Introduction

Brazil is home to vast water reservoirs and a remarkable diversity of native fish species, many of which are suitable for consumption. Despite this, the exotic Nile tilapia (Oreochromis niloticus) dominates the market, followed by native species such as tambaqui (Colossoma macropomum) and pacu (Piaractus mesopotamicus)1,2. Tilapia farming began around 1990 and has since become one of the most important freshwater aquaculture practices globally, with production spanning more than 125 countries. This rapid development has been supported by research institutions and international breeding programs3.

In aquaculture, sedation plays a critical role in reducing stress and minimizing injury during routine handling procedures such as transport, treatment baths, spawning, and other interventions. Proper sedation enhances both fish welfare and handler safety4,5. However, the limited availability of effective, safe, and affordable anesthetics for fish remains a significant challenge. Many commonly used anesthetics are costly, difficult to obtain, and may pose safety risks for the fish, making it essential to explore alternative products6,7.

Natural substances such as menthol8, eugenol9, and Ocimum basilicum2 have emerged as promising alternatives to synthetic anesthetics. Among these, camphor (CPR) is particularly noteworthy. CPR is a naturally occurring aromatic compound found in the essential oil of Cinnamomum camphora (L.) J. Presl and other plants. It is characterized by its white or transparent appearance, distinct aroma, and widespread use in traditional medicine. CPR has been shown to act on the central nervous system, exhibiting anesthetic potential in several animal models, including clownfish (Amphiprion ocellaris) (Cuvier, 1830)10,11,12,13. However, studies also report potential adverse effects, such as seizures during acute intoxication, which may impact autonomic functions13.

The literature already points out that sublethal exposure to CPR can pose a significant risk to the development of aquatic organisms. A study carried out on Danio rerio (zebrafish) embryos found substantial changes in oxidative stress, cardiotoxicity and alterations in cardiac physiology following sublethal exposure to CPR14. The effects included increased reactive oxygen species (ROS), reduced antioxidant activity, oxidative damage, arrhythmias, pericardial edema and changes in heart rate. These results highlight the risks of CPR for fish and reinforce the need for caution in its use and the need for more scientific research on the subject, since, this is the only study that addresses the issue of CPR cardiotoxicity in fish. However, there are no studies using CPR to evaluate muscle and cardiotoxic activity in the fish species O. niloticus.

The use of natural anesthetics like CPR offers several advantages over synthetic drugs such as tricaine (MS-222)15 and benzocaine16. These chemical agents can cause undesirable side effects, including severe metabolic disturbances, residual toxicity, and long-term health impacts on fish. Additionally, synthetic anesthetics may accumulate in aquatic environments, posing risks to local ecosystems and water quality17. In contrast, natural products are often more biodegradable, less toxic, and more environmentally sustainable. They also reduce residue accumulation in fish tissues, promoting safer consumption and enhancing biological compatibility.

In this context, this study aims to evaluate the safety and efficacy of different concentrations (150, 200, 250, and mg L⁻1) of CPR as an alternative anesthetic for Nile tilapia. This evaluation incorporates behavioral observations and electrophysiological techniques (EMG, ECG, and opercular activity recordings), providing a comprehensive assessment of CPR’s anesthetic potential in aquaculture practices.

Material and methods

Experimental animals

A total of 99 juveniles of Nile tilapia were used. The animals were kept in aquariums at the experimental vivarium of the Laboratory of Pharmacology and Toxicology of Natural Products at the Federal University of Pará (UFPA) in an environment with controlled temperature (25 to 28 °C) and photoperiod (12/12 h; light/dark). Feeding was carried out twice a day with commercial feed (32% protein) until satiation. After feeding, the aquariums were siphoned to remove food and feces residues, and the water was partially changed (approximately 20% of the volume) with water from the same source. During acclimation (20 days), water quality variables such as water temperature (°C), pH, and dissolved oxygen (DO) were monitored and maintained as follows: Temperature 26.4 °C; pH 7.6; DO > 5.0 mg L. All procedures were approved by the ethics committee (Ethics Committee on the Use of Animals UFPA- Federal University of Pará) (CEUA/UFPA N 1724310322). All the experiments were carried out using the ARRIVE guidelines and with the normal guidelines.

After all the experiments, euthanasia was carried out, where all the animals were overdosed with the anesthetic tricaine (Ethyl 3-aminobenzoate methanesulfonate) of Sigma- Aldrich (USA- Burlington/Vermont) at a concentration of 500 mg L−1 until death. The material resulting from euthanasia, that is, the sacrificed animals, was disposed of by the company that provides disposal services for biological materials at the Federal University of Pará (UFPA).

Chemical substance

CPR was obtained from the Êxodo Científica laboratory (Sumaré-São Paulo- Brazil). was diluted at a concentration of 10% (100 mg ml) in P.A ethyl alcohol (SouLab laboratory).

Experimental design

Behavioral assessment

The fish were individually exposed to CPR at doses of 150 mg L−1, 200 mg L−1, 250 mg L−1 and 300 mg L−1 and the latencies, that is, times until the loss of the reflex postural, characterized by recumbency and immobility. Subsequently, the fish were transferred to the aquarium water without CPR, where the latency for recovery of the postural reflex was recorded. The fish were observed for 5 min18,19. It is worth noting that all the experiments were carried out only after a pilot study had been conducted to determine the range of doses to be administered that did not cause immediate toxicity in these animals.

Electromyographic analysis (EMG)

To assess muscle activity, electrodes were implanted in the dorsal muscle, following the methodology of Vieira et al.20. Electrodes were affixed to the muscle, 5.0 mm below the dorsal fin. The fish were placed in aquariums inside a Faraday cage and submitted to 5 min baths with CPR solution at: (a) control (clean water), (b) vehicle control (ethanol only-added water) (c)150 mg L−1, (d) 200 mg L−1, (e) 250 mg L−1 and (f) 300 mg L−1. Thereafter, recovery responses were observed and evaluated in CPR-free aquarium water.

Electrocardiogram (ECG)

Tilapia juveniles (38.2 ± 5.5 g) were randomly distributed in the following treatments: (a) control (clean water), (b) vehicle control (ethanol only-added water), (c) 150 mg L−1 CPR, (d) 200 mg L−1 CPR, (e) 250 mg L−1 CPR, and (f) 300 mg L−1 CPR. Measurements were performed during exposure and recovery for each concentration. Each recording lasted five minutes and nine fish were used (n = 9/treatment).

Acquisition of registration (ECG)

For cardiac monitoring, the electrodes were made of stainless steel with 0.3 mm in diameter and 6.0 mm in length, the electrodes were insulated with self-curing acrylic. Fixation of the reference electrode followed the indication of the cardiac vector in lead D1, being positioned ventrally at 0.3 mm from the end of the opercular cavity (left) as a reference electrode, while the electrode for recording was fixed at 0.3 mm from the end of the right opercular cavity. Subsequently, the electrodes were connected to a high impedance amplifier. From the records, heart rate (HR) in beats per minute (BPM), QRS complex amplitude (mV), QRS complex duration (ms), PQ (ms), RR (ms) and QT (ms) intervals were analyzed.

Obtaining the opercular beat frequency

For the analysis and monitoring of respiratory function through the opercular beat per minute (OBM), conjugated electrodes were made 0.5 mm apart in stainless steel with a diameter of 0.3 mm and a length of 5.0 mm and were placed on the left operculum to record the activity during the immersion bath with the same groups of CPR concentrations. The intensity of force used for opercular closure was also recorded in the form of power during contact with CPR. Each recording lasted 5 min and the final ten seconds of each recording were analyzed.

Analysis of records

The electrodes were connected to a digital data acquisition system through a high impedance differential amplifier (Grass Technologies, Model P511), adjusted with 0.3 and 300 Hz filtering, with 2000X amplification and monitored by oscilloscope (Protek, Model 6510). Recordings were continuously digitized at a rate of 1 kHz using a data acquisition board (National Instruments, Austin, TX) and stored on a hard disk for further processing using a specialized software (LabVIEW express).

Statistical analysis

After verifying compliance with the assumptions of normality and homogeneity of variances using the Kolmogorov–Smirnov and Levene tests, respectively, comparisons were made between the mean power of the records using one-way ANOVA, followed by the post-hoc test of Turkey. The GraphPad Prism® 8 software was used for the analyzes and p-values of * < 0.05, ** < 0.01, or *** < 0.001 were considered statistically significant in all cases.

Results

As no differences were observed between the controls and the vehicle group treatment, only mean values of the former were used for comparison purposes against CPR-exposed fish.

CPR causes loss of postural reflex, with incomplete muscle relaxation

Behavioral analysis showed that CPR caused loss of the posture reflex, which was dose-dependent, and the higher the dose, the shorter the latency for the onset of the loss of the posture reflex. Fish exposed to 150 mg L−1 of CPR showed loss of posture in 247.0 ± 8.7 s while the group exposed to 200 mg L−1 took significantly less time for loss of reflex, in 218.6 ± 11.9 s. Fish treated with 250 mg L−1 of CPR showed a mean loss of the postural reflex of 184.8 ± 10.8 s, significantly faster than the groups exposed to 150 mg L−1 and 200 mg L−1 of CPR. For the group treated with 300 mg L−1 of CPR, the mean latency was even lower (167.0 ± 8.8 s) compared to the groups submitted to lower concentrations (Fig. 1A).

Fig. 1
figure 1

Mean latencies (s) for the onset of postural reflex loss during immersion baths with different concentrations of CPR (A). Recovery of the postural reflex after exposure to different concentrations of CPR (B) after ANOVA followed by Tukey’s test (*p < 0.05, **p < 0.01, ***p < 0.001, n = 9).

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The recovery of the postural reflex in the group exposed to 150 mg L−1 CPR occurred in 210.4 ± 7.2 s, being similar to the recovery time in fish exposed to 200 mg L−1 CPR (241.4 ± 11.6 s). The group exposed to 250 mg L−1 CPR showed an average recovery of 262.4 ± 8.6 s, greater than the recovery times of fish bathed at 150 mg L−1 or 200 mg L−1 CPR. Fish exposed to 300 mg L−1 CPR recovered the postural reflex in 287.9 ± 9.7 s, which was longer than the other groups (Fig. 1B).

Dorsal muscle contraction activity of O. niloticus in normal activity 300 s electromyographic (EMG) tracing, and frequency spectrogram showing power distribution at frequencies up to 40 Hz (Fig. 2A). The colorimetric scale shows signal strength (power) over time and at different frequencies (Fig. 2B–F).

Fig. 2
figure 2

Recording of muscle activity (EMG) of fish during swimming during treatment and after treatment with CPR and their respective spectrograms indicating the power of data analyzed up to 40 Hz. Evaluated for the following groups: control (A); vehicle (B), treated with 150 mg L−1 CPR (C); 200 mg L−1 CPR (D). 250 mg L−1 CPR (E) and 300 mg L−1 CPR (F). Average power of muscle contraction in the last 50 s of the 5-min records of contact with CPR (F). EMG during recovery after immersion baths with CPR at 150 mg L−1 (G); 200 mg L−1 (H). 250 mg L−1 (I); 300 mg L−1 (J). Average values of muscle contraction power in the last 50 s of the 5 min records during recovery (K). (ANOVA followed by Tukey’s test; ***p < 0.001; n = 9).

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During the final 50 s of immersion in 150, 200, 250 and 300 mg L−1 of CPR decreased muscle activity was observed, but irregular tracing patterns were observed for the treated group, as shown in Fig. 2B. However, the concentrations were sufficient to induce loss of posture (as shown in Fig. 1A). Frequency spectrograms (Fig. 2A–F) indicate energy levels according to low-intensity muscle contraction in the last 50 s of records when compared to the control group.

The normal muscle contraction of the controls had an average of 58.9 ± 11.8 mV2/Hz × 10–3, it was similar to the vehicle (p = 0.930), but they were higher than the average values of the groups exposed to 150 mg L−1, 200 mg L−1, 250 mg L−1 and 300 mg L−1 RCP (6.2 ± 0.5 mV2/Hz × 10–3, 3.4 ± 1.1 mV2/Hz × 10−3, 2.5 ± 0.7 mV2/Hz × 10–3 and 2.1 ± 0.1 mV2/Hz × 10–3, respectively), in any case, there was no statistical difference between the treated groups (p = 0.855) (Fig. 2G).

Although higher concentrations of CPR caused loss of the postural reflex with decreased muscle activity and lower latency, this occurred at the expense of a longer recovery of the postural reflex (Fig. 1B). The normal muscle contraction of the controls had an average of 58.9 ± 11.8 mV2/Hz × 10–3, showing no difference from the groups vehicle and exposed to 150, 200 and 250 mg L−1 CPR (p = 0.261) that presented respective powers of 71.7 ± 17.8 mV2/Hz × 10–3, 67.4 ± 8.3 mV2/Hz × 10–3, 59.5 ± 8.1 mV2/Hz × 10–3 (Fig. 2K). The group receiving 300 mg L−1 CPR, presented a mean value of 30.8 ± 10.8 mV2/Hz × 10–3, which was lower than those of the groups exposed to 150 mg L−1, 200 mg L−1 and 250 mg L−1 CPR and the control group (p = 0.312), however, regardless of concentration, all fish showed reversibility of the effect and loss of posture reflex (Fig. 2H–L) small variation in the EMG recording reveals muscle activity before full recovery, demonstrating that CPR has a partial myorelaxant effect.

Cardiorespiratory activity during and after immersion bathing reinforces camphor toxicity in fish

Cardiac activity in the controls showed a mean rate of 85.1 ± 3.1 bpm, in amplitudes around 0.8 ± 0.06 mV (Fig. 3A). It was similar to the vehicle group (p = 0.673) At 10 s magnification, all cardiac waves and complexes can be seen in sinus rhythm, with atrial activity represented by P waves, ventricular activity by QRS complexes, and ventricular repolarization by T waves (Fig. 3B).

Fig. 3
figure 3

Cardiac activity recording (ECG) in tilapia, with amplification of the recording in the final 10 s with identification of cardiac triggers P wave, QRS complex and T wave. With analysis of HR, amplitude (mV), RR interval (ms), PQ interval, QRS complex (ms) and QT interval. for the following groups: control (A); control amplification (B);Vehicle group (C); 150 mg L−1 (D); 200 mg L−1 (E) and 250 mg L−1 (F); 300 mg L−1 of CPR (G). Mean HR values (BPM) (H); Mean values of QRS complex amplitude (mV) (I), mean values of R-R (ms) (J), P-Q intervals (ms) (K), QRS complex duration (ms) (L) and QT interval (ms) (M) (ANOVA followed by Tukey’s test; *p < 0.05, **p < 0.01, ***p < 0.001; n = 9).

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During exposure to CPR at 150 mg L−1 and 200 mg L−1, the ECG showed sinus bradycardia (Fig. 3D,E), and HR decreased by 26.10% and in the group exposed to 150 mg L−1 and 29.5% in fish exposed to 200 mg L−1, compared to the controls (Fig. 3H). Bradycardia was dose-dependent as the fish were submitted to higher concentrations of CPR (250 mg L−1 and 300 mg L−1) (Fig. 3F,G), with more intense bradycardia compared to the controls (43.08% and 56.3%, respectively). Changes were observed in the T wave which showed a decreased amplitude (Fig. 3G, right).

HR was significantly affected by CPR. Controls averaged 85.1 ± 3.8 bpm, higher than the group treated with 150 mg L−1 (62.8 ± 3.1 bpm). Fish exposed to 200 mg L−1 had a mean HR of 60 ± 3.7 bpm, similar to that of the 150 mg L−1 CPR-exposed fish. The mean HR of animals exposed to 250 mg L−1 and 300 mg L−1 were 48.4 ± 2.4 bpm and 37.1 ± 2.4 bpm, respectively, being lower than those of the controls and from fish exposed to 150 mg L−1 and 200 mg L−1 (Fig. 3H). The group exposed to higher concentration of CPR showed a more pronounced change in the ECG, such as a decreased T wave (Fig. 3G,H).

The mean amplitude of the QRS complex in the control group was 0.8 ± 0.06 mV, it was similar to the vehicle group, (p = 0.999) showing no difference in relation to the other groups. The group treated with 150 mg L−1 showed a mean QRS amplitude of 0.7 ± 0.1 mV, while the groups exposed to 200 mg L−1 (0.7 ± 0.2 mV), 250 mg L−1 (0.7 ± 0.1 mV) and 300 mg L−1 CPR (0.6 ± 0.07 mV) had similar responses F (5,48) = 1.532 (p = 0197) (Fig. 3I).

The mean RR interval in the control group was 705.8 ± 33.6 ms, it was similar to the vehicle group, (p = 0.959), which was lower than those of the other groups. The group treated with 150 mg L−1 CPR had an average RR interval of 955.9 ± 48.5 ms, with no difference from the average of fish exposed to 200 mg L−1 (1033 ± 59.6 ms) (p = 0.587). The 250 mg L−1 CPR group had a mean interval of 1239 ± 60.09 ms, that is, greater than that of the controls, 150 mg L−1 and 200 mg L−1 groups. The mean RR interval for the 300 mg L−1 CPR group was 1622 ± 102.7 ms, and was longer than those of any other group (Fig. 3J).

The mean PQ interval for the control group was 94.5 ± 2.3 ms, similar to the vehicle group and 150 mg L−1 group (p = 0.951). Fish exposed to 200 mg L−1 of CPR showed an average of 107.6 ± 6.3 ms, with longer intervals than that of the control group, similar to the150 mg L−1 group (p = 0.057). The 250 mg L−1 CPR group had an average of 111.2 ± 15.9 ms, which was higher than that of the control group and the 150 mg L−1 CPR-exposed group, similar to 200 mg L−1 (p = 0.918). The 300 mg L−1 CPR-exposed fish presented an average of 151.8 ± 5.8 ms, differing from all other groups due to the increased P-Q interval (Fig. 3K).

The mean duration of the QRS complex for the control group during induction was 29.1 ± 1.5 ms, being similar to similar to vehicle and the 150 mg L−1 group (p = 0.176), but the control group and 150 mg L−1 was different from the 200 mg L−1 and 250 mg L−1 groups, which respectively presented 46.2 ± 2.2 ms and 46 ± 2.2 ms. The 300 mg L−1 group (53.4 ± 8.2 ms), however, treated with 200 mg L−1 and 250 mg L−1 they were similar (p = 0.999), showed an increase in QRS complex duration compared to all groups (Fig. 3L).

For the control group, the mean QT interval during induction was 326.0 ± 2.8 ms, which was similar to vehicle and 150 mg L−1 treated group (p = 0.869). The 200 mg L−1 group (390 ± 8.3 ms) showed an increase in the Q-T interval compared to the control and 150 mg L−1 group. The 250 mg L−1 group (417.6 ± 2.8 ms) showed an increase in the QT interval for the control, 150 mg L−1 and 200 mg L−1 groups. The 300 mg L−1 group (548.2 ± 26.4 ms) showed statistical difference compared to the other groups (Fig. 3M).

During recovery from exposure to 150 mg L−1, 200 mg L−1 and 250 mg L−1 CPR, reversibility of electrocardiographic changes were observed (Fig. 4B–D). The HR increased compared to the control group, 31.2% for the group treated with 150 mg L−1 and 15.9% for the fish treated with 200 mg L−1 and 10.18% for the group treated with 250 mg L−1, during 5 min of recovery. For the group treated with 300 mg L−1 it was 29.7% below the control group (Fig. 4E). This indicates a gradual recovery of cardiac activity which was dependent on the concentration (Fig. 4B–E).

Fig. 4
figure 4

Electrocardiogram (ECG) of tilapia during recovery from CPR treatment with their respective amplifications of the last 10 s of recording for the following groups: Vehicle (A); 150 mg L−1 (B); 200 mg L−1 (C); 250 mg L−1 (D) and 300 mg L−1 (E). Graph showing the means of mean HR in beats per minute (F), mean values of QRS complex amplitude (mV) (G), mean R-R values (ms) (H), P-Q intervals (ms) (I), QRS complex duration (ms) (J) and QT interval (ms) (K). (ANOVA followed by Tukey’s test; *p < 0.05, **p < 0.01, ***p < 0.001; n = 9).

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Fish that recovered from exposure to 150 mg L−1, 200 mg L−1 and 250 mg L−1 had mean HR of 112.1 ± 8.2 bpm, 98.6 ± 3.6 bpm and 93.7 ± 4.1 bpm, respectively, and presented a mean value above the control group (85.1 ± 3.8 bpm). The 300 mg L−1 CPR group showed partial recovery (59.7 ± 3.07 bpm) when compared to the control group (Fig. 4F). HR recovery was dose-dependent. The higher the concentration, the longer it took for bradycardia to reverse.

The amplitude of the QRS complex during recovery for the control group was 0.8 ± 0.06 mV, showing no difference for the other groups. The 150 mg L−1 (0.8 ± 0.08 mV), 200 mg L−1 (0.8 ± 0.1 mV), 250 mg L−1 (0.8 ± 0.1 mV) and 300 mg L−1 (0.7 ± 0.1 mV), showed no statistical difference between groups (F (5, 48) = 1.078; p = 0.384) (Fig. 4G).

The mean RR interval during recovery for the control group was 705.8 ± 33.66 ms similar to the vehicle group (p = 0.787), which was higher compared to the 150 mg L−1 (537.7 ± 41.7 ms), 200 mg L−1 (608.7 ± 22.7 ms) groups, 250 mg L−1 (636.3 ± 29.9 ms). The 200 mg L−1 and 250 mg L−1 groups showed a statistical difference for the control and 150 mg L−1 groups. The group treated with 300 mg L−1 with a mean of 995.6 ± 60.7 ms, showed a statistical difference from the other groups (Fig. 4H).

The PQ interval during recovery in controls was 94.5 ± 2.3 ms, 150 mg L−1 (88.5 ± 2.7 ms), 200 mg L−1 (84.1 ± 16.8 ms) and 250 mg L−1 (93.5 ± 9.4 ms) maintained no statistical difference (p = 0.156). The group treated with 300 mg L−1 presented an average of 111.7 ± 8.7 ms, demonstrating a statistical difference for the other groups (Fig. 4I).

The QRS complex duration during recovery did not differ between controls (29.1 ± 1.1 ms) and fish exposed to 150 mg L−1 CPR (29.1 ± 2.2 ms), 200 mg L−1 CPR (27.9 ± 2.6 ms), 250 mg L−1 CPR (29.2 ± 1.5 ms) (p = 0.7595). However, it foresees a statistical difference for the 300 mg L−1 CPR group (35 ± 3.9 ms), indicating that the recovery of cardiac activity takes longer for the animals submitted to the immersion bath with 300 mg L−1 (Fig. 4J).

During recovery, the QT interval for the control was 326.0 ± 28.28 ms, similar to that of fish exposed to 150 mg L−1 CPR (306.9 ± 44.5 ms), 200 mg L−1 CPR (301.0 ± 30.8 ms), 250 mg L−1 CPR (296.1 ± 21.2 ms), 300 mg L−1 CPR (294.6 ± 15.2 ms) (Fig. 4K) (F (5, 48) = 1.595; p = 0.179).

Tilapia respiratory activity in the control group demonstrated an average frequency of 50.8 ± 4.01 OBM, with amplitude maintained at 0.5 mV (Fig. 5A). In the 10 s amplification, the opercular movement rhythm can be observed, which are indicated by the red arrows (Fig. 5B).

Fig. 5
figure 5

Recording OBM in tilapia (A), Amplification of the recording over a period of 10 s (290–300 s) observed opercular beating rhythm (B) indicated by red arrows. Records OBM in tilapia, during the immersion bath with CPR, and amplification of the recording in the period 290–300 s: 150 mg L−1 (D); 200 mg L−1 (E); 250 mg L−1 (F); 300 mg L−1 (G). Graph demonstrating the average frequency of opercular movements for the groups during the immersion bath with CPR (H) and Graph demonstrating the power of opercular beats during the immersion bath with CPR (I). Records of respiratory activity of OBM in tilapia, after the immersion bath with CPR, in the return period (on the left), and amplification of the record in the period 290–300 s (on the right) in the return period after contact: 150 mg L−1 (J); 200 mg L−1 (K); 250 mg L−1 (L); 300 mg L−1 (M). Graph showing the average frequency of OBM for the groups after the immersion bath with CPR (N) and Graph showing the power of opercular beats after the immersion bath with CPR (O). (ANOVA followed by Tukey *p < 0.05; **p < 0.01; ***p < 0.001; n = 9).

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Opercular beating recordings at different CPR concentrations during the immersion bath demonstrated a decrease in respiratory activity, observed at higher concentrations (Fig. 5D–G).

Records of tilapia respiratory activity demonstrated that the control group had an average of 50.8 ± 4.01 OBM it was similar to thevehicle group (p = 0.649), a higher value than the other groups during the immersion bath with CPR: 150 mg L−1 (42.2 ± 2. 9 OBM), 200 mg L−1 (40.4 ± 2,1 OBM), 250 mg L−1 (35.5 ± 1.9 OBM) and 300 mg L−1 (30 ± 3,3 OBM). The 150 mg L−1 vs 200 mg L −1 group were similar (p = 0.951), however, they were higher than the 250 mg L−1 and 300 mg L−1 groups. The group submitted to the immersion bath with 250 mg L−1 it was similar to the 300 mg L−1 group (p = 0.885) (Fig. 5H).

Recording the power of the opercular beats demonstrated that the control group presented an average of 10.5 ± 3.4 mV2 /Hz × 10–3, with no difference to the other groups: 150 mg L−1 (12.2 ± 1.7 mV2/Hz × 10−3 ) (p = 0.770), 200 mg L−1 (11.4 ± 1.5 mV2/Hz × 10–3) (p = 0.979), 250 mg L−1 (13.5 ± 3.7 mV2 /Hz × 10−3) (p = 0.191) and 300 mg L−1 (9.1 ± 0.6 mV2/Hz × 10–3) (p = 0.883). The groups 150 mg L−1 versus 200 mg L−1 (p = 0.989), vs 250 mg L−1 (p = 0.910), versus 300 mg. L−1 (p = 0.171). The groups 200 mg L−1 vs 250 mg L−1 (p = 0.583), versus 300 mg L−1 (p = 0.475). The 250 mg L−1 versus 300 mg L−1 groups showed a difference (Fig. 5I).

During the recovery period from the immersion bath, the animals showed opercular hypermotility in the 150 mg L−1 groups; 200 mg L−1; 250 mg L−1 , which is related to the irritability of CPR, thus, with the recovery of reflexes, the animals showed reactions that facilitate their elimination, however this was not observed in the 300 mg L−1 groups as the recovery of reflexes was slower (Fig. 5J–M).

The mean opercular beat frequency for the control group (50.8 ± 4.01 OBM) to similar to vehicle group (p = 0.748), however, was lower than the groups 150 mg L−1 (76.2 ± 3.9 OBM), 200 mg L−1 (68.8 ± 2.2 OBM), 250 mg L−1 (60.8 ± 4.8 OBM).The control and vehicle control it was similar to the 300 mg L−1 group (47.5 ± 3.5 OBM) (p = 0.070). The 150 mg L−1 group was larger than the other groups. All treated groups were different, with a lower frequency of opercular beating during recovery for the group subjected to a higher concentration of CPR (Fig. 5N).

Recording the average power of opercular beats during the recovery period demonstrated that the control group had an average of 10.53 ± 3.45 mV2/Hz × 10–3 was lower than the groups: 150 mg L−1 (25.6 ± 3.5 mV2/Hz × 10−3), 200 mg L−1 (25.5 ± 5.4 mV2/Hz × 10–3), 250 mg L−1 (18.9 ± 7.5 mV2/Hz × 10–3). However, the recovering group with 300 mg L−1 (11.3 ± 8.2 mV2/Hz × 10−3) (p = 0.999) was similar. The groups 150 mg L−1 versus 200 mg L−1 (p = 0.999), versus 250 mg L−1 (p = 0.147). The groups 200 mg L−1 versus 250 mg L−1 (p = 0.160), 250 mg L−1 versus 300 mg L−1 (p = 0.065). The 300 mg L−1 group it was lower than the groups treated with 150 mg L−1 and 200 mg L−1 (Fig. 5O).

Discussion

The use of natural anesthetics such as CPR should be evaluated not only for their impact on individual fish, but also for environmental sustainability in large-scale aquaculture systems. Previous studies indicate that synthetic anesthetics, such as MS-22215,21,22, can remain for longer in the environment, before its degradation, compromising water quality and affecting non-target species23,24. CPR, being a natural compound, has the potential to degrade more quickly in the aquatic environment. However, its introduction into aquaculture systems must be carefully monitored to avoid sublethal concentrations that could cause chronic stress or alter aquatic communities.

The use of CPR as an anesthetic for fish, especially tilapia, has been little explored. In our data, we demonstrated the onset of muscle relaxation with loss of postural reflex (Fig. 1A), similar to previous studies in which CPR was used as an anesthetic during the handling of clownfish (A. ocellaris), inducing the animal to deep anesthetic planes11. The EMG data corroborated the described behavior, demonstrating that during the immersion bath with CPR, the animals initially exhibited intense muscular activity, which was followed by a reduction in the amplitude of the recordings. This progression ultimately led to the immobility of the animals. In this state, the potential difference between the electrodes diminished, indicating a decrease in muscle activity. However, the irregularities observed in the EMG tracings suggested that relaxation was not fully achieved.

Some studies have already demonstrated characteristics of the electromyogram in anesthetized fish, such as the Tambaqui, which has characteristics with graphoelements that reveal a decrease in muscle activity dependent on drug concentration and contact time in an immersion bath, features such as excitability and increased muscle activity during the initiation of contact followed by deep relaxation without EMG activity18,21,25,26. CPR did not present characteristics similar to other anesthetics studied, the recordings of muscle activity were not compatible for procedures in which there is a need for deep myorelaxation, at all treatment concentrations changes were observed in the tracing that indicated minimal muscle activity that caused the loss of the reflex postural. This low-intensity muscle activity was observed during fish treatment and recovery.

Although camphor has shown anesthetic potential in tilapia, its practical application should consider direct comparisons with conventional anesthetics such as eugenol9 and MS-22215,22. Both have been widely used in aquaculture, but have limitations, such as cumulative toxic effects and higher cost. In a recent study, eugenol showed rapid reversibility and minimal cardiovascular adverse effects in tilapia when used in adequate concentrations9. Directly comparing the physiological effects and cost-effectiveness of camphor with these anesthetics will help define its viability as a viable alternative in commercial aquaculture practices27.

The effects of camphor on the cardiovascular system observed in this study may be related to its direct impact on autonomic function and cardiac electrophysiology. Studies with other models, such as zebrafish (Danio rerio)14, have shown that camphor can alter ventricular repolarization and the duration of cardiac intervals, possibly due to interference with cardiac ion channels. These effects are critical because a prolongation of the QT interval, as observed at higher concentrations (300 mg L−1), can predispose fish to arrhythmias. In addition, the decrease in the amplitude of the T wave suggests a possible impairment in the repolarization process, which could be explored in future studies to identify whether camphor interacts specifically with potassium channels or other components of the cardiac excitatory circuit.

The ECG parameters correspond to important elements to evaluate anesthesia in any animal species, some works have been published with ECG monitoring in anesthetized or intoxicated fish25,28,29. The ECG is a tool that can indicate cardiac malfunction due to tissue damage or acute toxicity25,30. Recent electrocardiogram studies in juvenile tambaqui fish have reported bradycardia when exposed to different types of anesthetics, where a reduction in HR was observed during anesthetic induction with gradual recovery and no mortality5,18,25,28,30. CPR is known to cause CNS changes, including seizures, which can alter autonomic functions such as the heart27,13. Our data demonstrated that during the immersion bath in varied concentrations of CPR, cardiac depression was dose-dependent, as there was a decrease in HR, increase in the RR interval, increase in the PQ interval, increase in the duration of the QRS complex and increase in the QT interval, with changes in these parameters being more significant during the immersion bath at a concentration of 300 mg L−1. Notwithstanding, one of the parameters that did not show variation in the averages was the amplitude of the QRS complex. For the 300 mg L−1 group, a decrease in the amplitude of the T wave was observed, which may be related to the action of CPR acting directly on cardiac repolarization or indirectly by breaking homeostasis of the autonomic nervous system.

Electrocardiographic characterization should be systematically used to rule out the possibility of deleterious implications that may be involved throughout exposure, thus, we evaluated the recovery of cardiac activity in water without CPR, in relation to HR, in animals from groups 150, 200 and 250 mg L−1 showed sinus tachycardia during recovery, therefore, a decrease in the RR interval was observed for these groups. For the 300 mg L−1 group, the recovery was slower, the heart remained in sinusal bradycardia, the RR interval, the PQ interval and the duration of the QRS complex remained increased in relation to the control, demonstrating that high concentrations of CPR can be a limiting factor for its use in tilapia, therefore, during the recovery of the posture reflex and swimming activity, they can be influenced by changes in hemodynamics due to cardiac malfunction, which can lead to questions about the use of CPR in this species for anesthesia.

The respiratory rhythm can be significantly affected by anesthetic agents, which is why it is crucial to monitor the respiratory activity of the anesthetic to be tested31. Studies on monitoring respiratory activity in fish have sought to elucidate the action of these anesthetics, proving their effectiveness in induction and recovery18,22,31. Our results demonstrate a decrease in respiratory activity in all concentrations of CPR used in the work, with respiratory depression being more observed in groups with higher concentrations. The opercular beating power was also reduced during the immersion bath at a higher concentration. During the recovery period, an increase in the frequency of opercular beating was observed at concentrations between 150 mg L−1, 200 mg L−1 and 250 mg L−1, which is related to increased muscle tone and recovery of the postural reflex, this can be corroborated by the average power of the opercular beat.

The results presented in this study highlight that intermediate concentrations of CPR (200–250 mg L−1) seem to offer a balance between anesthetic efficacy and the reversibility of adverse effects. This dosage range may be ideal for short-term procedures, such as handling and transport, where it is necessary to minimize physiological stress in the fish. Future studies could investigate strategies to mitigate the residual effects of camphor at higher concentrations, such as combinations with other natural compounds that promote hemodynamic stability or reduce toxicity. Alternatively, adjustments to the exposure time and formulation of the anesthetic solution could further improve its safety and efficacy.

We concluded that during treatment with CPR the animals showed loss of postural reflex, but muscle activity was detected even at the highest concentrations. The changes in the ECG and respiratory movement indicate that CPR in the concentrations used in this work can be harmful to the hemodynamics of tilapia, causing electrocardiographic and respiratory changes for a long period, even after recovery of muscular activity such as the postural reflex.

This work reinforces the importance of further studies to define an ideal therapeutic window for the use of CPR in tilapia. Long-term assessments of the impacts on immunological and reproductive parameters would be valuable, considering the potential for hormonal interference of natural anesthetics. In addition, exploring inter-individual variability in the response to camphor could help predict risks associated with genetic or metabolic differences between fish populations. Finally, cost–benefit analyses and experiments under real management conditions should be conducted to confirm the commercial viability of camphor in aquaculture systems.