 2020; Zhong et al., 2021; Al-Mahariki et al., 2022; Li Pomi et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Sharma, 2024). Among its main components, 1,8-cineole (eucalyptol) stands out in experimental studies due to its ability to act on the central nervous system, activating transient cation channels that promote desensitization and result in anesthetic effects (Caceres, 2017; Khumpirapang et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Sadeh et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Cai et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Dhouibi, Flamini \u0026amp; Bouaziz, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In addition to its anesthetic properties, 1,8-cineole also influences autonomic cardiovascular regulation, demonstrating effects such as bradycardia and reduced blood pressure, underscoring its therapeutic potential (Lahlou et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Moon, 2014; Wang, 2021).\u003c/p\u003e\u003cp\u003eIn fish experiments, 1,8-cineole has proven to be an effective anesthetic, with induction and recovery times directly dependent on the concentration used. In common carp (\u003cem\u003eCyprinus carpio\u003c/em\u003e), results indicate that higher concentrations reduce induction time, highlighting the potential of 1,8-cineole as a promising natural anesthetic for use in aquaculture (Hoseini et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Mirghaed et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Mazandarani \u0026amp; Hoseini, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Furthermore, studies in rats have shown that intravenous administration of 1,8-cineole significantly reduces mean arterial pressure and heart rate in a dose-dependent manner, also indicating its effect on the cardiovascular system (Lahlou et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). Despite these advances, there remains a research gap concerning the cardiovascular effects of 1,8-cineole in different fish species, which justifies the need to expand its evaluation.\u003c/p\u003e\u003cp\u003eThe present study aims to investigate the anesthetic effect of \u003cem\u003eRosmarinus officinalis\u003c/em\u003e essential oil in \u003cem\u003eColossoma macropomum\u003c/em\u003e (tambaqui), one of the most widely farmed species in Brazilian aquaculture. It was sought to determine optimal dose-response relationships for different concentrations of the oil and to assess cardiovascular safety and behavioral effects in fish during the induction and recovery phases of anesthesia. This study aims to provide a safer and more sustainable alternative to synthetic anesthetics, contributing to animal welfare and promoting more ethical and sustainable practices in aquaculture.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Experimental Animals\u003c/h2\u003e\u003cp\u003eThe subjects used were \u003cem\u003eColossoma macropomum\u003c/em\u003e tambaqui (n\u0026thinsp;=\u0026thinsp;108), housed in aquariums in the Experimental Animal Facility of the Laboratory of Pharmacology and Toxicology of Natural Products at the Federal University of Par\u0026aacute; (ICB/UFPA). The environment was maintained at a controlled temperature (25\u0026ndash;27\u0026deg;C) with a 12 h light: 12 h dark photoperiod. The fish were fed commercial feed (32% protein) twice daily until satiation. Simultaneously, uneaten food and feces were removed by siphoning, and the water was partially renewed (approximately 40% of tank volume) with water from the same source. During the acclimatization period (20 days), water quality variables such as water temperature (\u0026deg;C) (26.5\u0026deg;C) and pH (pH\u0026thinsp;=\u0026thinsp;7.5) were monitored. The project was previously approved by CEUA-UFPA under registration 5846030624 (ID 002640).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Organoleptic Properties and Chromatographic Analysis\u003c/h2\u003e\u003cp\u003eRosemary essential oil (\u003cem\u003eRosmarinus officinalis\u003c/em\u003e) \u0026ndash; ROEO - was acquired from Harmonie Aromatherapy (Florian\u0026oacute;polis, SC, Brazil, CNPJ: 11.938.821/0001\u0026ndash;90). The oil was extracted by steam distillation, and analysis was conducted by Gas Chromatography using an AGILENT 7820A Gas Chromatograph under the following conditions: Column: RXi-5MS 30 m x 0.25 mm x 0.25 \u0026micro;m (Restek). Temperature program: Column at 50\u0026deg;C (0 min), increasing at 3\u0026deg;C/min to 200\u0026deg;C. Injector: 200\u0026deg;C, split ratio: 1/50. FID detector: 220\u0026deg;C. Injection volume: 1 \u0026micro;L (1% in ethyl acetate). The phytoconstituents of the oil were identified as follows: Eucalyptol (47.5%), Camphor (19.3%), α-pinene (12.2%), β-pinene (7.8%), and β-caryophyllene (4.6%).\u003c/p\u003e\u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e \u003cb\u003ehere\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eChemical composition \u003cem\u003eRosmarinus Officinalis\u003c/em\u003e essential oil\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRetention index\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eIdentification\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePercentage (%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003ePeak\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e913\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAlpha-pinene\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e12.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e925\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCamphene\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e950\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBeta-pinene\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e7.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e963\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMyrcene\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e995\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePara-cymene\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1000\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLimonene\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1003\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eEucalyptol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e47.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1031\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGamma terpinene\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1073\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLinalool\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e9\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1121\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCamphor\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e19.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1148\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBorneol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e11\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1159\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eTerpinen-4-ol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e12\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1174\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAlpha terpineol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e13\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1281\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBornyl acetate\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e14\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1425\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBeta-caryophyllene\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e4.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e15\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1461\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eHumolene\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOthers\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"4\"\u003eSource: Prepared by the authors, 2024\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eFigure \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e \u003cb\u003ehere\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3. Experimental Design\u003c/h2\u003e\u003cdiv id=\"Sec6\" class=\"Section3\"\u003e\u003ch2\u003e2.3.1. Experiment with Rosmarinus officinalis Essential Oil (ROEO)\u003c/h2\u003e\u003cp\u003eJuvenile \u003cem\u003etambaqui\u003c/em\u003e (26.18\u0026thinsp;\u0026plusmn;\u0026thinsp;3.5g) were randomly assigned to the following treatments: a) Control; b) vehicle group (fish subjected to immersion bath with 2 ml of 70% alcohol diluted in 1 liter of aquarium water); c) fish treated with ROEO 100 \u0026micro;L L⁻\u0026sup1;; d) 125 \u0026micro;L L⁻\u0026sup1;; e) 150 \u0026micro;L L⁻\u0026sup1;; f) 175 \u0026micro;L L⁻\u0026sup1;; and g) 200 \u0026micro;L L⁻\u0026sup1;. All fish underwent anesthetic induction with a contact period of 5 minutes, followed by observation during recovery for 5 minutes in water without ROEO. For each recording, \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;9 per treatment was used (immersion bath with ROEO and recovery post-immersion bath), totaling 108 animals.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section3\"\u003e\u003ch2\u003e2.3.2. Experiment 1 - Behavioral Analysis of Anesthetic Induction and Recovery\u003c/h2\u003e\u003cp\u003eConsidering the exposure time for the following ROEO treatments: a) 100 \u0026micro;L L⁻\u0026sup1;, b) 125 \u0026micro;L L⁻\u0026sup1;, c) 150 \u0026micro;L L⁻\u0026sup1;, d) 175 \u0026micro;L L⁻\u0026sup1;, and e) 200 \u0026micro;L L⁻\u0026sup1; (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;9 per treatment), the latency to the behavior of postural reflex loss (characterized by lateral recumbency) was evaluated. Following removal from ROEO contact, the latency for recovery of postural reflex was recorded.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section3\"\u003e\u003ch2\u003e2.3.3. Experiment 2 - Electrocardiogram (ECG) Analysis\u003c/h2\u003e\u003cp\u003eFor cardiac function analysis and monitoring, groups were divided as follows: a) Control group; b) vehicle group; c) treated group with ROEO 100 \u0026micro;L L⁻\u0026sup1;; d) 125 \u0026micro;L L⁻\u0026sup1;; e) 150 \u0026micro;L L⁻\u0026sup1;; f) 175 \u0026micro;L L⁻\u0026sup1;; and g) 200 \u0026micro;L L⁻\u0026sup1; (a total of 63 animals). Electrodes were made from 0.3 mm diameter, 10 mm long 925 silver electrodes, and subsequently isolated with liquid insulation. Electrodes were non-conjugated. The reference electrode positioning followed the cardiac vector indication (negative pole at the cardiac base and positive pole at the apex). The reference electrode was fixed to the ventral portion of the left opercular opening 0.2 mm past the opercular cavity. The recording electrode was inserted 2.0 mm from the right opercular opening, capturing the signal on lead D1. The electrodes were connected to a high-impedance amplifier (Grass Technologies, Model P511) for ECG recording, enabling analysis of heart rate (bpm), QRS complex amplitude (mV), QRS complex duration (ms), and R-R (ms), P-Q (ms), and Q-T intervals (ms).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section3\"\u003e\u003ch2\u003e2.3.4. Experiment 3 - Opercular Activity Recording\u003c/h2\u003e\u003cp\u003eFor opercular activity analysis, 925 silver electrodes were made with a 0.5 mm diameter and 15 mm length, conjugated at a 7 mm distance and isolated with liquid insulation. The electrodes were placed at the center of the right opercular opening to record opercular beating, assessing the frequency (beats per minute) and power of opercular beating (mV\u0026sup2;/Hz).\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.4. Recording and Analysis of Data\u003c/h2\u003e\u003cp\u003eThe electrodes were connected to a digital data acquisition system via a differential high-impedance amplifier (Grass Technologies, Model P511), set with 0.3 to 300 Hz filtering, 2000X amplification, and monitored by an oscilloscope (ProteK, Model 6510). Recordings were continuously digitized at a 1 KHz rate on a computer equipped with a data acquisition board (National Instruments, Austin, TX), and stored on a hard drive for further processing via specialized software (LabVIEW express). The acquired signals were analyzed using a tool developed in Python version 2.7. Numpy and Scipy libraries were used for mathematical processing, while Matplotlib was used for graph plotting. The graphical interface was created with the PyQt4 library (Da Paz, 2024).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.5. Statistical Analysis\u003c/h2\u003e\u003cp\u003eAfter confirming normality and homogeneity of variances with the Kolmogorov-Smirnov and Levene tests, respectively, comparisons of mean power values were made via one-way ANOVA, followed by Tukey\u0026rsquo;s test. GraphPad Prism\u0026reg; 8 software was used for the analyses, with p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, *p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, and **p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 considered statistically significant in all cases.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cp\u003eThe behavioral analysis showed that ROEO caused a concentration-dependent loss of postural reflex, with higher doses resulting in shorter latency for reflex loss onset. Fish exposed to 100 \u0026micro;L L⁻\u0026sup1; exhibited an average postural reflex loss at 214.1\u0026thinsp;\u0026plusmn;\u0026thinsp;16.66 s, which was similar to the group treated with 125 \u0026micro;L L⁻\u0026sup1; (p\u0026thinsp;=\u0026thinsp;0.060). However, these were longer than in the other groups. The group treated with 150 \u0026micro;L L⁻\u0026sup1; (151.4\u0026thinsp;\u0026plusmn;\u0026thinsp;18.76 s), 175 \u0026micro;L L⁻\u0026sup1; (131.2\u0026thinsp;\u0026plusmn;\u0026thinsp;8.89 s), and 200 \u0026micro;L L⁻\u0026sup1; (110.9\u0026thinsp;\u0026plusmn;\u0026thinsp;13.57 s) were significantly different (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe recovery of postural reflex in the group treated with 100 \u0026micro;L L⁻\u0026sup1; ROEO occurred in 87.0\u0026thinsp;\u0026plusmn;\u0026thinsp;11.75 s and was shorter than in the other groups: 125 \u0026micro;L L⁻\u0026sup1; (124.6\u0026thinsp;\u0026plusmn;\u0026thinsp;12.38 s), 150 \u0026micro;L L⁻\u0026sup1; (154.7\u0026thinsp;\u0026plusmn;\u0026thinsp;15.80 s), 175 \u0026micro;L L⁻\u0026sup1; (172.2\u0026thinsp;\u0026plusmn;\u0026thinsp;11.01 s), and 200 \u0026micro;L L⁻\u0026sup1; (208.1\u0026thinsp;\u0026plusmn;\u0026thinsp;16.13 s). All groups showed recovery time dependent on the concentration used, with higher concentrations resulting in longer recovery times, indicating a slower reversibility of the effect for groups that received higher concentrations (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003eFigure \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e \u003cb\u003e(A and B) here\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe normal electrocardiogram of the tambaqui exhibited a sinus rhythm with an average frequency of 86.89\u0026thinsp;\u0026plusmn;\u0026thinsp;2.47 bpm. The P wave, QRS complex, and T wave can be identified (Figures A, B, and C). The intervals between R-R deflections, as well as the P-Q and Q-T intervals, remained regular considering the normal cardiac parameters. Thus, the interference of increasing doses of ROEO was measured. For the control group, the heart rhythm was sinusoidal during the 5-minute recording (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFigure \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e \u003cb\u003e(A, B and C) here\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe cardiac activity in the control group showed a mean frequency of 86.89\u0026thinsp;\u0026plusmn;\u0026thinsp;2.47 bpm, which was similar to the vehicle group (p\u0026thinsp;=\u0026thinsp;0.999), with a sinus rhythm and all cardiac deflections present on the electrocardiogram (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). In the 30-second amplification, all graph elements were visible: P wave, QRS complex, and T wave in a 5-second amplification, allowing for the evaluation of intervals during the immersion bath in ROEO and their recovery. This enabled the identification of atrial activity represented by P waves, ventricular activity by QRS complexes, and ventricular repolarization represented by T waves (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, B, C, D, E, and F).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eDuring immersion treatment with 100 \u0026micro;L L⁻\u0026sup1;, the fish showed a 40.66% decrease in heart rate compared to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). The group treated with 125 \u0026micro;L L⁻\u0026sup1; of ROEO exhibited a 45.78% decrease (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC), while the group treated with 150 \u0026micro;L L⁻\u0026sup1; showed a 55.49% reduction (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). The 175 \u0026micro;L L⁻\u0026sup1; group decreased by 56.51% (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE), and the group treated with 200 \u0026micro;L L⁻\u0026sup1; exhibited a 56.77% reduction (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). The treated fish presented bradycardia while maintaining sinus rhythm (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, C, D, E, and F).\u003c/p\u003e\u003cp\u003eFigure \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e \u003cb\u003e(A, B, C, D, E and F) here\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe heart rate was significantly affected by the increasing concentrations of ROEO. The control group had an average heart rate of 86.89\u0026thinsp;\u0026plusmn;\u0026thinsp;2.47 bpm, and the vehicle group showed 86.67\u0026thinsp;\u0026plusmn;\u0026thinsp;3.46 bpm, both being similar (p\u0026thinsp;=\u0026thinsp;0.999). However, these values were higher than those in the treated groups. The group treated with 100 \u0026micro;L L⁻\u0026sup1; had an average heart rate of 51.56\u0026thinsp;\u0026plusmn;\u0026thinsp;2.60 bpm, which was higher than the other treated groups. The group treated with 125 \u0026micro;L L⁻\u0026sup1; had an average of 47.11\u0026thinsp;\u0026plusmn;\u0026thinsp;1.76 bpm, which was also higher than the groups treated with the higher doses. The group treated with 150 \u0026micro;L L⁻\u0026sup1; had an average of 38.67\u0026thinsp;\u0026plusmn;\u0026thinsp;2.44 bpm, which was like the groups treated with 175 \u0026micro;L L⁻\u0026sup1; and 200 \u0026micro;L L⁻\u0026sup1; (p\u0026thinsp;=\u0026thinsp;0.966) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe average amplitude of the QRS complex for the control group was 1.168\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27 mV, and for the vehicle group, it was 1.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27 mV, which were similar to the groups treated with 100 \u0026micro;L L⁻\u0026sup1;, 125 \u0026micro;L L⁻\u0026sup1;, and 150 \u0026micro;L L⁻\u0026sup1;, as well as 175 \u0026micro;L L⁻\u0026sup1; (p\u0026thinsp;=\u0026thinsp;0.747). The control and vehicle groups had greater amplitudes than the group treated with 200 \u0026micro;L \u0026middot;L⁻\u0026sup1;, which measured 0.713\u0026thinsp;\u0026plusmn;\u0026thinsp;0.134 mV (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003eThe average RR interval for the control group was 696.7\u0026thinsp;\u0026plusmn;\u0026thinsp;19.33 ms, which was similar to the vehicle group (p\u0026thinsp;=\u0026thinsp;0.999), and was shorter compared to the other treated groups. The group treated with 100 \u0026micro;L L⁻\u0026sup1; had an average RR interval of 1166\u0026thinsp;\u0026plusmn;\u0026thinsp;58.31 ms, like the group treated with 125 \u0026micro;L L⁻\u0026sup1; (p\u0026thinsp;=\u0026thinsp;0.1106) and was shorter than the other treated groups. The group treated with 150 \u0026micro;L L⁻\u0026sup1; had an RR interval of 1545\u0026thinsp;\u0026plusmn;\u0026thinsp;91.82 ms, which was similar to the groups treated with 175 \u0026micro;L L⁻\u0026sup1; and 200 \u0026micro;L L⁻\u0026sup1; (p\u0026thinsp;=\u0026thinsp;0.2725) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003eThe average PQ interval for the control group was 115.1\u0026thinsp;\u0026plusmn;\u0026thinsp;6.27 ms and showed no difference from the vehicle group (p\u0026thinsp;=\u0026thinsp;0.999), as well as from the groups treated with 100 \u0026micro;L L⁻\u0026sup1; and 125 \u0026micro;L L⁻\u0026sup1; (p\u0026thinsp;=\u0026thinsp;0.1060). The group treated with 150 \u0026micro;L L⁻\u0026sup1; had an average of 134.8\u0026thinsp;\u0026plusmn;\u0026thinsp;8.21 ms, and the group treated with 175 \u0026micro;L L⁻\u0026sup1; was similar (p\u0026thinsp;=\u0026thinsp;0.6530). The group treated with 175 \u0026micro;L L⁻\u0026sup1; was also like the group treated with 200 \u0026micro;L L⁻\u0026sup1; (p\u0026thinsp;=\u0026thinsp;0.2505) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD).\u003c/p\u003e\u003cp\u003eThe average duration of the QRS complex for the control group during induction was 27.22\u0026thinsp;\u0026plusmn;\u0026thinsp;2.72 ms, which was similar to the vehicle group (p\u0026thinsp;=\u0026thinsp;0.999) but shorter than the other groups. The groups treated with 100 \u0026micro;L L⁻\u0026sup1; (38.11\u0026thinsp;\u0026plusmn;\u0026thinsp;4.06 ms) were like the group treated with 125 \u0026micro;L L⁻\u0026sup1; (p\u0026thinsp;=\u0026thinsp;0.9999). The group treated with 150 \u0026micro;L L⁻\u0026sup1; (44.33\u0026thinsp;\u0026plusmn;\u0026thinsp;3.87 ms) was like the group treated with 175 \u0026micro;L L⁻\u0026sup1; (p\u0026thinsp;=\u0026thinsp;0.6513). The group treated with 200 \u0026micro;L L⁻\u0026sup1; (51.67\u0026thinsp;\u0026plusmn;\u0026thinsp;6.083 ms) was greater than the other groups but was like the group treated with 175 \u0026micro;L L⁻\u0026sup1; (p\u0026thinsp;=\u0026thinsp;0.2251) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE).\u003c/p\u003e\u003cp\u003eFor the control group, the average QT interval during induction was 290.3\u0026thinsp;\u0026plusmn;\u0026thinsp;10.51 ms, which was similar to the vehicle group (p\u0026thinsp;=\u0026thinsp;0.9967) and shorter than the other groups. The group treated with 100 \u0026micro;L L⁻\u0026sup1; had an average QT interval of 375.7\u0026thinsp;\u0026plusmn;\u0026thinsp;13.59 ms, and the 125 \u0026micro;L L⁻\u0026sup1; group was not significantly different (p\u0026thinsp;=\u0026thinsp;0.999). The group treated with 150 \u0026micro;L L⁻\u0026sup1; (435.7\u0026thinsp;\u0026plusmn;\u0026thinsp;18.91 ms) was like the group treated with 175 \u0026micro;L L⁻\u0026sup1; (p\u0026thinsp;=\u0026thinsp;0.9525). The group treated with 200 \u0026micro;L L⁻\u0026sup1; (474.1\u0026thinsp;\u0026plusmn;\u0026thinsp;16.44 ms) was greater than the other groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF).\u003c/p\u003e\u003cp\u003eFigure \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e \u003cb\u003e(A, B, C, D, E and F) here\u003c/b\u003e\u003c/p\u003e\u003cp\u003eDuring the recovery period following exposure to the ROEO concentrations of 100 \u0026micro;L L⁻\u0026sup1;, 125 \u0026micro;L L⁻\u0026sup1;, 150 \u0026micro;L L⁻\u0026sup1;, 175 \u0026micro;L L⁻\u0026sup1;, and 200 \u0026micro;L L⁻\u0026sup1;, a reversibility of the electrocardiographic alterations was observed (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, B, C, D, and E), occurring more slowly in the groups treated with higher concentrations. The cardiac rhythm remained sinusoidal, with a gradual increase in heart rate approaching levels observed in the control group.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFor the group treated with 100 \u0026micro;L L⁻\u0026sup1; of ROEO, the heart rate recovered 99.48% compared to the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). The recovery percentages for the other groups were as follows: 125 \u0026micro;L L⁻\u0026sup1; (96.16%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB), 150 \u0026micro;L L⁻\u0026sup1; (74.68%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC), 175 \u0026micro;L L⁻\u0026sup1; (68.54%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD), and 200 \u0026micro;L L⁻\u0026sup1; (66.49%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE).\u003c/p\u003e\u003cp\u003eDuring the recovery period, no arrhythmias were observed, and the reversal of ROEO effects on the heart was gradual and concentration dependent.\u003c/p\u003e\u003cp\u003eFigure \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e \u003cb\u003e(A, B, C, D and E) here\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe amplitude of the QRS complex during recovery for the control group was 1.168\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27 mV, which was similar to the other groups (F (6, 56)\u0026thinsp;=\u0026thinsp;0.375, p\u0026thinsp;=\u0026thinsp;0.891) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe average RR interval during recovery for the control group was 686.7\u0026thinsp;\u0026plusmn;\u0026thinsp;19.33 ms, showing no statistical difference compared to the vehicle, 100 \u0026micro;L L⁻\u0026sup1;, and 125 \u0026micro;L L⁻\u0026sup1; groups (p\u0026thinsp;=\u0026thinsp;0.7572); however, it was shorter than the other groups. The groups treated with higher concentrations, 150 \u0026micro;L L⁻\u0026sup1; (926.1\u0026thinsp;\u0026plusmn;\u0026thinsp;43.80 ms), were shorter than the 175 \u0026micro;L L⁻\u0026sup1; (1009\u0026thinsp;\u0026plusmn;\u0026thinsp;57.90 ms) and 200 \u0026micro;L L⁻\u0026sup1; (1043\u0026thinsp;\u0026plusmn;\u0026thinsp;85.05 ms) groups. The 175 \u0026micro;L L⁻\u0026sup1; and 200 \u0026micro;L L⁻\u0026sup1; groups were similar (p\u0026thinsp;=\u0026thinsp;0.5171) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003eThe PQ interval during recovery for the control group was 115.1\u0026thinsp;\u0026plusmn;\u0026thinsp;6.27 ms, which was like the vehicle, 100 \u0026micro;L L⁻\u0026sup1;, 125 \u0026micro;L L⁻\u0026sup1;, and 150 \u0026micro;L L⁻\u0026sup1; groups (p\u0026thinsp;=\u0026thinsp;0.1260). The group treated with 175 \u0026micro;L L⁻\u0026sup1; (125.3\u0026thinsp;\u0026plusmn;\u0026thinsp;4.062 ms) was like the group treated with 200 \u0026micro;L L⁻\u0026sup1; (p\u0026thinsp;=\u0026thinsp;0.9543) but was longer than the other groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD).\u003c/p\u003e\u003cp\u003eThe duration of the QRS complex during recovery for the control group averaged (27.22\u0026thinsp;\u0026plusmn;\u0026thinsp;2.72 ms) and was like the other groups (F (6, 56)\u0026thinsp;=\u0026thinsp;2.88; p\u0026thinsp;=\u0026thinsp;0.0161) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE).\u003c/p\u003e\u003cp\u003eDuring recovery, the QT interval for the control was 290.3\u0026thinsp;\u0026plusmn;\u0026thinsp;10.51 ms, which was like the vehicle, 100 \u0026micro;L L⁻\u0026sup1;, 125 \u0026micro;L L⁻\u0026sup1;, and 150 \u0026micro;L L⁻\u0026sup1; groups (p\u0026thinsp;=\u0026thinsp;0.1525). The groups treated with 150 \u0026micro;L L⁻\u0026sup1; (311.8\u0026thinsp;\u0026plusmn;\u0026thinsp;14.06 ms) were like the 175 \u0026micro;L L⁻\u0026sup1; group (p\u0026thinsp;=\u0026thinsp;0.1142). The group treated with 200 \u0026micro;L L⁻\u0026sup1; (354.9\u0026thinsp;\u0026plusmn;\u0026thinsp;22.27 ms) was like the group treated with 175 \u0026micro;L L⁻\u0026sup1; (p\u0026thinsp;=\u0026thinsp;0.1894) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eF).\u003c/p\u003e\u003cp\u003eFigure \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e \u003cb\u003e(A, B, C, D, E and F) here\u003c/b\u003e\u003c/p\u003e\u003cp\u003eDuring the treatment with ROEO at concentrations of 100 \u0026micro;L L⁻\u0026sup1;, 125 \u0026micro;L L⁻\u0026sup1;, 150 \u0026micro;L L⁻\u0026sup1;, 175 \u0026micro;L L⁻\u0026sup1;, and 200 \u0026micro;L L⁻\u0026sup1;, the frequency of opercular movement decreased with higher concentrations (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA, B, C, D, E, F, and G). The control group exhibited a mean of 98.67\u0026thinsp;\u0026plusmn;\u0026thinsp;6.92 movements per minute (mpm), which was similar to the vehicle group (p\u0026thinsp;=\u0026thinsp;0.9282) but higher than the other treatment groups. The group treated with 100 \u0026micro;L L⁻\u0026sup1; showed a reduction in opercular beats of 22.52%. The group treated with 125 \u0026micro;L L⁻\u0026sup1; exhibited a decrease of 21.17%, while the group treated with 150 \u0026micro;L L⁻\u0026sup1; showed a reduction of 31.08%. The group treated with 175 \u0026micro;L L⁻\u0026sup1; reduced opercular activity by 32.43%, and the group treated with 200 \u0026micro;L L⁻\u0026sup1; demonstrated a decrease of 37.95%.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe group treated with 100 \u0026micro;L L⁻\u0026sup1; (76.44\u0026thinsp;\u0026plusmn;\u0026thinsp;3.97 mpm) was like the group treated with 125 \u0026micro;L L⁻\u0026sup1; (p\u0026thinsp;=\u0026thinsp;0.9980), but higher than the other treatment groups. The group treated with 150 \u0026micro;L L⁻\u0026sup1; (68.00\u0026thinsp;\u0026plusmn;\u0026thinsp;4.123 mpm) was like the groups treated with 175 \u0026micro;L L⁻\u0026sup1; and 200 \u0026micro;L L⁻\u0026sup1; (p\u0026thinsp;=\u0026thinsp;0.0995) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eH).\u003c/p\u003e\u003cp\u003eThe decrease in power in the opercular movement records was observed during the induction of anesthesia. The control group showed a mean power of 0.3165\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0609 mV\u0026sup2;/Hz, which was similar to the vehicle group (p\u0026thinsp;=\u0026thinsp;0.134) but greater than that of the other treatment groups. The group treated with 100 \u0026micro;L L⁻\u0026sup1; showed a mean of 0.189\u0026thinsp;\u0026plusmn;\u0026thinsp;0.025 mV\u0026sup2;/Hz, which was like the group treated with 125 \u0026micro;L L⁻\u0026sup1; (p\u0026thinsp;=\u0026thinsp;0.5722). The group treated with 125 \u0026micro;L L⁻\u0026sup1; (0.155\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0301 mV\u0026sup2;/Hz) was like the groups treated with 150 \u0026micro;L L⁻\u0026sup1; and 175 \u0026micro;L L⁻\u0026sup1; (p\u0026thinsp;=\u0026thinsp;0.1882). The group treated with 200 \u0026micro;L L⁻\u0026sup1; (0.04063\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0087 mV\u0026sup2;/Hz) was lower than the other groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eI).\u003c/p\u003e\u003cp\u003eFigure \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e \u003cb\u003e(A, B, C, D, E, F, G, H and I) here\u003c/b\u003e\u003c/p\u003e\u003cp\u003eDuring the recovery period from anesthesia, opercular movement showed recovery (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eA, B, C, D, and E). The control group had a mean of 98.67\u0026thinsp;\u0026plusmn;\u0026thinsp;6.92 mpm, which was like the vehicle group, the group treated with 100 \u0026micro;L L⁻\u0026sup1;, the group treated with 125 \u0026micro;L L⁻\u0026sup1;, and the group treated with 150 \u0026micro;L L⁻\u0026sup1; (p\u0026thinsp;=\u0026thinsp;0.1338), but higher than the other treated groups. The group treated with 175 \u0026micro;L L⁻\u0026sup1; (81.67\u0026thinsp;\u0026plusmn;\u0026thinsp;5.52 mpm) was like the group treated with 200 \u0026micro;L L⁻\u0026sup1; (p\u0026thinsp;=\u0026thinsp;0.2669) (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eF).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAn increase in power in the opercular movement recordings was observed during recovery from anesthesia. The control group had a mean power of 0.3165\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0609 mV\u0026sup2;/Hz, which was like the recovery groups of the vehicle, treated with 100 \u0026micro;L L⁻\u0026sup1;, 125 \u0026micro;L L⁻\u0026sup1;, and 150 \u0026micro;L L⁻\u0026sup1; (p\u0026thinsp;=\u0026thinsp;0.5085). The group treated with 125 \u0026micro;L L⁻\u0026sup1; was like the group treated with 200 \u0026micro;L L⁻\u0026sup1; (p\u0026thinsp;=\u0026thinsp;0.0549). The group treated with 200 \u0026micro;L L⁻\u0026sup1; was like the groups treated with 150 \u0026micro;L L⁻\u0026sup1; and 175 \u0026micro;L L⁻\u0026sup1; (p\u0026thinsp;=\u0026thinsp;0.0603) (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eG).\u003c/p\u003e\u003cp\u003eFigure \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e \u003cb\u003e(A, B, C, D, E, F and G) here\u003c/b\u003e\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe results of this study demonstrate that \u003cem\u003eRosmarinus officinalis\u003c/em\u003e essential oil (ROEO) exerts a concentration-dependent anesthetic effect on \u003cem\u003eColossoma macropomum\u003c/em\u003e (tambaqui). The group treated with 200 \u0026micro;L L⁻\u0026sup1; of essential oil showed a significantly faster loss of postural reflex compared to groups with lower concentrations, such as 100 \u0026micro;L L⁻\u0026sup1;, indicating a faster anesthetic action at higher concentrations. These findings align with the study by Bianchini et al. (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), which evaluated the anesthetic effects of essential oil compounds like carvacrol and thymol and observed a clear relationship between concentration and anesthetic response, with significantly shorter induction times as concentrations increased across all induction stages.\u003c/p\u003e\u003cp\u003eUehara et al. (2018), in a comparative study of synthetic and natural anesthetic agents, highlighted that clove oil exhibited effective anesthetic action across all tested concentrations, with dose-dependent effects on both anesthetic induction and recovery time. Similarly, the lower concentrations of \u003cem\u003eR. officinalis\u003c/em\u003e essential oil evaluated in this experiment, such as 100 \u0026micro;L L⁻\u0026sup1; and 150 \u0026micro;L L⁻\u0026sup1;, required a longer latency time for anesthetic action than the other concentrations. Meanwhile, postural reflex recovery time was longer in groups treated with higher doses, demonstrating the dose-dependent and gradual reversibility of the anesthetic effect - a characteristic often described in the literature for natural anesthetics (Hoseini et al., 2019).\u003c/p\u003e\u003cp\u003eThese observed effects may be attributed to the composition of ROEO, particularly its main component, 1,8-cineole, whose action on the central nervous system is well-documented in the literature. High-performance gas chromatography analysis revealed that 1,8-cineole comprises 47.5% of the oil composition, followed by camphor (19.3%) and α-pinene (12.2%). These terpenoid compounds are known for their biological properties, including antioxidant, anti-inflammatory activities, and synergistic effects in various applications. The anesthetic effect of \u003cem\u003eR. officinalis\u003c/em\u003e is closely linked to 1,8-cineole, which modulates GABA-A receptors in the central nervous system and blocks voltage-dependent sodium channels in peripheral nerves, resulting in reduced neuronal excitability (Dougnon \u0026amp; Ito, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Ferreira et al., 2015; Uusi-Oukari \u0026amp; Korpi, 2010). Studies such as Khumpirapang et al. (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) have demonstrated the potent depressant effect of 1,8-cineole on the central nervous system, explaining the rapid anesthetic induction observed at higher ROEO concentrations.\u003c/p\u003e\u003cp\u003eAdditionally, 1,8-cineole significantly impacts the cardiovascular system, inducing bradycardia and modulating cardiac electrical activity. In this study, prolonged R-R and QT electrocardiographic intervals were observed, similar to effects reported in fish treated with other essential oils containing 1,8-cineole or camphor (Mazandarani \u0026amp; Hoseini, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The reduction in QRS complex amplitude at higher doses (200 \u0026micro;L L⁻\u0026sup1;) may be associated with ventricular depression caused by \u003cem\u003eR. officinalis\u003c/em\u003e, indicating a direct effect on heart contractility. Studies with essential oils, such as eucalyptus oil, have also reported reduced QRS complex amplitude, suggesting that these compounds may modulate cardiac electrical activity, affecting myocardial cell function (Taheri Mirghaed et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe extension of the R-R interval observed at higher ROEO doses suggests modulation of the interval between heartbeats, consistent with induced bradycardia. These findings indicate that ROEO affects the cardiac cycle in a concentration-dependent manner, similar to findings in studies with clove oil and lemongrass (Bianchini et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Furthermore, the increase in the P-Q interval and prolonged QRS complex duration at higher ROEO doses indicate delayed atrioventricular conduction, possibly related to the action of 1,8-cineole on calcium channels and the cardiac conduction system (Aktop et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). This effect is observed in other natural anesthetics and should be considered a critical parameter to determine safe doses, avoiding cardiac blocks or prolonged alterations in fish cardiac electrical conduction.\u003c/p\u003e\u003cp\u003eProlongation of the QT interval at higher ROEO doses suggests increased ventricular repolarization time, which may indicate a risk of arrhythmias if used at inappropriate concentrations. This finding is relevant because anesthetics that prolong the QT interval are often associated with a higher risk of arrhythmias (Schroeder et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). However, the reversibility of the effect after recovery, without arrhythmias, demonstrates that ROEO is safe within the tested dose range.\u003c/p\u003e\u003cp\u003eThe electrocardiographic data show that, for all concentrations of \u003cem\u003eR. officinalis\u003c/em\u003e, there was a significant dose-dependent reduction in heart rate, but sinus rhythm was maintained, indicating that \u003cem\u003eR. officinalis\u003c/em\u003e did not compromise the normal electrical function of the heart. This suggests that ROEO can be considered a safe anesthetic for use in fish, with a cardioprotective effect and no arrhythmias. These results support the findings of Krasteva, Yankova, \u0026amp; Ivanova (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), who evaluated \u003cem\u003eR. officinalis\u003c/em\u003e oil in \u003cem\u003eCyprinus carpio\u003c/em\u003e L. and found that, in addition to dose-dependent anesthetic induction, all oil concentrations were safe for fish transport for up to 4 hours without causing harm to the animals.\u003c/p\u003e\u003cp\u003eThe complete reversibility of the electrocardiographic changes observed during recovery with ROEO reinforces its safety profile for aquaculture, with sinus rhythm being progressively restored. This is especially advantageous, as natural anesthetics, like ROEO, often exhibit slower recovery due to their prolonged bioavailability (Can \u0026amp; S\u0026uuml;mer, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The near-total recovery of heart rate at lower concentrations also suggests that moderate ROEO doses can induce effective anesthesia without compromising long-term cardiovascular function. In contrast, MS-222 causes an abnormal respiratory response in fish, with rapid and irregular gill movements, indicating respiratory depression and stress. This abrupt behavior contrasts with essential oils, which tend to promote a smoother, more controlled anesthetic induction, reducing stress during the process and highlighting the safety of essential oils as natural anesthetics in aquaculture.\u003c/p\u003e\u003cp\u003eThis study demonstrated that \u003cem\u003eRosmarinus officinalis\u003c/em\u003e essential oil exerts a dose-dependent anesthetic effect on \u003cem\u003eColossoma macropomum\u003c/em\u003e. The findings also indicated that the main component of ROEO, 1,8-cineole, plays a key role in modulating the central nervous system and regulating cardiac activity. Electrocardiographic data revealed changes such as bradycardia and prolongation of the R-R and QT intervals, effects that were gradually reversed during recovery, reinforcing the safety of R. officinalis as a natural anesthetic. R. officinalis provided a gentler anesthetic induction with low impacts on the respiratory function of the fish, providing a more controlled anesthetic transition. These findings indicate that R. officinalis, in addition to being effective, is a safer and more sustainable alternative for fish management in aquaculture, offering an option that minimizes the side effects associated with synthetic anesthetics.\u003c/p\u003e\u003cp\u003eGiven the demonstrated efficacy and safety of R. officinalis, future studies may focus on assessing its impact on other fish species and examining its use under long-term conditions, such as for prolonged fish transport and handling. Research on the elimination of R. officinalis compounds and potential bioaccumulation may also provide important insights to ensure the safety of continued use. Furthermore, further studies on optimizing R. officinalis concentrations may help establish clear guidelines for its routine use in aquaculture, consolidating its potential as a natural anesthetic.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank the Laboratory of Pharmacology and Toxicology of Natural Products and the postgraduate program at the Federal University of Par\u0026aacute; for the support given in carrying out the experiments in the article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical trial number\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was experimental with fish and does not involve clinical trials on humans or companion animals, therefore is not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo human participants were involved; the study was conducted under institutional ethics approval (CEUA/UFPA), which already covers the consent requirement, therefore it is not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent publish\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors have read and approved the final version of the manuscript and consent to its publication.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003eEthical approval and ARRIVE statement\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eAll experimental procedures were conducted in accordance with national and institutional guidelines for animal care and use, following the principles of the Brazilian legislation (Law No. 11.794/2008) and the recommendations of the National Council for Animal Experimentation Control (CONCEA). The study protocol was approved by the Ethics Committee on Animal Use of the Federal University of Par\u0026aacute; (CEUA-UFPA, protocol No. 5846030624/2024). Experimental design, randomization, and sample size were defined a priori to minimize the number of animals used and ensure reproducibility, in compliance with the ARRIVE 2.0 guidelines (Percie du Sert et al., 2020). Each animal constituted an independent experimental unit, and observers responsible for behavioral and electrophysiological recordings were blinded to treatment groups during data collection and analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval and animal welfare statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experimental procedures involving fish were conducted in strict accordance with the ethical principles and guidelines for animal experimentation established by the Brazilian National Council for the Control of Animal Experimentation (CONCEA; Law No. 11.794/2008). The experimental protocol was reviewed and approved by the Ethics Committee on Animal Use of the Federal University of Par\u0026aacute; (CEUA-UFPA, protocol No. 5846030624/2024).\u003c/p\u003e\n\u003cp\u003eEfforts were made to minimize the number of animals used and to reduce their suffering. Anesthetic and handling procedures were designed to ensure fish welfare throughout the experimental period, following the ARRIVE 2.0 guidelines (Percie du Sert et al., 2020).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDAS statement request\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article. No supplementary files are available.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eThis work was supported by the Federal University of Par\u0026aacute; (UFPA), through the Laboratory of Pharmacology and Toxicology of Natural Products, and by internal research grants from the Graduate Program in Biological Sciences (ICB-UFPA). No external funding agencies influenced the study design, data collection, or interpretation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCRediT authorship contribution statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceived and designed the experiments: \u003cstrong\u003eLorena Cristina Nunes de Almeida\u003c/strong\u003e\u003cstrong\u003e,\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;Priscille Fidelis Pacheco Hartcopff\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eand Mois\u0026eacute;s Hamoy\u003c/strong\u003e. Performed the experiments: \u003cstrong\u003eDaniella Bastos de Ara\u0026uacute;jo\u003c/strong\u003e\u003cstrong\u003e, Axell Lins,\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eTaissa Viana Damasceno, Luciana Esquerdo Cerqueira, Thaysa de Sousa Reis\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eand Mois\u0026eacute;s Hamoy\u003c/strong\u003e. Writing-original draft and editing: \u003cstrong\u003eAxell Lins,\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eMaria Klara Otake Hamoy, Marcelo Victor dos Santos Brito, Luana Vasconcelos de Souza, Sarah Farias C\u0026acirc;mara, Luciana Eiro Quirino and Nilton Akio Muto\u003c/strong\u003e. Financial support and administrative support: \u003cstrong\u003eMois\u0026eacute;s Hamoy\u003c/strong\u003e. All authors have read and agreed to the published version of the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eThe datasets generated and analyzed during the current study (raw ECG traces, opercular activity records, and chromatographic profiles) are available from the corresponding author upon reasonable request. Processed data and scripts for signal analysis (Python 2.7, NumPy, SciPy, and Matplotlib) are stored in the institutional repository of the Federal University of Par\u0026aacute; and can be provided for review or replication purposes.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAktop Y, Aydın B, \u0026Ccedil;ağatay İT (2019) Usability of coriander oil (\u003cem\u003eCoriandrum sativum\u003c/em\u003e) as a herbal anesthetic on fish. \u003cem\u003eTurkish Journal of Agriculture \u0026ndash; Food Science and Technology\u003c/em\u003e 7(3): 23\u0026ndash;26. https://doi.org/10.24925/turjaf.v7isp3.23-26.3128\u003c/li\u003e\n\u003cli\u003eAkrout A, Mighri H, Krid M, Thabet F, Turki H, El-Jani H, Neffati M (2012) Chemical composition and antioxidant activity of aqueous extracts of some wild medicinal plants in southern Tunisia. \u003cem\u003eInternational Journal of Life Sciences and Medical Sciences\u003c/em\u003e 2: 1\u0026ndash;4.\u003c/li\u003e\n\u003cli\u003eAl-Maharik N, Jaradat N, Hawash M, Al-Lahham S, Qadi M, Shoman I, Issa L (2022) Chemical composition, antioxidant, antimicrobial and anti-proliferative activities of \u003cem\u003eRosmarinus officinalis\u003c/em\u003e essential oils from five sites in Palestine. \u003cem\u003eSeparations\u003c/em\u003e 9(11): 339. https://doi.org/10.3390/separations9110339\u003c/li\u003e\n\u003cli\u003eAl Zuhairi JJMJ, Kashi FJ, Rahimi-Moghaddam A, Yazdani M (2020) Antioxidant, cytotoxic and antibacterial activity of \u003cem\u003eRosmarinus officinalis\u003c/em\u003e L. essential oil against bacteria isolated from urinary tract infection. \u003cem\u003eEuropean Journal of Integrative Medicine\u003c/em\u003e 38: 101192. https://doi.org/10.1016/j.eujim.2020.101192\u003c/li\u003e\n\u003cli\u003eAndrade JM, Faustino C, Garcia C, Ladeiras D, Reis CP, Rijo P (2018) \u003cem\u003eRosmarinus officinalis\u003c/em\u003e L.: an update review of its phytochemistry and biological activity. \u003cem\u003eFuture Science OA\u003c/em\u003e 4(4): FSO283. https://doi.org/10.4155/fsoa-2017-0124\u003c/li\u003e\n\u003cli\u003eAydın B, Barbas LAL (2020) Sedative and anesthetic properties of essential oils and their active compounds in fish: a review. \u003cem\u003eAquaculture\u003c/em\u003e 520: 734999. https://doi.org/10.1016/j.aquaculture.2020.734999\u003c/li\u003e\n\u003cli\u003eBarbas LAL, Torres MF, Costa BMP, Feitosa MJM, Maltez LC, Amado LL, Hamoy M (2021) Eugenol induces body immobilization yet evokes increased neuronal excitability in fish during short-term baths. \u003cem\u003eAquatic Toxicology\u003c/em\u003e 231: 105734. https://doi.org/10.1016/j.aquatox.2020.105734\u003c/li\u003e\n\u003cli\u003eBianchini AE, Garlet QI, Da Cunha JA, Bandeira Junior G, Brusque ICM, Salbego J, Heinzmann BM, Baldisserotto B (2017) Monoterpenoids (thymol, carvacrol and S-(+)-linalool) with anesthetic activity in silver catfish (\u003cem\u003eRhamdia quelen\u003c/em\u003e): evaluation of acetylcholinesterase and GABAergic activity. \u003cem\u003eBrazilian Journal of Medical and Biological Research\u003c/em\u003e 50(1): 1\u0026ndash;8. https://doi.org/10.1590/1414-431x20176346\u003c/li\u003e\n\u003cli\u003eBr\u0026oslash;nstad A (2022) Good anesthesia practice for fish and other aquatics. \u003cem\u003eBiology\u003c/em\u003e 11(9): 1355. https://doi.org/10.3390/biology11091355\u003c/li\u003e\n\u003cli\u003eCaceres AI, Liu B, Jabba SV, Achanta S, Morris JB, Jordt SE (2017) TRPM8 channels mediate the anti-inflammatory effects of eucalyptol. \u003cem\u003eBritish Journal of Pharmacology\u003c/em\u003e 174(9): 867\u0026ndash;879. https://doi.org/10.1111/bph.13760\u003c/li\u003e\n\u003cli\u003eCai ZM, Peng JQ, Chen Y, Tao L, Zhang YY, Fu LY, Shen XC (2021) 1,8-Cineole: a review of source, biological activities, and applications. \u003cem\u003eJournal of Asian Natural Products Research\u003c/em\u003e 23(10): 938\u0026ndash;954. https://doi.org/10.1080/10286020.2020.1839432\u003c/li\u003e\n\u003cli\u003eCan E, S\u0026uuml;mer E (2019) Anesthetic and sedative efficacy of peppermint (\u003cem\u003eMentha piperita\u003c/em\u003e) and lavender (\u003cem\u003eLavandula angustifolia\u003c/em\u003e) essential oils in blue dolphin cichlid (\u003cem\u003eCyrtocara moorii\u003c/em\u003e). \u003cem\u003eTurkish Journal of Veterinary and Animal Sciences\u003c/em\u003e43: 334\u0026ndash;341. https://doi.org/10.3906/vet-1809-22\u003c/li\u003e\n\u003cli\u003eDhouibi I, Flamini G, Bouaziz M (2023) Comparative study on the essential oils extracted from Tunisian rosemary and myrtle: chemical profiles, quality, and antimicrobial activities. \u003cem\u003eACS Omega\u003c/em\u003e 8: 6431\u0026ndash;6438. https://doi.org/10.1021/acsomega.2c06713\u003c/li\u003e\n\u003cli\u003ede Ara\u0026uacute;jo ERL, Silva JDS, Lopes LM, Torres MF, Costa BMA, Amarante CB, Sampaio LA (2021) Geraniol and citronellol as alternative and safe phytoconstituents to induce immobilization and facilitate handling of fish. \u003cem\u003eAquaculture\u003c/em\u003e537: 736517. https://doi.org/10.1016/j.aquaculture.2021.736517\u003c/li\u003e\n\u003cli\u003eDougnon G, Ito M (2020) Inhalation administration of the bicyclic ethers 1,8- and 1,4-cineole prevent anxiety and depressive-like behaviours in mice. \u003cem\u003eMolecules\u003c/em\u003e 25: 8184. https://doi.org/10.3390/molecules25081884\u003c/li\u003e\n\u003cli\u003eElmas E, Karadal O (2022) Anesthetic and sedative effects of plant-derived essential oils on red swamp crayfish (\u003cem\u003eProcambarus clarkii\u003c/em\u003e) at different concentrations and temperatures. \u003cem\u003eNauplius\u003c/em\u003e 30: e2022035. https://doi.org/10.1590/2358-2936e2022035\u003c/li\u003e\n\u003cli\u003eGonzalez-Minero F, Bravo-Diaz L, Ayala-Gomez A (2020) \u003cem\u003eRosmarinus officinalis\u003c/em\u003e L. (rosemary): an ancient plant with uses in personal healthcare and cosmetics. \u003cem\u003ePlants\u003c/em\u003e 7(1): 1\u0026ndash;17. https://doi.org/10.3390/cosmetics7040077\u003c/li\u003e\n\u003cli\u003eHoseini SM et al. (2020) Antioxidant and immune responses of common carp (\u003cem\u003eCyprinus carpio\u003c/em\u003e) anesthetized by cineole: effects of anesthetic concentration. \u003cem\u003eAquaculture\u003c/em\u003e 520: 734680. https://doi.org/10.1016/j.aquaculture.2019.734680\u003c/li\u003e\n\u003cli\u003eKheawfu K, Pikulkaew S, Rades T, M\u0026uuml;llertz A, von Gersdorff J\u0026oslash;rgensen L, Okonogi S (2021) Design and optimization of self-nanoemulsifying drug delivery systems of clove oil for efficacy enhancement in fish anesthesia. \u003cem\u003eJournal of Drug Delivery Science and Technology\u003c/em\u003e 61: 102241. https://doi.org/10.1016/j.jddst.2020.102241\u003c/li\u003e\n\u003cli\u003eKizak V, Can E, Danabaş D, Can ŞS (2018a) Evaluation of anesthetic potential of rosewood (\u003cem\u003eAniba rosaeodora\u003c/em\u003e) oil as a new anesthetic agent for goldfish (\u003cem\u003eCarassius auratus\u003c/em\u003e). \u003cem\u003eAquaculture\u003c/em\u003e 493: 296\u0026ndash;301. https://doi.org/10.1016/j.aquaculture.2018.05\u003c/li\u003e\n\u003cli\u003eKizak V, Guner Y, Kayim M, Can E (2013b) The effects of clove oil on adult males and females of rainbow trout (\u003cem\u003eOncorhynchus mykiss\u003c/em\u003e) and brown trout (\u003cem\u003eSalmo trutta fario\u003c/em\u003e). \u003cem\u003eJournal of Food, Agriculture \u0026amp; Environment\u003c/em\u003e 11: 1542\u0026ndash;1545.\u003c/li\u003e\n\u003cli\u003eKhumpirapang N et al. (2018) In vivo anesthetic effect and mechanism of action of active compounds from \u003cem\u003eAlpinia galanga\u003c/em\u003e oil on \u003cem\u003eCyprinus carpio\u003c/em\u003e. \u003cem\u003eAquaculture\u003c/em\u003e 496: 1\u0026ndash;7. https://doi.org/10.1016/j.aquaculture.2018.07.017\u003c/li\u003e\n\u003cli\u003eKondo S, El Omri A, Han J, Isoda H (2015) Antidepressant-like effects of rosmarinic acid through mitogen-activated protein kinase phosphatase-1 and brain-derived neurotrophic factor modulation. \u003cem\u003eJournal of Functional Foods\u003c/em\u003e 14: 758\u0026ndash;766. https://doi.org/10.1016/j.jff.2015.03.001\u003c/li\u003e\n\u003cli\u003eKrasteva V, Yankova M, Ivanova A (2021) Anesthetic effect of rosemary essential oil (\u003cem\u003eRosmarinus officinalis\u003c/em\u003e L.) and its use for transport of common carp (\u003cem\u003eCyprinus carpio\u003c/em\u003e L.) stocking material. \u003cem\u003eInstitute of Fisheries and Aquaculture Reports\u003c/em\u003e, Plovdiv, Bulgaria.\u003c/li\u003e\n\u003cli\u003eLahlou S et al. (2002) Cardiovascular effects of 1,8-cineole, a terpenoid oxide present in many plant essential oils, in normotensive rats. \u003cem\u003eCanadian Journal of Physiology and Pharmacology\u003c/em\u003e 80(12): 1125\u0026ndash;1131. https://doi.org/10.1139/Y02-142\u003c/li\u003e\n\u003cli\u003eLi Pomi F, Papa V, Borgia F, Vaccaro M, Allegra A, Cicero N, Gangemi S (2023) \u003cem\u003eRosmarinus officinalis\u003c/em\u003e and skin: antioxidant activity and possible therapeutical role in cutaneous diseases. \u003cem\u003eAntioxidants\u003c/em\u003e 12(3): 680. https://doi.org/10.3390/antiox12030680\u003c/li\u003e\n\u003cli\u003eMazandarani M, Hoseini SM (2017) Menthol and 1,8-cineole as new anaesthetics in common carp (\u003cem\u003eCyprinus carpio\u003c/em\u003e). \u003cem\u003eAquaculture Research\u003c/em\u003e 48: 3041\u0026ndash;3051. https://doi.org/10.1111/are.13136\u003c/li\u003e\n\u003cli\u003eMirghaed A et al. (2018) Anaesthetic efficacy and biochemical effects of 1,8-cineole in rainbow trout (\u003cem\u003eOncorhynchus mykiss\u003c/em\u003e). \u003cem\u003eAquaculture Research\u003c/em\u003e 49: 2156\u0026ndash;2165. https://doi.org/10.1111/are.13671\u003c/li\u003e\n\u003cli\u003eMocho JP (2024) Anaesthesia, analgesia and zebrafish: a practical guide to husbandry, welfare and research methodology. In: \u003cem\u003eZebrafish: A Practical Guide to Husbandry, Welfare and Research Methodology\u003c/em\u003e, p. 427.\u003c/li\u003e\n\u003cli\u003eMoon HK, Kang P, Lee HS, Min SS, Seol GH (2014) Effects of 1,8-cineole on hypertension induced by chronic exposure to nicotine in rats. \u003cem\u003eJournal of Pharmacy and Pharmacology\u003c/em\u003e 66(5): 688\u0026ndash;693. https://doi.org/10.1111/jphp.12195\u003c/li\u003e\n\u003cli\u003eNeiffer DL (2021) Anesthesia and analgesia. In: \u003cem\u003eClinical Guide to Fish Medicine\u003c/em\u003e. Wiley-Blackwell, pp. 198\u0026ndash;212. https://doi.org/10.1002/9781119259886.ch10\u003c/li\u003e\n\u003cli\u003ePercie du Sert N et al. (2020) The ARRIVE guidelines 2.0: Updated guidelines for reporting animal research. \u003cem\u003ePLOS Biology\u003c/em\u003e 18(7): e3000410. https://doi.org/10.1371/journal.pbio.3000410\u003c/li\u003e\n\u003cli\u003ePurbosari N, Warsiki E, Syamsu K, Santoso J (2019) Natural versus synthetic anesthetics for transport of live fish: a review. \u003cem\u003eAquaculture and Fisheries Studies\u003c/em\u003e 4: 129\u0026ndash;133. https://doi.org/10.1016/j.aaf.2019.03.002\u003c/li\u003e\n\u003cli\u003eRairat T, Chi Y, Hsieh CY, Liu YK, Chuchird N, Chou CC (2021) Determination of optimal doses and minimum effective concentrations of tricaine methanesulfonate, 2-phenoxyethanol and eugenol for laboratory management in Nile tilapia (\u003cem\u003eOreochromis niloticus\u003c/em\u003e). \u003cem\u003eAnimals\u003c/em\u003e 11: 1521. https://doi.org/10.3390/ani11061521\u003c/li\u003e\n\u003cli\u003eReis TDS, Ara\u0026uacute;jo DBD, Paz CAD, Santos RG, Barbosa ADS, Souza LVD, Hamoy M (2024) Etomidate as an anesthetic in \u003cem\u003eColossoma macropomum\u003c/em\u003e: behavioral and electrophysiological data complement each other as a tool to assess anesthetic safety. \u003cem\u003ePLOS ONE\u003c/em\u003e 19(8): e0305093. https://doi.org/10.1371/journal.pone.0305093\u003c/li\u003e\n\u003cli\u003eRoohi Z, Imanpoor MR (2015) The efficacy of spearmint and methyl salicylate oils as new anesthetics and their effect on glucose levels in common carp (\u003cem\u003eCyprinus carpio\u003c/em\u003e). \u003cem\u003eAquaculture\u003c/em\u003e 439: 1\u0026ndash;6. https://doi.org/10.1016/j.aquaculture.2014.12.019\u003c/li\u003e\n\u003cli\u003eRucinque DS, Ferreira PF, Leme PRP, Lapa-Guimaraes J, Viegas EMM (2021) \u003cem\u003eOcimum americanum\u003c/em\u003e and \u003cem\u003eLippia alba\u003c/em\u003eessential oils as anaesthetics for Nile tilapia: induction, recovery and sensory analysis of fillets. \u003cem\u003eAquaculture\u003c/em\u003e 531: 735902. https://doi.org/10.1016/j.aquaculture.2020.735902\u003c/li\u003e\n\u003cli\u003eSadeh D et al. (2019) Interactive effects of genotype, seasonality and extraction method on chemical compositions and yield of rosemary essential oil. \u003cem\u003eIndustrial Crops and Products\u003c/em\u003e 138: 111240. https://doi.org/10.1016/j.indcrop.2019.05.068\u003c/li\u003e\n\u003cli\u003eSchroeder P, Lloyd R, McKimm R, Metselaar M, Navarro J, O\u0026rsquo;Farrell M, Readman GD, Speilberg L, Mocho JP (2021) Anaesthesia of laboratory, aquaculture and ornamental fish: proceedings of the first LASA-FVS symposium. \u003cem\u003eLaboratory Animals\u003c/em\u003e 55(4): 317\u0026ndash;328. https://doi.org/10.1177/0023677221998403\u003c/li\u003e\n\u003cli\u003eSeyidoglu N, Yagcilar C (2020) The anesthetic role of some herbal oils for zebrafish. \u003cem\u003eErciyes \u0026Uuml;niversitesi Veteriner Fak\u0026uuml;ltesi Dergisi\u003c/em\u003e 17(3): 209\u0026ndash;214. https://doi.org/10.32707/ercivet.828319\u003c/li\u003e\n\u003cli\u003eSharma AD, Kaur I, Kaur J, Chauhan A (2024) Chemical profiling and in-vitro antioxidant, anti-diabetic, anti-inflammatory, anti-bacterial and anti-fungal activities of essential oil from \u003cem\u003eRosmarinus officinalis\u003c/em\u003e L. \u003cem\u003eNotulae Scientia Biologicae\u003c/em\u003e 16(1): 11756.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Rosmarinus officinalis, Colossoma macropomum, fish anestesia, electrocardiography, aquaculture welfare, natural anesthetics, eucalyptol","lastPublishedDoi":"10.21203/rs.3.rs-7964102/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7964102/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAnesthetic management plays a crucial role in maintaining the welfare and physiological stability of fish during routine aquaculture and research procedures. The essential oil of \u003cem\u003eRosmarinus officinalis\u003c/em\u003e (ROEO) has emerged as a promising natural anesthetic, yet its cardiophysiological effects in Amazonian species remain underexplored. This study assessed the behavioral, electrocardiographic, and respiratory responses of \u003cem\u003eColossoma macropomum\u003c/em\u003e(tambaqui) exposed to ROEO, aiming to define safe and effective concentrations for use as a plant-derived anesthetic in aquaculture. Juvenile tambaqui were exposed to immersion baths containing 100\u0026ndash;200 \u0026micro;L L⁻\u0026sup1; of ROEO for 5 min. Behavioral endpoints (latency to loss and recovery of postural reflex), electrocardiographic parameters (heart rate, QRS amplitude, R\u0026ndash;R, P\u0026ndash;Q, and Q\u0026ndash;T intervals), and opercular frequency were monitored. The oil composition was determined by gas chromatography. Statistical analyses were performed using one-way ANOVA followed by Tukey\u0026rsquo;s test (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). ROEO produced a clear dose-dependent anesthetic response, characterized by faster induction and prolonged recovery times at higher concentrations. Cardiac monitoring revealed reversible bradycardia (up to \u0026minus;\u0026thinsp;56%), elongation of R\u0026ndash;R and Q\u0026ndash;T intervals, and a slight reduction in QRS amplitude without arrhythmias. Opercular frequency decreased by up to 38% at 200 \u0026micro;L L⁻\u0026sup1;, indicating moderate respiratory depression. All effects were reversible within the recovery period. Eucalyptol (47.5%), camphor (19.3%), and α-pinene (12.2%) were the main phytoconstituents identified. \u003cem\u003eRosmarinus officinalis\u003c/em\u003e essential oil provides a safe, reversible, and concentration-dependent anesthetic effect in \u003cem\u003eC. macropomum\u003c/em\u003e, maintaining sinus rhythm and physiological recovery. These results highlight ROEO as a biodegradable, low-toxicity, and effective alternative to synthetic anesthetics, reinforcing its applicability for sustainable and welfare-oriented aquaculture practices.\u003c/p\u003e","manuscriptTitle":"Rosmarinus officinalis essential oil components promote anesthesia in Colossoma macropomum (Cuvier, 1818) through behavioral and cardiorespiratory modulation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-18 15:00:21","doi":"10.21203/rs.3.rs-7964102/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d13fd820-ae03-4da8-8648-9c1caf1502b3","owner":[],"postedDate":"November 18th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-12-03T08:54:54+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-18 15:00:21","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7964102","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7964102","identity":"rs-7964102","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}