Essential oils from Cymbopogon citratus and Lippia sidoides in the anesthetic induction and transport of ornamental fish Pterophyllum scalare

This study verified the effects of essential oils from Lippia sidoides (EOLS) and Cymbopogon citratus (EOCC) on the anesthesia of freshwater angelfish (Pterophyllum scalare) of two different sizes (juveniles I (0.82 g) and II (2.40 g)) and the transport (8 h) of juveniles II. Fish were exposed to different concentrations of EOLS and EOCC: 0, 10, 25, 50, 75, 100, 150, 200, and 250 mg L−1. Ventilatory rate (VR) and transport for 8 h with 0, 10, and 15 mg L−1 of each essential oil were evaluated in juveniles II. The major components found in EOLS and EOCC were carvacrol (44.50%) and α-citral (73.56%), respectively. The best sedation and anesthesia times for both essential oils were obtained with 10 and 25 mg L−1 and 200 and 250 mg L−1 for juveniles I and II, respectively. Fish sedated with EOLS had lower VR values than the other treatments. Blood glucose levels were higher in ornamental fish transported with 10 and 15 mg EOLS L−1 and 15 mg EOCC L−1. Hepatic glycogen values were higher in the control group. In general, fish transported with 10 mg EOLS L−1 showed fewer gill histological alterations than other transported fish. When the type of lesion was evaluated, the highest gill alterations occurred in fish transported with EOCC. In conclusion, 10 mg EOLS L−1 could be used to transport of juveniles II because although this concentration increased blood glucose levels, it decreased the VR and muscle glycogen levels and caused only mild alterations to the gills.


Introduction
Freshwater angelfish (Pterophyllum scalare) is one of the most attractive species of ornamental fish from continental waters for the aquarium market (Chapman et al. 1997), mainly because of its morphological diversity and color patterns (Oliveira et al. 2019a). It is an omnivorous fish with a varied diet, ingesting plankton, plants, and small invertebrates. In its natural environment (Amazon Basin), it can reach Abstract This study verified the effects of essential oils from Lippia sidoides (EOLS) and Cymbopogon citratus (EOCC) on the anesthesia of freshwater angelfish (Pterophyllum scalare) of two different sizes (juveniles I (0.82 g) and II (2.40 g)) and the transport (8 h) of juveniles II. Fish were exposed to different concentrations of EOLS and EOCC: 0,10,25,50,75,100,150,200, and 250 mg L −1 . Ventilatory rate (VR) and transport for 8 h with 0, 10, and 15 mg L −1 of each essential oil were evaluated in juveniles II. The major components found in EOLS and EOCC were carvacrol (44.50%) and α-citral (73.56%), respectively. The best sedation and anesthesia times for both 1 3 Vol:. (1234567890) a length of up to 15 cm and inhabits slightly acidic waters with low hardness (Cacho et al. 1999). The species is sensitive to handling and transportation procedures (Oliveira et al. 2019a).
In this sense, anesthetics are used to reduce stress during handling and transport (short or long duration) procedures, preventing damage to the health of the fish (Hohlenwerger et al. 2016;Aydin and Barbas 2020). During transport, fish can be exposed to high-stress levels, the accumulation of excreted compounds, and reduced available dissolved oxygen (Sampaio and Freire 2016;Teixeira et al. 2018;Oliveira et al. 2019b), impairing their welfare. Fish subjected to these conditions can have undesirable side effects such as a compromised immune system, reduced growth, and mortality (Ross and Ross 2008;Souza et al. 2019).
Many recent studies have recommended essential oils from medicinal plants as anesthetics as they appear to be a safe alternative and have contributed to improving fish welfare during handling and transport Teixeira et al. 2017;Limma-Netto et al. 2018;Aydin and Barbas 2020;He et al. 2020;Brandão et al. 2021;Hoseini et al. 2021;Mirzargar et al. 2022;Yousefi et al. 2022). At lower concentrations, they can sedate fish, increasing their tolerance to more extended periods of transport (e.g., 8 h) (Teixeira et al. 2018;Oliveira et al. 2019a). The ventilatory rate (VR) evaluation is used to confirm the sedative effects of essential oils, where opercular movements are reduced in fish exposed to an anesthetic/sedative (Hohlenwerger et al. 2017;Becker et al. 2018;Teixeira et al. 2018).
Pepper-rosmarin (Lippia sidoides Cham. ((syn. L. origanoides)) is a shrub plant of the Verbenaceae family and endemic to northeastern Brazil. It is used in traditional medicine as an infusion or decoction due to its pharmacological actions as a sedative, anxiolytic, antifungal, antiseptic, anti-inflammatory, and muscle-relaxing compound (Dos Santos et al., 2015). In aquaculture, essential oil from L. sidoides (EOLS) is effective in transporting, treating, and combating parasites (10-20 mg L -1 ) and as an anesthetic (5-70 mg L -1 ) to mitigate damage associated with stress from routine procedures (weighing, aquarium change, and harvesting) (Soares et al. 2017;Ventura et al. 2019;Brandão et al. 2021).
Although essential oils are already widely known as anesthetics, they are foreign substances (xenobiotics) to fish organs and tissues, altering their physiological functions. The gill tissue has an extensive epithelial surface area and is in direct contact with the environment (Fernandes et al. 2007), making it the primary target of essential oils. Therefore, gill histological studies should be used to detect possible stressful effects of xenobiotics (Araujo et al. 2019;Fiedler et al. 2020). In addition, metabolic parameters such as blood glucose and muscle glycogen can be assessed in small fish (~ 2 g) and provide insight into physiological changes (Oliveira et al. 2019a). This is the first study evaluating the effects of EOLS and EOCC as anesthetics for freshwater angelfish, which have shown promising results in previous studies for other fish species (Kizak et al. 2018;Hacke et al. 2020;Monteiro et al. 2021;Brandão et al. 2021). The time taken to achieve anesthesia can vary depending on the concentration of the anesthetic (Teixeira et al. 2017) and fish weight (Tarkhani et al. 2016). Based on these facts, the present study evaluated the effects of different concentrations of EOLS and EOCC on sedation, induction, and anesthetic recovery in freshwater angelfish juveniles of two different sizes (juveniles I and II). Furthermore, we also evaluated the effects of these essential oils for juveniles II as sedatives on the transport (gill and metabolic changes) and VR reduction.

Essential oils
The plants to produce EOLS and EOCC were cultivated in Petrolina, PE, Brazil. The collections of the fresh leaf samples were carried out in autumn 2018.

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Vol.: (0123456789) The extraction of essential oils was carried out by steam distillation using a Clevenger-type apparatus (British Pharmacopoeia 2007) for 2 h in the Natural Products Laboratory of the Instituto Federal do Sertão Pernambucano, Petrolina, PE, Brazil. The determination of their chemical compounds was carried out by gas chromatography/mass spectrometry (GC-MS) in the Multi-User Characterization and Analysis Laboratory of the Universidade Federal da Paraíba, João Pessoa, PB, Brazil.
The GC-MS analysis was performed using a gas chromatograph coupled to a mass selective detector (Shimadzu GCMS-QP2010 Ultra-Barueri, SP, Brazil). The capillary column was DB-5MS (Agilent technologies; 30 m × 0.25 mm × 0.25 µm). Helium (99.99%) was used as the gas carrier. The conditions used were as follows: a constant flow of 1.1 mL min -1 ; injection volume of 1.0 µL with injector split ratio of 1:10 (250 °C); electron impact mode at 70 eV; ion source temperature at 280 °C; and transfer line temperature at 260 °C. The oven temperature was programmed to 60 °C, with a progressive increase of 3 °C min -1 until it reached 240 °C. A mixture of linear hydrocarbons (C8H18-C20H42) was injected under the same experimental conditions. The essential oils' constituents were identified by comparing retention indices in the equipment database (NIST, 2008) and Kovats retention index. The essential oils used in this study were the same as those used by Felix e Silva et al. (2022). The major components found for EOLS were carvacrol (44.50%), p − cymene (14.06%), and thymol (7.99%); for EOCC, they were α − citral (73.56%), myrcene (12.65%), and geraniol (3.51%).

Location and animals
Two different sizes of fish were tested, termed here as juveniles I (0.82 ± 0.02 g; 3.24 ± 0.05 cm) and juveniles II (2.40 ± 0.08 g; 4.78 ± 0.05 cm). Initially, we evaluated sedation, anesthesia induction, and anesthetic recovery times with EOLS or EOCC in juveniles I and II (n total = 144). Afterward, a second experiment was performed to evaluate the effects of these essential oils on the transportation of juveniles II (n total = 48). Finally, a third experiment, evaluating VR, was conducted with juveniles II (n = 40). Only juveniles II were selected for the second and third experiments because juveniles I were smaller, severely impairing the analysis of blood glucose, muscle glycogen, gill histology, and VR.
The experiments were carried out in the Laboratory of Study and Physiology of Aquatic Fauna of the Universidade Federal da Bahia (UFBA) under registration no. 03-2019 at the Ethical Committee of the Biology Institute of the UFBA. A total of 232 juveniles from a commercial fish farm (Lauro de Freitas, BA, Brazil) were used.
Before testing, the fish were kept in two 500-L tanks with constant aeration and mechanical and biological filters. Juveniles were fed four times a day (commercial diet, 50% crude protein, Sera Discus Color Red, Germany) for an acclimation period of 10 days until apparent satiety. Feeding was suspended 24 h before the beginning of the experiments.

Induction and anesthetic recovery
The procedure involved transferring juveniles (I and II) to aquaria containing 2 L of water and EOLS or EOCC at concentrations of 10,25,50,75,100,150,200, and 250 mg L −1 diluted 1:10 with absolute ethanol (1:10). Densities for EOLS and EOCC were, respectively, 0.93 and 0.90 g cm −3 . A control group was transferred to aquaria containing only ethanol (2250 mg L −1 ) at a concentration equivalent to the dilution used for 250 mg L −1 of both essential oils. A second control group with fish kept in aquariums containing only water was also performed.
In each treatment, eight juveniles I and eight juveniles II were used. For sedation (partial loss of balance and erratic swimming) and anesthesia (complete loss of balance and cessation of swimming) evaluation, two fish were simultaneously placed in each aquarium (adapted from Oliveira et al. 2019a). The fish were evaluated for up to 30 min, and each fish was used only once. Subsequently, the juveniles were transferred to a 5-L aquarium containing anesthetic-free dechlorinated water to measure the postanesthetic recovery time (swimming, equilibrium, and behaviors similar to those of the fish kept in the maintenance tanks) (adapted from Sena et al. 2016). Survival was monitored up to 72 h after anesthetic induction.

Transportation procedure
Another 48 freshwater angelfish juveniles II were used. Five different treatments (n = 8 fish per treatment) in quadruplicate were evaluated: fish transported by 8 h without essential oil (control) or with 10 and 15 mg L −1 of EOLS or EOCC. In a pilot experiment, concentrations of 10, 15, 20, and 25 mg L −1 in plastic bags were used, and 20 and 25 mg L −1 of both essential oils showed the potential to induce fish to anesthesia after 8 h of transport. Therefore, it was decided to use only the concentrations of 10 and 15 mg L −1 in this experiment, which triggered sedation, but not anesthesia, in this same transport time. Another eight juveniles were not subjected to transport and were designated 'before transport'.
The fish (n = 2 fish per bag) were placed in 2-L plastic bags (20 × 40 cm) closed with elastic straps containing 0.5 L of water, and the remainder was filled with pure oxygen. The plastic bags were stored in plastic boxes for transportation in a closed car under ambient temperature (30 ºC), simulating the routine transport of ornamental fish.
The physical and chemical parameters of the water were measured before (0 h) and after transportation (8 h) in quadruplicate. Dissolved oxygen and temperature were measured with an oxygen meter (YSI 200A, Yellow Springs, USA) and pH meter (HmPA-210P, Piracicaba, Brazil). The hardness, carbon dioxide, chlorine, and nitrite were measured with a commercial kit (JBL GmbH and Co. KG, Neuhofen, Germain) and total ammonia with another commercial kit (Alfatecnoquímica, Florianópolis, Brazil). Un-ionized ammonia levels were calculated according to a conversion table specific to freshwater.

Biochemical analysis
Blood, muscle, and gill samples were collected from eight fish per treatment before (0 h) and after transportation (8 h). Blood was collected by venocaudal puncture using heparinized syringes to determine blood glucose levels, which were analyzed using microfilm strips and a digital glucometer (Accu-Chek, Mannheim, Germany) immediately after blood collection. Then, juveniles II were euthanized with a lethal dose of benzocaine hydrochloride (250 mg L -1 ) (CONCEA, 2013) to collect gills and muscle.

Gill analysis
Gills destined for histopathologic evaluation were fixed in 10% neutral buffered formalin for 18 h and then washed and conditioned in 70% ethanol. Subsequently, a gill arch in each fish was removed and subdivided into three samples for histological processing (n = 8 fish per treatment). The samples were then dehydrated in an increasing sequence of ethyl alcohol and xylol for 1 h, immersed in paraffin for 3 h, and then embedded in paraffin blocks at 56 °C. The blocks were stored in a refrigerator (4 °C) for 12 h to cut posteriorly in a microtome (Zeiss Hyrax M15, Germany) with cross-Sects. (3 µm). After this, they were fixed on slides and submitted to staining using the hematoxylin-eosin technique (Luna 1968).
The slides were mounted in Entellan® (Merck, Darmstadt, Germany) and analyzed with a × 40 objective light microscopy. Ten microphotographs per replicate were examined, totaling 80 micrographs per treatment. A digital camera (Axio Cam ICc3, Zeiss, Germain) coupled to the microscope (Axio Scope, Zeiss) and a computer was used; for analysis, we used the Axion Vision Le® 4.8.2 (Zeiss) software.
Analysis and quantification of the microphotographed samples were performed using the Cavalieri method (Mandarim-de-Lacerda 2003;Fiedler et al. 2020), which consists of an isotropic point counting system derived from a grid of points superimposed on the histological image to be analyzed. Then, the volume density (Vv) of the histological components was estimated using the following equation (adapted from Mandarim-de-Lacerda 2003): V V (%) = P P /P T × 100, where P P is the part number of test points counted on a section in relation to the total points, and P T is the total number of test points lying over the references space.
A 21.0 × 29.7-cm rectangular grid of stitches was used to count the points, with 1 cm between the points to estimate the V V of gill tissue, secondary lamella (SL), and filament epithelium. The following V V of gills lesions were also estimated using the same grid size, according to Monteiro et al. (2008): hyperplasia, SL fusion, epithelial displacement, ionocyte proliferation, mucous cell proliferation, edema, hemorrhage, aneurysm, fibrosis (fibrous connective tissue), and necrosis.
Photomicrographs were also used to quantify the histopathology of gills, which were classified into three stages: (I) (alterations that do not affect organ functions), (II) (mild to moderate alterations that do not affect organ functions), and (III) (severe or irreversible alterations). The histopathological alteration index (HAI) was calculated according to Poleksic and Mitrovic − Tutundzic (1994), using the following equation: where HAI is the degree of alterations in a single gill, a = first-stage alterations, b = second-stage alterations, and c = third-stage alterations.

Ventilatory rate (VR)
Juveniles II (n = 40, one individual per 5-L tank), which were not used in the previous experiments, were used to assess the VR. The fish were exposed for 8 h to the same concentrations used in the transport procedure (0 (control), 10, and 15 mg L −1 of each essential oil). The evaluation times were: 0, 0.5, 1, 2, 3, 4, 5, 6, 7, and 8 h. Each fish was used only once. The VR was quantified by visually counting 20 successive opercular/mouth movements and measuring the elapsed time with a stopwatch (adapted from Hohlenwerger et al. 2017).

Statistical analysis
All data are expressed as the mean ± SEM (standard error of the mean). All data were subjected to Levene's test and the Shapiro-Wilk test to verify the homogeneity of the variances and the normality of the data, respectively. Sedation and anesthetic activities were evaluated by power regression analysis (concentration × time). Two-way analysis of variance (ANOVA) was performed (time × treatment), followed by Tukey's post hoc test for VR analysis. Comparisons between different treatments and times of transport were made using one-way ANOVA followed by Tukey's post hoc. Significance was set at a critical level of 95% (p < 0.05).

Anesthetic induction
Freshwater angelfish juveniles I and II showed no mortality after 72 h of exposure to both essential oils. The application of 2.250 mg L −1 of ethanol alone (the concentration equivalent to the dilution used for the 250 mg L −1 EOLS or EOCC treatment) did not induce sedation or anesthesia. The regression results showed that higher concentrations of EOLS or EOCC resulted in a shorter time being required for sedation and anesthesia in Juveniles I and II (Figs. 1 and 2). Juveniles I exposed to 10 mg L −1 EOLS or EOCC were sedated (Figs. 1A and 2A). In juveniles II, sedation occurred at concentrations above 25 mg L −1 for both essential oils (Figs. 1B and 2B).
For EOLS, juveniles I ( Fig. 1C) and II (Fig. 1D) were anesthetized at concentrations above 25 and 50 mg L −1 , respectively. For EOCC, anesthesia occurred from a concentration of 50 mg L −1 , regardless of the size of the juveniles (Fig. 2C, D). No relationship was observed between the EOLS or EOCC concentrations and the duration of anesthetic recovery in juveniles I (Figs. 1E and 2E) and II (Figs. 1F and 2F).
In juveniles I, the concentration of 200 mg L −1 induced the shortest anesthesia and anesthetic recovery times for both essential oils. These times were, respectively, 115.38 (Fig. 1C) and 343.25 s (Fig. 1E) for EOLS and 239.25 (Fig. 2C) and 349.75 s (Fig. 2E) for EOCC. In juveniles II, the shortest times for anesthesia and anesthetic recovery occurred at a concentration of 250 mg L −1 for both essential oils. These times were, respectively, 128.88 (Fig. 1D) and

Transportation of juveniles II
The water temperature was significantly lower in the pre-transport group compared to the transported groups. The other water quality parameters did not differ significantly between treatments (Table 1). Blood glucose levels were significantly higher in the fish sedated with 10 and 15 mg EOLS L −1 (p < 0.001 and p < 0.008, respectively) and 15 mg EOCC L −1 (p = 0.036) in relation to the group control 8 h after transport. In addition, blood glucose levels were significantly higher in the fish sedated with 10 mg EOLS L −1 (p = 0.025) than in the fish before transport (Fig. 3A). Fish that were not transported (p = 0.008) or transported with 10 and 15 mg EOLS L −1 (p < 0.001 and, 0.001, respectively) or 10 and 15 mg EOCC L −1 (p = 0.002 and, 0.001, respectively) had significantly lower muscle glycogen values than fish that were transported without the addition of essential oil (control group) (Fig. 3B).
The V V of gills was significantly higher in treatments with 10 mg EOLS L −1 and 10 and 15 mg EOCC L −1 compared to values before transport (p = 0.005, 0.002 and, 0.041, respectively) or values in animals transported without essential oil (p = 0.001, < 0.001 and, 0.007, respectively) or with the addition of 15 mg EOLS L −1 (p = 0.006, 0.002 and, 0.049, respectively). The V V of the secondary lamellae was significantly higher in the groups before transport or transported without the addition of essential oil or with the addition of 15 mg EOLS L −1 than in the fish sedated with 10 mg EOLS L −1 (p = 0.049, 0.004, and 0.043, respectively). The V V of the filament epithelium in fish transported with 10 mg EOLS L −1 and 10 and 15 mg EOCC L −1 was significantly higher than that in animals transported with 15 mg EOLS L −1 (p = 0.043, 0.008, and 0.017, respectively) ( Table 2).
In all animals submitted to transport, the HAI of gills was significantly higher than those not transported in relation to stages I and II (p < 0.001 for both stages). In addition, for stage I, juveniles transported without the addition of essential oil (p < 0.001) or with the addition of 15 mg EOLC L −1 (p < 0.004) and 10 and 15 mg EOCC L −1 (p < 0.001 for both) had significantly higher HAI values than those transported with 10 mg EOLS L −1 . For stage III, fish transported without the addition of essential oil (p = 0.012) or with the addition of 15 mg EOLS L −1 , 10 and 15 mg EOCC L −1 (p = 0.001 for all) had significantly higher HAI values than fish not transported (Table 3, Fig. 4).
Fish submitted to transport with 10 mg L −1 of EOLS or EOCC had significantly higher percentages Table 1 Water parameters of plastic bags with freshwater angelfish (Pterophyllum scalare) juveniles II without essential oil before and after transport (8 h) and with essential oil from Lippia sidoides (EOLS) and Cymbopogon citratus (EOCC) added to the water after transport (8 h) Temperature (temp) is expressed as °C. Dissolved oxygen (DO) is expressed as mg L −1 O 2 . Hardness is expressed as mg L −1 CaCO 3 . Chlorine (Cl) is expressed as mg L −1 Cl 2 . Carbon dioxide (CO 2 ) is expressed as mg L −1 CO 2 . Nitrite is expressed as mg L −1 N-NO 2 . Total ammonia (TAM) is expressed as mg L −1 N-NH 3 . Un-ionized ammonia UIA) is expressed as μg L −1 N-NH 3 . Data are expressed as mean ± SEM (n = 4 plastic bags per treatment). Different letters indicate significant differences between treatments (p < 0.05)

Variables
Before transport After transport (8 h for secondary lamella fusion than those not transported (p = 0.013 and < 0.001, respectively) or transported without the addition of essential oil (p = 0.005 and < 0.001, respectively). In addition, the 10 mg EOCC L −1 treatment had significantly higher percentages of secondary lamella fusion than the 15 mg L −1 of EOLS or EOCC treatments (p < 0.001 for both). Juveniles transported with 10 mg L −1 of EOLS or EOCC had significantly higher percentages of epithelial displacement than those belonging to the groups without the addition of essential oil before (p = 0.001 and 0.002, respectively) or after transport (p < 0.001 for both). The 10 and 15 mg EOCC L −1 treatments had significantly higher edema percentages than the pre-transport group (p < 0.006 and 0.011, respectively) ( Table 4).
Fish transported with 10 mg EOCC L −1 had significantly higher fibrosis percentages when compared to those not transported or transported without the addition of essential oil or with the addition of 15 mg EOCC L −1 (p < 0.003, 0.011, and < 0.006, respectively). There were no significant differences for hyperplasia, ionocytes proliferation, mucous cell proliferation hemorrhage, aneurysm, and necrosis (p > 0.05) ( Table 4).

Ventilatory rate
Juveniles sedated with EOLS (10 and 15 mg L −1 ) had significantly lower VR values than those in the other treatments (p < 0.023) (except between 10 mg EOLS L −1 and 15 mg EOCC L −1 in 8 h). In animals sedated with 15 mg EOLS L −1 , the VR was significantly higher at time 0 h compared to times between 5 and 8 h (p < 0.018). Additionally, for this same treatment, the VR was significantly higher at times 0.5 and 1 h Fig. 3 Blood glucose (a) and muscle glycogen (b) in freshwater angelfish (Pterophyllum scalare) juveniles II without essential oil before and after transport (8 h) and with essential oil from Lippia sidoides (EOLS) and Cymbopogon citratus (EOCC) added to the water after transport (8 h). Data are expressed as mean ± SEM (n = 8 fish per treatment). Different letters indicate significant differences between treatments (p < 0.05) Table 2 Gill volume density (V V , %) of freshwater angelfish (Pterophyllum scalare) juveniles II without essential oil before and after transport (8 h) and with essential oil from Lippia sidoides (EOLS) and Cymbopogon citratus (EOCC) added to the water after transport (8 h) V V = volume density, GT = gill tissue, SL = secondary lamella, FE = filament epithelium. Data are expressed as mean ± SEM (n = 8 fish per treatment). Different letters indicate significant differences between treatments (p < 0.05) Before transport After transport (  than at 8 h (p < 0.001). For fish exposed to 10 mg EOCC L −1 , the VR was significantly higher at times 0 and 0.5 h compared to times between 3 and 8 h (p < 0.028). For juveniles subjected to 15 mg EOCC L −1 , the VR was significantly higher at time 0 than at times between 2 and 8 h (p < 0.046) and at time 0.5 h than at times between 5 and 8 h (p < 0.004). In addition to this same treatment, at time 1 h, the VR was significantly higher than at time 8 h (p = 0.003) (Fig. 5).

Discussion
The anesthetic effects of EOLS and EOCC must be attributed to their major chemical compounds (they can act together with the other compounds present), which were carvacrol and α-citral, respectively. Previous studies carried out with essential oil from Lippia alba (chemotype citral) and EOCC (citral as the major component) in mammals demonstrated its anxiolytic and sedative action through the inhibition of the GABA receptor system (γ − aminobutyric acid), an essential neurotransmitter in the brain (Costa et al. 2011;Heldwein et al. 2012;Wang and Heinbockel 2018). In addition, the evaluation of anxiolytic properties of EOCC in zebrafish indicated that GABA receptors are possibly related to the mechanism of action of their constituents (Hacke et al. 2020). Similarly, in a recent study, citral induced sedation and anesthesia in silver catfish (Rhamdia quelen) (15-40 and 50-600 µL L −1 , respectively) and grass carp (Ctenopharyngodon idella) (15-40 and 75-450 µL L −1 , respectively) (Dos Santos et al. 2022).
Likewise, carvacrol exerted anxiolytic and antidepressant activity and minimized excitability in the peripheral nervous system in rodents (Mello et al. 2010;Gonçalves et al. 2010). Carvacrol affects the central nervous system through GABAergic neurons through the GABA receptor, where activation of these channels promotes central nervous system depression (Mello et al. 2010;Gonçalves et al. 2010;Bianchini et al. 2017). In addition, carvacrol proved to be an efficient anesthetic for doctor fish (Garra rufa) since it did not affect electrocardiographic parameters in these fish (Aydın and Orhan 2021). Thus, α-citral and carvacrol could be involved in sedative and anesthetic action in fish (Heldwein et al. 2012;Bianchini et al. 2017).
Body size is a determining factor in defining the best anesthetic concentration, where younger individuals, and therefore with lower body weight, would be more easily anesthetized (Tarkhani et al. 2016). This was verified in the current study, where smaller individuals (juveniles I) were sedated with 10 mg L −1 for both essential oils. Still larger individuals (juveniles II) were sedated with only 25 mg L −1 of EOLS Table 4 Volume density (V V , %) of gills lesions of freshwater angelfish (Pterophyllum scalare) juveniles II without essential oil before and after transport (8 h) and with essential oil from Lippia sidoides (EOLS) and Cymbopogon citratus (EOCC) added to the water after transport (8 h) SLF = secondary lamella fusion, DE = epithelium displacement. IP = ionocytes proliferation, MCP = mucous cell proliferation. Data are expressed as mean ± SEM (n = 8 fish per treatment). Different letters indicate significant differences between treatments (p < 0.05) Before transport After transport (8 h or EOCC. Anesthesia occurs in fish when the minimum effective concentration of the anesthetic is met in its blood (Rairat et al. 2021). Therefore, the induction time is expected to decrease as the anesthetic concentration increases (Hoseini et al. 2021). Accordingly, the concentrations of both essential oils that showed the best results (based on anesthesia and recovery times) for juveniles I and II were 200 and 250 mg L −1 , respectively. As verified for the sedation stage, again, the smaller fish required a lower concentration of essential oil to be anesthetized. This can be explained by the fact that the gill surface of smaller fish is proportionally larger in relation to other body tissues than in larger fish (Javahery et al. 2012;Hoseini et al. 2013;Chambel et al. 2015). According to Ross and Ross (2008), there is usually an inverse association between metabolic rate and body weight. Therefore, larger fish require a longer exposure time and higher concentrations to be anesthetized than smaller ones. This was verified in the present study, as an increase in the concentration of EOLS or EOCC caused a reduction in anesthesia times for freshwater angelfish. Similar to our study, previous studies also evaluated the anesthesia time using the power equation. As verified for the current study, these studies found that as the anesthetic concentration increases, there is a reduction in anesthesia time. These studies evaluated the use of eugenol in freshwater angelfish and Amazon catfish (Pseudoplatystoma reticulatum × Leiarius marmoratus) (Oliveira et al. 2019a, b), and myrcene and linalool (Mirghaed et al. 2016), menthol and 1,8-cineole (Mazandarani and Hoseini 2017), and citronellal and linalool in common carp (Cyprinus carpio) (Yousefi et al. 2018). However, concentrations above 250 mg L −1 of both essential oils evaluated in our study should be avoided. The use of an excessive amount of an anesthetic could affect the welfare or even cause the death of the fish (Summerfelt and Smith 1990;Park et al. 2008). Furthermore, an anesthetic concentration above required represents a waste of essential oil and unnecessary expenses (Teixeira et al. 2017).
Previous studies indicate that the ideal period for anesthesia should be close to or less than 3 min, followed by periods between 5 and 10 min for anesthetic recovery (Gilderhus and Marking 1987;Small 2003;Ross and Ross 2008). In the present study, the recommended concentrations of EOLS presented anesthesia periods around 2 min, below the periods recommended by the previously mentioned authors. The recommended concentrations for EOCC induced anesthesia periods are between 3 and 4 min, close to the times suggested by those authors. In addition, EOLS and EOCC can be used as anesthetics in these concentrations in biometrics and another rapid handling in these juveniles when followed by the recovery period in anesthetic-free water, as the anesthetic recovery times of both essential oils reached the recommended times.
Essential oils added to water in low concentrations can be used as sedatives during transport, reducing swimming and reaction to external stimuli in fish Teixeira et al. 2018). In the present study, the concentrations of both essential oils determined to transport freshwater angelfish juveniles II for 8 h were 10 and 15 mg L −1 since exposure to these concentrations only caused sedation in the fish, with no risk of anesthesia. Since fish are less agitated due to sedative substances used in the transport, essential oils can prevent more sudden changes in the water's physical-chemical parameters. Thus, fish could reduce dissolved oxygen consumption and the production of toxic nitrogen compounds (Hohlenwerger et al. 2017), which should consequently contribute to their welfare during transport.
In our study, however, except for the increase in water temperature observed in all treatments subjected to 8 h transport, the water physical-chemical parameters were not influenced by the treatments. An increase in ammonia (ionized and un-ionized) and a decrease in dissolved oxygen are expected during fish transport Hohlenwerger et al. 2017;Teixeira et al. 2018;Oliveira et al. 2019a;Mirzargar et al. 2022). When pure oxygen is inserted into the plastic bag, water dissolved oxygen over-saturates because of pressure caused by the bag . This explains the high concentration of dissolved oxygen found in all treatments performed in this study. Transportation can also increase ammonia excretion in fish, and sedative substances added to the water could reduce this problem (Teixeira et al. 2018). However, EOLS and EOCC were not effective in reducing ammonia excretion in the juveniles evaluated in our study. On the contrary, ammonia excretion was decreased in the transport of tambacu, and Nile tilapia with essential oil from L. alba Hohlenwerger et al. 2017;Mirzargar et al. 2022), common carp with linalool ) and freshwater angelfish with eugenol (Oliveira et al. 2019a) added to the water.
In addition, water temperature is one of the main parameters that must be monitored in fish transportation and affects the efficacy of an anesthetic (He et al. 2020;Hoseini et al. 2021). Transportation by car exposes the fish to the ambient temperature ). In the present study, this transport (by car) explains the higher water temperature recorded after transportation compared to the treatment before transport. An increase in water temperature can reduce the anesthetic induction time, as verified in previous studies with common carp (Mirghaed et al. 2016;, due to increased metabolism and VR of fish (Mylonas et al. 2005). However, we believed that this hypothesis is not supported in our study because although an increase in blood glucose was recorded, the temperature rise did not affect anesthetic induction after transporting the fish for 8 h, as the opercular movements remained stable or were reduced in fish exposed to EOCC and EOLS, respectively.
In stressful situations resulting from transport, fish tend to spend more energy maintaining physiological homeostasis, which can be intensified by external factors (Teixeira et al. 2017(Teixeira et al. , 2018, such as heat and vehicle movement. Therefore, the increased blood glucose levels observed in fish exposed to 10 (mainly) and 15 mg EOLS L −1 and 15 mg EOCC L −1 may have been triggered as a physiological response secondary to stress (Pankhurst 2011). The rise in blood glucose levels occurs immediately after the nervous system receives an adverse stimulus that will activate the hypothalamic-pituitary-interrenal axis, releasing cortisol and catecholamines into the bloodstream (Barton and Iwama 1991;Ventura et al. 2019). These hormones could have initiated metabolic processes for the use of energy reserves (Oliveira et al. 2019a) during transport in the animals of the current study. However, an increase in glucose levels does not directly correspond to the rise in blood cortisol levels. This has been verified in previous studies, with L. alba added to the transport water of tambacu (10 µL L −1 ; Sena et al. 2016) and Nile tilapia (10 µL L −1 ; Hohlenwerger et al. 2017) and Aloysia triphylla added to Nile tilapia transport water (20 µL L −1 ; Teixeira et al. 2018), where the fish showed increased levels of glucose but not plasma cortisol after transport of 8 h compared to the group not transported. Therefore, an increase in blood glucose levels could result from the hepatic glycogen breakdown by glycogenolysis (Inoue et al. 2005), which is released as energy glucose to supply the increased metabolic demand (Perry and Capaldo 2011) during transport. The liver and, to a lesser degree, the kidney, are the major sites of gluconeogenesis in fishes, and this process is unlikely to occur in skeletal muscle (Knox et al. 1980). In addition, we verified there was a reduction in muscle glycogen levels in juveniles transported with essential oil added to the water compared to the control group.
The glycogen level in muscle is changeable: it is spent as required and stored up again at extra glucose intake with food (Scarabello et al. 1991). However, the elevated plasma cortisol level inhibits muscle glycogenesis (Milligan 2003), which may have occurred in fish sedated with EOLS and EOCC, and muscle glycogen mobilized for muscular activity is not replenished. This physiological adjustment will consume energy without compromising the individuals' survival. In contrast, there was no mobilization of muscle glycogen in the control group. As plasma glucose levels remained stable in this treatment, excess glucose may be stored as hepatic glycogen, which could be used for other physiological functions in fish or be converted into lipids (Walker et al. 2020). Unfortunately, our study found it impossible to assess other critical physiological variables, such as hepatic glycogen and cortisol plasma levels, due to the small body size typical of freshwater angelfish. Despite this, our study's VR and gill histology analysis contributed to a better integrative interpretation of the results.
According to Balasch and Tort (2019), activation of the cerebrum-pituitary-interrenal cell metabolic axis also activates other rapid stress responses, increasing ventilatory and cardiac rates, which confer greater blood volume to the gills and muscle (Hohlenwerger et al. 2017). Therefore, a reduction in VR would reduce the possible metabolic stress in fish (Becker et al. 2018). Another possible consequence of reduced blood flow would be an energy supply to the cells, with the consequent accumulation of glucose in the blood. Both essential oils effectively reduced VR in the present study, where EOLS was more efficient than EOCC. Similarly, the use of essential oils from L. alba as a sedative in Nile tilapia (10-20 µL L −1 ) (Hohlenwerger et al. 2017) and silver catfish (5-10 µL L −1 ) (Becker et al. 2018) and from A. triphylla as a sedative in Nile tilapia (20-30 µL L −1 ) (Teixeira et al. 2018) also reduced VR.
The gills are in direct contact with the environment and thus are the main target of external elements present in the water. When fish are subjected to stressful handling or transport conditions, their gills can also undergo modifications to adjust to the environment and the conditions imposed on them, guaranteeing the maintenance of their functions ). In our study, the groups transported without the addition of essential oil or with 15 mg EOLS L −1 had no increase in the V V of the gills. The V V maintenance may have been influenced by an adjustment in the gill surface in these treatments. For example, an increase in the length of the lamellae and a reduction in the diffusion barrier may favor the increase in the tissue's energy demand (Perry 1998).
Fish exposed to 10 mg EOLS L −1 during transport showed lower V V to the secondary lamella, gas exchange surface, and ion exchange in the gills (Fiedler 2020), which may indicate lower demand for oxygen and ion exchange during transport. Respiratory efficiency decreases in anesthetized fish because of slowed opercular movements (Hoseini et al. 2021). Additionally, the reduction in V V in the filament epithelium can reduce the water/blood barrier distance, reflected in the facilitation of the diffusion of respiratory gases (Mallatt 1985). In our study, a reduction in the V V of the filament epithelium was observed only in fish transported with 15 mg EOLS L −1 added to water.
Early tissue damage in the gills can impair physiological functions (Barbieri and Bondioli 2015; 1 3 Vol.: (0123456789) Brandão et al. 2021). In the present study, transported fish presented stage III lesions (severe and irreversible lesions that can severely affect vital organ functions), with HAI values higher than stages I (reversible, without damaging the gill tissues) and II (lesions severe but reversible, with effects on associated tissue function) (Poleksic and Mitrvic-Tutundzic 1994). Nevertheless, some stage II and III lesions were dispersed and not concentrated in specific gills areas, with no indication of severe physiological damage to the fish (Poleksic and Mitrovic-Tutundzic 1994). In addition, these lesions were less severe in fish treated with 10 mg EOLS L −1 . Similarly, Soares et al. (2017) found that therapeutic baths with EOLS (10 and 20 mg L −1 ) in tambaqui triggered some injuries, ranging from severe to irreparable for stages II and III.
The histological lesions demonstrated that the gill tissue could withstand changes according to the transport conditions in a general context. Fish transported with 10 mg L −1 of EOLS or EOCC had a higher V V of fusion in the secondary lamellae and a higher V V of epithelial displacement, indicating that respiratory gas diffusion in these concentrations may be reduced. Similar responses have been verified by Brandão et al. (2021) with the use of EOLS (50 mg L −1 ) in tambaqui juveniles, however, without irreversible damage to the gills.
In the presence of xenobiotics, the gills can exhibit alterations that are considered defense responses (e.g., proliferation and displacement of epithelial cells, secondary lamella fusion), which can increase the water/blood distance (Monteiro et al. 2008). Therefore, when fish are exposed to environmental changes, physiological adjustments can occur in the gills to maintain the homeostasis of vital functions, such as osmoregulation, acid-base balance, excretion of nitrogen compounds, and gas exchange (Barbieri and Bondioli 2015; Brandão et al. 2021). In the current study, fish exposed to 10 mg L −1 of EOLS or EOCC had higher V V values for epithelial fusion and epithelial displacement, indicating that these alterations may have been essential to guarantee the functionality of the gills.
Another critical response to environmental changes is the proliferation of mucous cells and ionocytes covering the gill surface (Wolf et al. 2015). These gill alterations can protect the pillar cells, preserving the lamellar vascular channels from external damage. However, such proliferation can also trigger an increase in the diffusion distance of O 2 between respiratory epithelial cells and erythrocytes, impairing respiratory functions (Oliveira et al. 2019a). In addition to these changes, edema and fibrosis lesions are also responses to xenobiotics in the water, being considered biomarkers of acute toxicity (Monteiro et al. 2008). In our study, alterations in the proliferation of epithelial cells were not verified in the different treatments. However, in juveniles transported with EOCC, there was an increase in edema (10 or 15 mg L −1 ) and fibrosis (15 mg L −1 ) in the gills. Therefore, EOCC as a sedative for freshwater angelfish transport is not recommended. Other studies have also found unfavorable results for essential oils in fish transport. Benevoit et al. (2012) found that essential oil from Aloysia gratissima elicited side effects during anesthesia and mortality during transport in Brazilian flounder (Paralichthys orbignyanus).  verified that linalool is not beneficial for common carp transportation because it increases stress in fish and has no benefits in preventing ion loss. He et al. (2020) found that eugenol induced stress and high metabolism in spotted sea bass after transportation, leading to the accumulation of ammonia and CO 2 .
Finally, in the present study, although fibrosis was a severe lesion verified in animals exposed to 15 mg EOCC L −1 , other severe alterations such as hemorrhage, aneurysm, and necrosis (Poleksic and Mitrovic-Tutundzic 1994) did not differ between transported juveniles (regardless of treatment) and those before transport, indicating the regenerative potential of gill tissues. Understanding the regenerative mechanisms of gills in fish exposed to anesthetics requires further elucidation (Kumari et al. 2018). Therefore, a concentration that minimizes gill damage should be preferential when selecting an essential oil with a sedative capacity for transporting fish. Thus, in our study, the treatment that achieved these premises was 10 mg EOLS L −1 , allowing freshwater angelfish juvenile II transport for up to 8 h without causing irreparable damage to the gills.

Conclusions
We found that the developmental stage of fish can influence the action of an anesthetic, in which larger juvenile freshwater angelfish require a higher concentration of EOLS or EOCC to be sedated or anesthetized. For rapid handling, 200 and 250 mg L −1 of both essential oils are indicated for juveniles I and II, respectively. We also found that for the transport of juveniles II for 8 h, the best treatment was 10 mg EOLS L −1 . Nevertheless, this concentration increased the blood glucose levels, decreased the VR and muscle glycogen levels, and recorded no occurrences of irreversible damage to the gill tissue.