Routine operational processes such as vaccination, handling and transportation in aquaculture cause a stressful environment that compromises animal welfare (1). Ultimately, as the fish’s response to stressors, resistance to diseases decreases and physiological disruptions occur (2). In order to eliminate such negative effects in aquaculture, anesthetic substances are used before any operation to fish (3). Anesthetic agents are used in a wide range of applications, from light sedation to reduce stress to full anesthesia to eliminate pain during surgery (4). This is an ethical and scientific obligation as well as a legal obligation (5). For this purpose, various synthetic [tricaine methanesulfonate (MS-222), benzocaine, 2-phenoxyethanol etc.] and natural anesthetic (herbal essential oils) agents are used in aquaculture (6). Although MS-222 is the only approved anesthetic agent, it has some disadvantages such as inducing hyperglycemia and oxidative stress (7). The potential adverse effects of synthetic anesthetic agents on the environment and organisms have triggered the search for alternative anesthetics of herbal origin (8). The sedative effects of various herbal essential oils on fish have been previously investigated (9–13). Commercial anesthetic mixtures prepared with plant essential oils that can replace synthetic anesthetics also need to be developed.

The effectiveness of an anesthetic agent is evaluated by many factors such as induction time on the organism, physiological effects and cost analysis (14, 15). The physiological, histological, oxidative and hematological effects of a newly applied anesthetic agent on fish provide an idea in terms of welfare and health. Since each species has its own physiological and behavioral responses, it would not be correct to generalize all fish to anesthetic agents. In this context, it is a correct approach to evaluate different species, especially when using agents that have not been researched before. Due to differences in physiological demands, survival standards vary according to the trophic level of the fish. Each organism’s response to any anesthetic agent is different and therefore the effectiveness of the anesthetic agent varies from fish to fish (8). In this context, it is necessary to perform multivariate modeling with different types of fish.

Common carp (Cyprinus carpio), one of the fish with the largest share in the aquaculture sector, is a warm water fish with a low trophic level. It is a potential candidate for aquaculture with its high adaptability to environmental conditions (16). Common carp accounts for more than 80% of total fish production in many European countries (17). On the other hand, Danube sturgeon (Acipenser gueldenstaedtii) is an important fish species with high potential for world aquaculture due to its high resistance to environmental mechanisms and high economic added value (commercial value of its meat and caviar) (18). Additionally, Danube sturgeon is one of the vulnarable fish species in the Red List of Threatened Species by IUCN (19). Therefore, it is important to use the most appropriate anesthetic agent for the fish in all cultivation operations. And, the rainbow trout (Oncorhynchus mykiss), which lives at a higher trophic level than the other two species, is the one of the most cultured cold-water fish in the world with its significant market value and meat quality (20). Rainbow trout, a fish that is very sensitive to environmental conditions such as temperature, dissolved oxygen (DO), and organic nutrients, is subjected to sedation or deep anesthesia during operational processes. When all these species are evaluated together due to their different trophic levels and possible different responses to anesthetic agents, they will reveal the overall effect of the target anesthetic agent. In this context, the current study focused on the physiological effect of a new commercial anesthetic on fish species belonging to three different trophic levels. The specific objectives of the current study are:

The ideal anesthetic agent should not be physiologically toxic to the fish and should not adversely affect human health during operating, and should have an appropriate induction and recovery time for the fish (29). Because long-term or high-concentration exposure to the anesthetic substance is important both in terms of operating cost and stress for the fish (30). In the current study, studies were first conducted with 80–100 mg L⁻¹, which was determined as the suitable concentration of the anesthetic mixture. However, the determined concentrations did not provide effective application in fish. Although the concentration was increased, the anesthetic concentration of 250 mg L⁻¹ for carp and 240 mg L⁻¹ for rainbow trout did not produce the expected induction effect. In accordance with the preliminary studies conducted for Danube sturgeon, a mild sedation effect was not observed within 5 min for 240, 360, and 480 mg L⁻¹. Therefore, the lowest concentration for Danube sturgeon has been increased to 600 mg L⁻¹. The expected anesthetic effect in fish was achieved only at this concentration. This issue reveals Danube Sturgeon’s resistance to the anesthetic mixture. As a result, HC concentrations were induced for all fish in less than 3 min. MC concentrations generally represent the minimum possible concentration for anesthesia. HC concentrations for common carp, rainbow trout, and Danube sturgeon were determined as 480, 500, and 750 mg L⁻¹, respectively. Common carp and rainbow trout showed similar anesthesia behavior at similar concentrations. However, although similar concentrations were used, the induction time was faster in rainbow trout than in common carp. Similarly, a slower induction time has been reported for common carp than rainbow trout (14, 31, 32). On the other hand, even the lowest anesthesia concentration of Danube sturgeon was recorded higher than the highest concentration of other groups. Although the anesthetic effect of chamomile oil was determined for rainbow trout in our previous study, similar concentrations did not show an anesthetic effect for Danube sturgeon (11). Wide range in anesthetic concentration is associated with fish species, size and environmental conditions (33–35). Therefore, we focused on the effect of fish species on anesthetic concentration determination, keeping fish sizes and environmental conditions constant. Smaller fish have a faster induction rate, while larger fish show a faster recovery time (4). Thus, combination anesthesia may be safer because this allows a reduction in dose, which is generally reflected in better recovery and lower mortality rates along with reduced adverse side effects in some cases (36). The anesthetic mixture should have a pleasant smell for the users (29). The commercial anesthetic mixture used in the current study has an aromatic odor and is not irritating to users. Its smell has an intense scent of thyme and clove. However, the current anesthetic mixture can be added directly to water due to unlike essential oils, it has the ability to dissolve in water. Therefore, it does not create a synergistic effect on fish like water-insoluble essential oils. The anesthetic agent suitable for fish should not cause death even after a 30-min induction period. According to the reliability test applied in this study, no mortality was observed in fish for optimum concentrations. As expected, recovery times increased significantly in all fish due to increasing concentration.

Anesthetics first come into contact with the gills in fish and are distributed throughout the body via this route. Therefore, histopathological studies are critical to confirm sensitivity to the gill epithelium, which is the predominant site of gas exchange and osmoregulation (37). In the present study, rainbow trout and common carp gill tissues were little affected by low anesthetic concentration. However, the commercial anesthetic mixture had a histological effect on Danube sturgeon gills even at low concentrations. Hypertrophic cells were observed moderately in the gill tissues of Danube sturgeon. In the MC groups, while rainbow trout and common carp gill tissues showed similar signs of moderate hyperplasia and necrosis, severe hyperplasia was observed in Danube sturgeon. For HC groups, severe and/or very severe hyperplasia and necrosis were observed in the gill tissues of all fish species. In general, the more severe histological output of Danube sturgeon than other species has been attributed to the higher anesthetic agent concentration. The contribution of increasing anesthetic agent concentration to histological degradation has been discussed previously (11). Hypertrophy of the secondary lamella and hyperplasia of the primary lamella in the current study are a common and nonspecific response to subacute and chronic damage to the gills (38, 39). These cell responses are among the first defense mechanisms and are classified as mild and reparable (40). The potential hyperplasia and hypertrophy effects of different herbal anesthetic essential oils, such as the content of the commercial anesthetic substance in our study, on gill tissues have been reported (26, 41).

Exposure to anesthesia slows down operculum movement in fish and causes hypoxia. The increase in hypoxia affects catecholamine in rainbow trout, resulting in greater RBC release from the spleen as well as an increase in hematocrit and hemoglobin (42). Similarly, higher RBC, HGB, and HCT were observed in the anesthesia groups at the beginning of experiment than in the control, and blood parameters increased further depending on increasing anesthesia concentration. Blood parameters returned to control-like levels over time. However, while significant differences were observed over time in the MANOVA test, fewer differences were observed depending on concentrations. It has been inferred that that this increase in blood parameters negatively affects fish welfare in the acute period after anesthesia and that the first 24 h in anesthetized individuals are critical. In one study, the lymphocyte ratio reached initial levels within 24 h after anesthesia, supporting our hypothesis (43). It has been previously discussed that clove oil, an herbal anesthetic agent, increases RBC (44). It may cause malfunctions in the hematopoietic system of fish exposed to the potentially toxic substance, resulting in increased WBC and HGB (45). The interaction effect showed significant differences between groups depending on one and/or two variables only for WBC, RBC, and HGB in carp. For instance, although there was no significant difference for the time variable, significant differences in concentration put pressure on the time variable.

PROMETHEE decision model is widely used in many fields due to its mathematical properties and easy applicability. Because it is a very well designed model for ranking a limited number of alternatives according to conflicting criteria (46). Although it has been applied in many areas, the PROMETHEE decision model is a design that has not yet been applied in the field of aquaculture. In the current study, three anesthesia concentration alternatives were evaluated for three different fish species based on 10 criteria. While MC showed the best results for rainbow trout and Danube sturgeon, LC group provided the top rank for common carp. Here, the weight values of the criteria have an important share and these values are determined according to expert opinion. C1 and C2 are the criteria that reveal the effectiveness of the anesthetic agent, and the weight value is determined as 15 and 10% for induction and recovery time, respectively. Because while the induction time is expected to be <3 min, the recovery time has a wider range of <5 min (28). C3 represents a part of the operating cost, which is one of the most important items for the employer in the aquaculture industry. As the running cost, the amount of anesthetic mixture is a significant input cost in full-scale facilities, depending on the volatility of the essential substance, the size of the water volume and the amount of fish. From a public policy perspective, potential positive or negative impacts should be considered on a cost–benefit basis (47). As physiological indicators of fish, criteria between C4 and C7 represent blood parameters that can provide rapid results (48). The weight values of WBC, which is a representative of the defense mechanism of the fish, and RBC, which transports oxygen in the blood, were determined higher. Finally, C8, C9, and C10 are criteria that indicate histological changes. The reason for choosing a higher weight value for necrosis (C8) is that it represents a significant and irreversible histological change in which the cell in particular begins to disappear. HC was seen as the least acceptable alternative for all groups, which was attributed to its high cost and potential to cause necrosis.

The effectiveness of an anesthetic agent depends on the active ingredients it contains (49). Since the current study focuses on the effect of a commercial anesthetic mixture on three different fish species, the content of anesthetic mixture will provide insight into possible components. In the evaluated anesthetic mixture primarily composed of monoterpenes, 1-propanol serves as the component that enhances water solubility, allowing for improved integration of the formulation in aqueous environments (50). Eugenol is the active ingredient of clove oil, which is frequently used as an anesthetic agent. On the other hand, linalool and linalyl acetate are the active ingredients of lavender essential oil. Clove oil has long been used both commercially and scientifically as an anesthetic for many species (35, 51–54). In general, it is an environmental friendly alternative to synthetic anesthetic agents, even though its use in high concentrations on fish may cause physiological effects. Lavender oil is an essential oil that has recently been shown to have an anesthetic effect on different species (12, 55, 56). Although both essential oils are important alternatives to synthetic agents, their physiological output on fish is likely to be reduced if used together. The current study provides a guide for comparison.

The effect of an anesthetic agent on fish depends on many parameters. The trophic level of the fish determines the concentration/effectiveness of the anesthetic agent. This study showed that, under current environmental conditions, Danube sturgeon is more resistant to a commercial anesthetic mixture than common carp and rainbow trout. However, this is not a direct comparison since the living conditions of these fish species differ under normal circumstances, and the possible anesthetic effect varies depending on both the current environmental parameters and fish biology (age, size, and weight). Under the present conditions, the effective concentration of the anesthetic mixture was higher for the Danube sturgeon. This means that sturgeon was exposed to higher anesthetic concentrations, which resulted in more pronounced histological damage. A lower anesthetic concentration was deemed unacceptable, as it significantly prolonged the induction time. In conclusion, the anesthetic concentration varies depending on the fish species. In this context, the best concentrations were determined by evaluating 10 different criteria using the PROMETHEE decision model. While these criteria are sufficient for assessing the suitability of an anesthetic agent, more detailed studies are needed to investigate stress indicators in fish. In future studies, different anesthetic agents can be compared using a multi-criteria decision-making model, and a combined anesthetic agent can be developed. In addition to synthetic substances, although natural essential oils are used individually, a mixture of different essential oils as an anesthetic agent is a topic worth investigating.