Monoterpenes as Natural Anesthetics to Mitigate Stress in Fish: Advances Using the Zebrafish Larvae Model

Abstract

During production, fish are exposed to multiple environmental, physiological, and physical stressors, which compromise development, productivity, and welfare and urge the implementation of effective and safe stress-mitigating strategies, particularly during early developmental stages. Larval zebrafish (Danio rerio) constitute a powerful model for studying acute stress responses due to the numerous advantages they offer, such as developmental transparency, a conserved hypothalamic–pituitary–interrenal (HPI) axis, and suitability for high-throughput screening. This review examines the potential of natural monoterpenes as stress-reducing compounds and compares their performance with conventional synthetic anesthetics. Evidence from vortex-flow stress paradigms, behavioral profiling and biochemical assays shows that acute stress in zebrafish larvae triggers metabolic disruption, behavioral hyperactivity and enzyme imbalance, with cortisol responses depending on stimulus intensity. Monoterpenes such as thymol and menthol consistently reduce stress-induced hyperactivity, support redox homeostasis and display favorable safety profiles at low doses and short exposures. Nevertheless, as research into these substances is still recent, evidence of any potential adverse effects is still limited. Although individual monoterpenes may act on different subsets of molecular targets, their multimodal mechanisms, including gamma-aminobutyric acid (GABA)ergic enhancement, voltage-gated ion channel and transient receptor potential (TRP) modulation, suggest broader and potentially safer actions compared to single-target anesthetics as tricaine methane sulfonate (MS-222). Collectively, these findings suggest that monoterpenes offer promising natural alternatives for stress mitigation in aquaculture and the refinement of research procedures involving early life stages.

1. Introduction

Aquaculture is the fastest-growing food-production sector worldwide, yet routine procedures such as grading, transport, vaccination or sampling, frequently expose fish to acute or chronic stress, which significantly impacts both welfare and performance. Therefore, strategies to mitigate stress responses are essential for both ethical and practical reasons. In this context, zebrafish (Danio rerio) larvae have become powerful models to investigate stress responses and anesthetic mechanisms in fish, due to their conserved HPI axis, optical transparency, and high-throughput suitability, allowing for a broader range of studies that are difficult to perform in commercial aquaculture species.

Chemical anesthetics, such as tricaine methanesulfonate (MS-222) and benzocaine, are widely used as standard fish anesthetics. However, these are often associated with adverse effects and regulatory restrictions, prompting the search for natural alternatives. Among these, monoterpenes, including eugenol, thymol and menthol, have emerged as promising candidates due to their reported anesthetic, anxiolytic, antioxidant and anti-inflammatory properties. Nevertheless, the diversity of compounds and the variability in experimental conditions require a systematic assessment of their mechanisms and applicability before their implementation as effective and safe anesthetics.

This review synthesizes current evidence on (1) stress physiology in fish, (2) the use of zebrafish larvae as a model for stress and anesthesia research, and (3) the potential of monoterpenes as natural anesthetics to mitigate stress in zebrafish larvae up to 5 days post-fertilization (dpf), in line with the European Directive 2010/63/eu. By restricting the scope to zebrafish larvae, this review allows for a more robust and standardized comparison of results across studies. In addition, mechanistic insights, comparative efficacy, limitations of existing studies and key research gaps to guide future applications in aquaculture and welfare-oriented practices are also addressed.

2. Stress Physiology in Fish

2.1. Functioning and Description of the Hypothalamus–Pituitary–Interrenal (HPI) Axis

The hypothalamus–pituitary–interrenal (HPI) axis is the central neuroendocrine pathway regulating the stress response in fish and is functionally analogous to the hypothalamus–pituitary–adrenal (HPA) axis in mammals [1,2,3]. Environmental or physiological stressors are first detected by the central nervous system (CNS) and integrated at the level of the hypothalamus, which responds by releasing the corticotropin-releasing hormone (CRH) and associated peptides [4,5] (Figure 1). These neurohormones stimulate the anterior pituitary to synthesize and secrete the adrenocorticotropic hormone (ACTH) into the circulation [6,7]. ACTH then targets the interrenal cells located in the anterior kidney (head kidney), where it activates steroidogenic pathways leading to the production and release of cortisol, the principal glucocorticoid in teleost fish [1,7]. The activity of the HPI axis is tightly regulated through negative feedback mechanisms, whereby elevated circulating cortisol acts on both the hypothalamus and pituitary to inhibit further CRH and ACTH release and prevent prolonged or excessive activation of the stress response [3,6]. In addition to cortisol secretion, stress exposure also activates the brain–sympathetic–chromaffin (BSC) system, which promotes the rapid release of catecholamines, mainly adrenaline and noradrenaline, from chromaffin tissue associated with the head kidney [2,8]. While catecholamines mediate the immediate “fight-or-flight” response, cortisol exerts longer-lasting effects by modulating gene transcription and coordinating metabolic, osmoregulatory, and immune processes [1,7].

Through the integrated action of these hormonal components, the HPI axis orchestrates a cascade of physiological adjustments that allows fish to cope with challenging conditions [2,9]. These adjustments are traditionally categorized into three hierarchical levels of response: the primary response, involving the release of stress hormones; the secondary response, encompassing downstream physiological and biochemical changes; and the tertiary response, which reflects whole-organism consequences such as altered growth, reproduction, and behavior [1,2,7].

2.2. Physiological Responses to Stress

Together, the HPI and the BSC axis regulate the primary, secondary and tertiary responses to stress. Primary responses involve neuroendocrine activation, particularly of the HPI axis, resulting in the release of stress hormones such as cortisol and catecholamines [10]. Secondary responses comprise downstream physiological and biochemical alterations that include changes in energy metabolism and ion regulation such as increased plasma glucose, lactate, and hematocrit, as well as reduced chloride (Cl⁻), sodium (Na⁺), and potassium (K⁺) levels [2,7]. In turn, tertiary responses arise as a consequence of prolonged or repeated secondary responses and may manifest as impaired growth, reproductive dysfunction, alterations in immune function, increased susceptibility to disease, behavioral changes, and even mortality [9,11]. Therefore, these effects are frequently associated with chronic or continuous stress rather than with acute stress exposure.

These responses can be triggered by a wide variety of stressors, including environmental, biological, and husbandry-related factors, whose nature and intensity determine the magnitude and duration of the stress response. Understanding the physiological mechanisms underlying these responses is therefore essential for identifying and mitigating key stress-inducing conditions, particularly during critical procedures such as fish transport.

2.3. Stress-Inducing Factors in Aquaculture

In aquaculture, fish are exposed to multiple stressors from early developmental stages to adulthood, as cultured fish are continuously subjected to husbandry-related, environmental, physical and physiological challenges [12]. These stressors are usually divided into three broad categories: environmental, biological, and physical stress [13,14] (Figure 2). Although this classification is useful, it is important to note that responses to stress may differ between species.

Environmental stressors are frequently encountered under farming conditions, to which ornamental species such as zebrafish are often exposed [12,15]. Exposure to stressors is particularly relevant during early development stages, as it may lead to long-term physiological and behavioral alterations that can be epigenetically transmitted to offspring, thereby influencing stress sensitivity in subsequent generations and highlighting the importance of managing stress at both the individual level and within breeding programs [16,17,18,19,20]. Nevertheless, while some stressors can be mitigated through improved management practices, such as gentle handling, optimized water quality, and appropriate stocking densities, others remain unavoidable due to production cycles and seasonal environmental variability and must be addressed through alternative strategies [21,22].

Among the various stressors encountered in aquaculture, transportation represents one of the most critical challenges and is consistently shown to induce osmotic imbalance and ion loss in several freshwater fish species [23,24,25]. Fish, particularly ornamental species, are typically transported in plastic bags filled with water often pre-treated with chemical additives or medications (e.g., salt and artificial or natural anesthetics) to reduce metabolic activity and stress and saturated with pure oxygen, sealed and placed in insulated containers [26,27]. Transport duration ranges from short periods (2 to 4 h) [28] to extended journeys lasting up to 72 h [29], with road transport being the most common mode [30,31]. Notably, the outcomes of transport are highly dependent on context, and its success depends on several interacting factors, including journey duration, water quality, fish size and density, temperature, physiological condition, and the depuration period prior to transport [32,33]. Moreover, fish are exposed to multiple stress-inducing conditions, such as agitation, noise, environmental fluctuations, and deterioration of water quality, particularly in terms of oxygen availability. These factors can trigger rapid endocrine responses characterized by elevated cortisol levels [2], disruption of ion homeostasis, increased oxidative stress [30], altered energy metabolism, accumulation of stress metabolites, and behavioral disturbances [32,34,35,36] that activate compensatory physiological mechanisms aiming to restore internal homeostasis [37]. Physical injuries, such as scale loss and mucus depletion, are also common during transport and consequently increase susceptibility to bacterial and fungal infections [38]. Although most transport-related studies have been conducted in different species, these stressors are also very important for zebrafish, which are widely transported both within the ornamental fish trade and between research facilities [39]. As a result, recent studies have emphasized the urgent need to improve welfare standards during transport of aquatic animals [40,41,42,43,44], considering transport-related stress is associated with compromised health and increased mortality rates, especially in ornamental species subjected to repeated handling and prolonged confinement [33,45].

Given the complexity and intensity of stress responses induced by transportation and other husbandry practices, the use of suitable experimental models is essential to investigate stress physiology and develop effective mitigation strategies. In this context, zebrafish have emerged as a valuable model organism for studying stress and anesthesia-related processes [46] under controlled laboratory conditions.

3. Zebrafish as a Model for Stress and Anesthesia Research

3.1. Advantages of the Zebrafish Model for Aquaculture Research

The use of zebrafish as an animal model for aquaculture studies has grown exponentially over the years [47,48,49,50]. This fish model offers numerous advantages, including low costs and relatively easy maintenance, large number of offspring and replicates per assay, and a lack of legal restrictions up to 120 h post-fertilization (hpf) [51], as well as the fact that major neurotransmitters can be found by 72 hpf [48,52], making the larvae a useful model in the stress and anesthesia field [48]. Zebrafish also exhibit advantageous biological characteristics that are representative of a broader range of aquatic species [53] and have thus been used to investigate the neurological, behavioral, and metabolic pathways highly conserved across them [38,48,54,55,56]. Although other species are commercially relevant, their larger size, slower development, and more complex husbandry requirements often limit their applicability in fundamental anesthesia research, making zebrafish an invaluable model for both scientific exploration and practical applications in fish procedures [46].

Zebrafish are used in the most diverse areas ranging from nutrition to immunological studies [6,49,50,53,57,58]. In addition, zebrafish have increasingly been used as models to assess welfare in aquaculture and for testing compounds aimed at reducing stress and improving welfare [51,59,60,61,62]. Early developmental stages have received particular attention due to their high sensitivity to compounds and external stimuli [63,64] and taking into account that stress at this stage can have lasting effects on growth and health [65,66,67,68]. Some studies corroborate this by demonstrating that zebrafish larvae are sensitive to stimuli such as vortex and agitation from 96 hpf, exhibiting behavioral, metabolic, and even genetic changes (

Table 1. Evolution of studies and key findings regarding different stress stimuli and their associated effects in zebrafish (Danio rerio) larvae across different development stages (0 to 120 h post-fertilization (hpf)).

Stage	Stimulus	Objective	Observed Effect	Findings	Reference
25–97 hpf	Different stressors (unspecified)	Development of corticosteroid axis	Cortisol synthesis and HPI activation from ~72 hpf No acute response until 97 hpf	HPI axis becomes functional before 97 hpf	[6]
0–97 hpf	Acute stress (unspecified)	Onset of functional HPI response	Basal cortisol levels present at hatch Stress-induced response from 97 hpf	Confirms timing of HPI activation in larvae	[70]
2, 24, 48 and 96 hpf	Acute swirling stressor and light stimulus	Role of GR and MR in stress behavior	Loss of GR/MR abolished hyperactivity after light stress Cortisol increases at 96 hpf	GCR and MR both mediate stress-related behavior	[66]
96 hpf	Acute swirling stressor	Mechanisms involving stress response	CRH/CRHR1 system induces hyperactivity GR/MR sustain response Cortisol increases	Establishes link between CRH and cortisol signaling	[71]
120 hpf	Vortex flow (medium strength)	Prolonged forced swimming and HPI activation	Whole-body cortisol increases from the moment of exposure up until 4 h later; return to baseline at 6 h Positive rheotaxis	Early life stress reconfigures cortisol secretion	[72]

) [6,64,69].

3.2. Zebrafish Larvae as a Model for Stress Research

One of the first studies using the zebrafish larvae model to study stress was conducted by Alsop and Vijayan [6], aiming to verify the development of the corticosteroid axis and the expression of corticoid receptors using different types of stress in larvae of different ages, with the earliest stage tested being 25 hpf [6]. Although the production of cortisol and activation of the HPI axis were clear from the moment of hatching, acute cortisol responses to stressors were not detected until 97 hpf, indicating that the HPI axis only becomes functionally responsive around 72 hpf. These findings were confirmed in another study, demonstrating that despite the early expression of all HPI axis components and the synthesis of cortisol at hatch, a stressor-induced cortisol response only becomes apparent at the same time (97 hpf) [70]. Collectively, these studies highlight the discrepancy in responses between larvae stages and reinforce the importance of considering development stages when interpreting stress physiology in zebrafish.

Faught and Vijayan [66] later established that this model was also a good basis for studying the mineralocorticoid receptor and the regulation of the HPI axis, by quantifying cortisol levels at different development stages, 2, 24, 48, and 96 hpf, with the latest stage being subjected to an acute swirling stressor. The study showed that both glucocorticoid (GCR) and mineralocorticoid (MR) receptors are involved in stress-related behaviors, as the loss of receptors abolished the glucocorticoid-mediated hyperactivity in response to a light stimulus [66]. In a subsequent study, the author confirmed that the CRH/CRHR1 system induces hyperactivity, while cortisol production and subsequent GCR/MR signaling are required to sustain this response [71]. Additional findings by Castillo-Ramírez et al., (2019) [72] also demonstrated that vortex speed might also induce speed-dependent stress responses with activation of cortisol-independent pathways in larval zebrafish using high-throughput forced swimming tests. These results align with mammalian models and offer an important framework for additional research on the early-life cortisol responses and feedback of zebrafish larvae, which are perfect for high-throughput screening and non-invasive imaging [73].

Recently, in a study conducted using vortex-flow methods with adaptations, it was possible to observe that acute stress in 96 hpf larvae increases metabolic rate, distance swum and speed [74]. Yet cortisol, glucose, and lactate levels remain unaltered [74]. At the biochemical level, the activity of both ATPase and glutathione S-transferase (GST) decreased, potentially due to alterations in osmoregulation associated with the hypothetical adrenocorticotropic hormone downregulation. Further advances have been made using molecular markers of stress, including associated immediate early genes and components of the stress axis, as well as urotensin 1, corticotropin-releasing hormone-binding protein, and succinate dehydrogenase [68]. This work was recently reviewed and compared with De Marco et al., (2025) [65], demonstrating that zebrafish larvae exhibit detailed cortisol response patterns, rapid regulation mediated by GC receptors, developmental modulation of the HPI axis function, and reduced cortisol reactivity. Collectively, these findings support the use of the vortex stimuli in zebrafish larvae as an innovative and reproducible option that respects ethical principles and fits within the 3Rs policy to study stress mechanisms and adaptive responses in vertebrate models [65]. In addition, the use of the zebrafish larvae model to assess changes in the HPI axis with the potential to interfere at the behavioral, metabolic, and physiological levels is increasingly justified by the literature. Adding to the similarity between the HPI and HPA axes, this model offers versatility and allows for coherent and clear results on what happens when fish are subjected to a stressful situation. Nevertheless, selecting any model to evaluate the effects of stress in aquatic animals must still consider several factors, such as its sensitivity to the stimulus and the extrapolation across species [75].

As these findings clearly establish that the HPI axis is functionally active and responsive to environmental stimuli from early development stages, developing and implementing effective anesthesia strategies becomes critical. Nevertheless, research on the topic is very limited when it comes to embryos or larvae.

3.3. Zebrafish Larvae as a Model for Anesthesia Research

To answer the urgent need for strategies to minimize stress in fish species, zebrafish larvae represent an important model to investigate both the efficacy of anesthetic substances, as well as their potential to modulate stress-related responses. In aquaculture and research settings, anesthesia is often implemented to reduce stress arising from multiple sources. Yet, since the effects of these substances depend on concentration, exposure time, mechanism of action, and even development stage, it becomes essential to understand the balance between all these factors to ensure welfare-oriented practices.

Responses to anesthetic compounds and stressors can vary depending on the development stage [76,77,78,79], reflecting differences in metabolic rate, oxygen consumption, and sensitivity to drugs during ontogeny [80]. As a result, effective concentration, induction time, and recovery of anesthetics, as well as their absorption, also differs between embryo–larval stages and more developed stages, such as juvenile and adult [76,81]. Using larvae for initial screening allows the evaluation of potential side effects, optimization of concentrations, and assessment of physiological and molecular responses, thereby serving as a replacement for other, more complex models [63,82]. In this context, a wide range of anesthetic agents—synthetic and natural—have been explored in zebrafish larvae [77,83,84,85]. The use of this model has also become relevant in other fields, such as analgesia and euthanasia, highlighting its versatility [86,87,88].

These findings position zebrafish larvae as a relevant and robust model to further explore the role of conventional synthetic anesthetics as stress-mitigating strategies, while allowing for the investigation of emerging natural alternatives.

4. Synthetic vs. Natural Anesthetics for Fish

4.1. Anesthesia as a Strategy to Reduce Stress

During fish procedures, different strategies have been explored to reduce stress during handling and transportation. Most approaches, including control of osmoregulatory balance, levels of dissolved oxygen and carbon dioxide, suspended solids and ammonia, temperature, and carrying capacity, as well as reducing physical handling and fish acclimatization, have been applied primarily to the juvenile and adult stages, as the small size and fragility of larvae have traditionally limited their use. Despite this, recent research indicates that early developmental stages may also serve as reliable models, given the comparable sensitivity of larvae and adults to stressors [76,81]. Within this scope, multiple natural and synthetic substances have been investigated for use during anesthetic procedures, with variations in licensing across countries. Currently, only MS-222 is approved for use on food fish by the United States Food and Drug Administration’s Centre for Veterinary Medicine, with a required 21-day withdrawal period [89]. However, this varies internationally and may extend up to 70 days [90]. In contrast, eugenol has been licensed as an aquatic anesthetic in several countries, including South Africa and New Zealand where no withdrawal period is necessary, and Japan where it has a 7-day withdrawal requirement [90].

Anesthesia has three levels, sedation, general anesthesia, and deep anesthesia [46]. While sedation is characterized by depression of awareness, suppression of the fish receptiveness to external stimuli, and limited arousal responses, the remaining phases result in inhibition of reflex activity and reduced skeletal muscle tone [88,91,92,93]. An ideal anesthetic should be environmentally friendly, non-toxic to fish, effective in under three minutes while facilitating immediate recovery (<5 min), easily accessible, reasonably priced, and leave no remnant in tissues [94,95].

The use of anesthetics has gained attention as a practical strategy given their ability to reduce metabolism, oxygen consumption, carbon dioxide and ammonia production, thereby reducing stress from handling and capture and improving animal welfare overall [33,96]. While some compounds with sedative properties exhibit tranquilizing, anxiolytic, and in some cases analgesic capacities [97], others of natural origin may provide additional benefits, such as antioxidant and antiseptic effects, and may even improve immune function [98,99]. However, the use of these substances in larvae remains unclear, particularly regarding the appropriate dosage which can vary considerably depending on species, size, and development stage [100]. In addition, most anesthetic substances used are often associated with notable adverse effects that should not be overlooked.

4.2. Aversive Properties of Conventional Anesthetics

The most common anesthetics in early developmental stages include MS-222, benzocaine, etomidate, propofol, quinaldine sulfate, eugenol and its isomer, isoeugenol (

Table 2. Overview of synthetic anesthetic substances tested in zebrafish (Danio rerio) embryos (6 to 72 h post-fertilization (hpf)) and larvae (3 to 9 days post-fertilization (dpf)).

Compound	Concentration (mg L−1)	Stage	Mechanism of Action	Main Observations	Side Effects	Reference
MS-222	78.4–2612.9	4–4.5 dpf	Na+ channel blockade	Limited CNS depression at higher concentrations ↓ Brain activity after light stimulation ↓ Atrial and ventricular rates * ↓ Stroke volume * ↓ Cardiac output * ↑ Thigmotaxis in light phase at higher concentrations ↓ Locomotion at lower concentrations	Cardiovascular stress Anxiogenesis	[103]
	500–1000	3–5 dpf		↑ Sensitivity with age due to maturation of ionoregulatory function	Stage-dependent sensitivity
	168	5–7 dpf		Rapid loss of tactile response and righting reflex * Rapid recovery of tactile response * Interference with optokinetic behavior Stable heart rate	Behavioral alterations	[104]
Propofol	1–5	6 hpf–3 dpf	GABA modulation Conductance of Cl− channels	↓ Heart rate ↓ Body length ↓ Hatchability ↑ Expression of casp3a, casp3b, casp9 and baxb Edema, spinal deformities, fin dysplasia, light pigmentation, hemorrhage	Cardiovascular stress Developmental toxicity Malformations Apoptosis	[105]
Quinaldine sulfate	193–1351.1	4–4.5 dpf	Unclear	Limited CNS depression at higher concentrations Aversion to the administration site ↓ Brain activity after light stimulation ↓ Atrial and ventricular rates * ↓ Cardiac output * ↑ Thigmotaxis response	Avoidance behavior Cardiovascular stress Anxiogenesis	[103]
Etomidate	29.3–537.4	4–4.5 dpf	GABA modulation	Limited reduction in brain activity ↓ Atrial and ventricular rates * ↑ Thigmotaxis response at intermediate concentrations	Minimal cardiovascular stress Anxiogenesis	[103]
Benzocaine	≥49.6	4–4.5 dpf	Na+ channel blockade	Widespread CNS depression at higher concentrations ↓ Atrial and ventricular rates * ↓ Cardiac output* ↑ Thigmotaxis in dark phase at higher concentrations	Cardiovascular stress Anxiogenesis	[103]

↑ Increase. ↓ Decrease. * Concentration-dependent effects.

). However, with increased use and consequent research regarding conventional anesthetic agents, the literature is being filled with articles that address the adverse effects of these compounds [99,101,102].

MS-222 exhibits its anesthetic effects through a voltage-gated Na+ channels blockade [104]. This compound has an acidic nature and requires buffering before it can be used, ultimately increasing preparation time and potentially inducing physiological alterations in fish depending on the buffering agent [100,106]. Although this substance is described as a better option than gradual cooling, another commonly used anesthetic method [107], MS-222 is also associated with adverse cardiac effects [104], namely concentration-dependent reductions in both atrial and ventricular rates, stroke volume and cardiac output, which corroborates the cardiovascular stress typically observed in conventional anesthetics [103]. In addition, alterations in locomotion and optokinetic behavior have also been documented. Interestingly, zebrafish larvae reveal differential sensitivity to this anesthetic that increases with developmental stage and reinforces the pressing need to establish stage-specific dosing regimens for this compound. Nevertheless, other relevant effects manifest on the toxicological level, as exposure to this anesthetic in zebrafish larvae reveals a range of physiological, biochemical, teratogenic and behavioral effects [104].

Benzocaine is a low-cost local anesthetic that provides light anesthesia by blocking voltage-sensitive Na+ channels [83,108,109,110]. However, its role as an anesthetic for larvae remains debatable. Dimitriadou et al., (2025) [103] reported that this substance depresses atrial and ventricular rates and cardiac output, significantly impacting cardiovascular function. Additional findings demonstrate that benzocaine induces anxiety-like behavior as confirmed through increased thigmotaxis in the dark phase, which ultimately overtakes its practicability.

Propofol, a short-acting sedative-hypnotic agent commonly used for induction and maintenance of anesthesia, modulates GABA receptors and increases conductance of CL-channels [103]. Studies in zebrafish embryos have demonstrated that propofol exposure induces stage-dependent developmental toxicity, as well neurological and cardiovascular effects [105,111].

Regarding quinaldine sulphate, although its precise mechanism of action remains unclear, its anesthetic and adverse effects in zebrafish larvae have been reported [112,113]. Notably, in the study by Dimitriadou et al., (2025) [103] this compound was the only one to elicit clear aversion behavior to the administered site. In the same study, etomidate, another GABAergic anesthetic, produced a limited anesthetic effect but still revealed significant adverse effects. This suggests that even moderate anesthesia from synthetic substances may still impact the cardiovascular system in larvae.

In general, the use of synthetic anesthetic agents is associated with prolonged induction and recovery times, as well as a numerous list of adverse effects that mainly affect the cardiorespiratory system [103]. These effects can lead to states of bradycardia, hypoxia, and blood acidosis, alter glucose, lactate, cortisol, and other biochemical parameters, and cause enzymatic, behavioral, and developmental changes. Nevertheless, it is important to acknowledge that the magnitude of these effects may be dependent on experimental conditions, including different anesthetic concentrations and exposure times. For instance, in the study by Leyden et al., (2022) [104], zebrafish were exposed to MS-222 for 7 min, whereas a longer exposure duration were applied in another similar study [103], which may contribute to differences in the reported severity of adverse effects. Considering the primary focus of most studies is on the biological effects of the compounds rather than on standardized exposure protocols, this leads to divergent exposure duration and administered dosage across studies, consequently limiting direct comparisons of the reported effects. Additionally, although all studies included here were conducted in zebrafish larvae, it is also important to note that sensitivity to these compounds may also vary across species and that both dose- and species-dependent effects are important aspects in toxicological assessment.

Over the years, the focus has shifted to replacing these compounds and others similar to them with compounds of natural origin [111,113,114]. Among these, the most extensively investigated are monoterpenes eugenol and isoeugenol, the main components of clove oil (Syzygium aromaticum). These substances exhibit a wide range of biological properties [37,99,115,116], as well as good anesthetic capacity in both zebrafish embryos and larvae [117,118,119,120,121]. Nevertheless, their use may also be associated with adverse effects at some concentrations, urging the need to expand research into other monoterpenes [76,122].

4.3. Monoterpenes as Alternative Stress-Reducing and Anesthetic Agents

The use of herbal compounds with anesthetic properties has recently gained attention, mainly due to the need for alternatives to conventional synthetic anesthetics and the numerous advantages they offer, including minimal aversion and behavioral changes [46]. Multiple plant species contain compounds with potential anesthetic properties, such as Lippia alba, Melaleuca alternifolia, Aloysia triphylla, Ocimum americanum, Ocimum gratissimum, Thymus vulgaris, and Mentha piperita, among others, as reviewed by Minaz and Félix, (2026) [46]. However, studies evaluating their efficacy in early stages of development, particularly in zebrafish larvae, are still very limited [46]. In this context, monoterpenes—volatile compounds often present in the essential oils of these plants—have been the most extensively investigated substances, including cineole, menthol, thymol, eugenol, myrcene, linalool, geraniol, citronellal, citral and terpinene-4-ol [76,84,122,123,124].

The mechanisms of action of monoterpenes are closely associated with their chemical structure, high lipophilicity, and interactions with biological receptors [90,125,126]. Their ability to integrate into the neuronal lipid bilayer and alter membrane fluidity may complement their direct binding to transmembrane proteins, potentially influencing the conformational transitions of ion channels [126]. Because they share structural similarities, monoterpenes generally exhibit comparable modes of action across different bioactivities (Figure 3) [127,128,129]. In terms of analgesic capacity, phenolic monoterpenes show affinity for the gamma-aminobutyric acid (GABA) [84,129,130] and transient receptor potential vanilloid-1 (TRPV1) receptors [131]. GABA receptors play a key role in pain processing, analgesia, depression, and anesthesia [132,133,134]. Specifically, positive allosteric modulation of GABA receptors, which mediate the main inhibitory neurotransmission in the CNS, suppresses neuronal activity and leads to CNS depression and anesthesia [132,135]. Recent evidence in fish models suggests that monoterpenes like thymol may interact with the GABA_A receptor complex at sites distinct from those of benzodiazepines, as their effects are often sensitive to picrotoxin but not flumazenil [125,136]. Monoterpenes with anesthetic properties act in a similar manner, primarily through modulation of the GABA receptor complex [132,135,137,138,139], and modulate various ion channels, including TRPV1 [140,141,142,143,144], and voltage-gated Na⁺ and Ca²⁺ channels [145]. The interaction with voltage-gated Na⁺ channels typically involves the stabilization of the inactivated state, preventing the rapid depolarization required for nociceptive signaling [146]. This multimodal action, characterized by the simultaneous inhibition of excitatory pathways and enhancement of inhibitory neurotransmission, resembles that of local anesthetics such as benzocaine and lidocaine [119,135,137,147]. Furthermore, in aquatic species, the rapid onset and recovery associated with these compounds are attributed to their efficient absorption across the gill epithelium and subsequent passage through the blood–brain barrier [94,125].

Despite advancements, research on the molecular mechanisms through which monoterpenes exhibit their anesthetic effects is still fresh, highlighting the need for further studies to validate their use in fish and improve our understanding of their physiological impacts in zebrafish larval stages [92,93,135,148]. Nevertheless, research into this topic is growing—supported by the inherently favorable effects of these compounds [90].

4.4. Physiological Effects and Applications of Monoterpenes

In a recent study, Capparucci et al., (2022) [123] demonstrated that both clove oil, which is rich in eugenol, and AQUI-S, constituted by 50% of isoeugenol, produced effective sedation in zebrafish embryos and larvae, although differences in induction and recovery times indicated a developmental dependence on physiological responses (

Table 3. Overview of plant-based substances with anesthetic properties tested in zebrafish (Danio rerio) embryos (24 to 96 h post-fertilization (hpf)) and larvae (3 to 5 days post-fertilization (dpf)).

Substance	Concentration (mg L−1)	Stage	Mechanism of Action	Main Observations	Side Effects	Reference
Clove oil	40, 60, 90, 120, and 150	24 hpf	GABA modulation; Na+/Ca2+ channel blockade	Rapid anesthesia * Prolonged recovery * ↑ Mortality with concentration	-	[76]
	90, 120, and 150	5 dpf		Rapid anesthesia * Prolonged recovery * ↑ Mortality at 90 and 150 mg/L
Isoeugenol	32.8–394.1	4–4.5 dpf		CNS depression at higher concentrations ↓ Atrial and ventricular rates * ↓ Stroke volume * ↓ Cardiac output * ↑ Thigmotaxis response at intermediate concentrations	Cardiovascular stress Anxiogenesis	[103]
AQUI-S	40, 100, 150 and 200	24 hpf		Rapid anesthesia * Prolonged recovery * ↑ Mortality with concentration; Lethal at 200 mg/L Heart edema at higher concentrations	Teratogenesis	[76]
	100 and 200	5 dpf		↑ Mortality with concentration Lethal at 200 mg/L	-
Basil essential oil (Ocimum basilicum)	50, 100 and 200	24 hpf–4 dpf	Unclear	↑ Mortality at both higher concentrations ↓ Hatch rate * ↓ Heart rate at both higher concentrations Pericardial edema, blood congestion and un-looped heart at both higher concentrations	Bradycardia at higher concentrations Teratogenesis	[123]
Thymol	15	3 dpf	GABA modulation; Na+/Ca2+ channel blockade	↓ Heart rate ↓ ATPase activity ↓ Stress	-	[124]
	100, 200 and 300	3 dpf		↓ ATPase activity Differential activities of antioxidant enzymes ↑ Swimming	Bradycardia on anesthesia induction	[122]
Menthol	50	3 dpf	TRPV1 agonist; Na+/Ca2+ channel blockade	↓ Heart rate ↓ ATPase activity Stress Modulates Nrf2 expression	-	[124]
	200, 400 and 500	3 dpf		Differential activities of antioxidant enzymes ↑ Speed at 500 mg/L	Bradycardia on anesthesia induction	[122]

↑ Increase. ↓ Decrease. * Concentration-dependent effects.

).

Similarly, Capparucci et al., (2022) [123] evaluated the essential oil of basil (Ocimum basilicum), mainly constituted by linalool, eugenol, geraniol, citronellol and limonene, and reported that, in addition to producing anesthetic effects comparable to synthetic agents, these compounds only demonstrated significant adverse effects when higher concentrations were administered. In addition, this study also demonstrated antioxidant and cytoprotective properties with potential to reduce stress-related oxidative damage. Vieira et al., (2025) [124] investigated thymol and menthol in 3 dpf zebrafish larvae, revealing behavioral and metabolic responses similar to conventional anesthetics, which supports their suitability as anesthetics for zebrafish larvae. Moreover, exposure to these monoterpenes also has a marked effect on other relevant parameters, such as reduced glucose and heart rate peaks [122]. Although studies on the effects of natural substances during early developmental stages are still limited, complementary studies in adult zebrafish have further established the potential of menthol and thymol as effective anesthetics [124]. These results, combined with similarities in chemical structure and mechanisms of action, suggest a broader anesthetic potential inherent to all monoterpenes that further reinforce their overall valuable role as natural alternatives for stress mitigation [136,149]. In addition, monoterpenes are often accompanied by a list of equally interesting biological properties. In addition to improving zebrafish appetite, development and growth [124,150], monoterpenes also exhibit antioxidant, anti-inflammatory and anti-apoptotic effects [151,152,153] that make them particularly relevant for application during fish procedures and in experimental contexts [84,124,153].

Collectively, these findings highlight the potential of herbal anesthetics to serve as effective and less harmful substitutes for conventional compounds. Moreover, their use aligns with the 3Rs principle, promoting welfare-oriented practices and encouraging broader application in larval stages. Nevertheless, studies on their concentrations, pharmacokinetics, and long-term safety in early developmental stages remain scarce, reinforcing the need for further research to standardize and regulate the use of these alternatives in fish procedures and experimental settings [46,90]. Moreover, the potential effects associated with their application remain poorly described in zebrafish larvae. In other fish species, monoterpene exposure has been associated with cardiovascular and haemato-biochemical alterations that typically return to baseline during recovery, minimal changes in behavior, and reduced cellular stress markers [90]. While these findings may be applicable to zebrafish, this area is still in need of further investigation.

5. Limitations and Future Directions

Despite growing interest in plant-based anesthetics, some important limitations and challenges remain. The lack of standardized protocols for concentration, duration of exposure, and recovery assessment constitutes one of the most important limitations of using monoterpenes as anesthetic in zebrafish larvae, complicating cross-study comparison, regulatory approval, and implementation in routine practices [123,124]. Environmental factors, such as temperature, pH, and water salinity, can influence the efficacy of natural anesthetics, yet few studies systematically address these interactions [154,155,156,157]. Moreover, data on short-term exposures, long-term safety, potential bioaccumulation, and metabolization of monoterpenes are also largely underexplored, underscoring the need for harmonized methodologies and integrated biochemical and molecular analyses.

Research on monoterpenes in the early stages of development is still lacking. Yet studies on juvenile and adults show that essential oils and some isolated monoterpenes may cause dose-dependent toxicity, neurotoxicity, developmental problems, pro-oxidant activity and apoptosis when applied outside of the anesthesia area [32,33]. In addition, knowledge of the mechanisms of action of monoterpenes, particularly concerning receptor-level interactions and potential sensitivity across developmental stages, requires further clarification.

Due to their composition, essential oils may be associated with limitations related to solubility, which may subsequently interfere with the mechanisms of action [154,155,156,157]. The vast majority of essential oils have lipophilic properties which contribute to a rapid dispersion across biological membranes, including the blood–brain barrier, allowing for modulation of the CNS [37,158]. However, these substances also have hydrophobic characteristics that can alter their bioavailability and stability, potentially reducing efficacy. Encapsulating these compounds may help with absorption and improve passage into the bloodstream and through the blood–brain barrier, ultimately increasing bioavailability in the brain and ensuring the intended mechanisms of action [37,93,159]. Although there are works conducted in the adult zebrafish model on this topic [139,160], information on this process using zebrafish larvae is still lacking.

6. Conclusions

Natural monoterpenes have shown significant promise as sustainable anesthetic alternatives in aquaculture, as evidenced by their ability to modulate stress associated with handling and transporting in zebrafish larvae. Although standardization and long-term safety assessments are still required, these compounds may provide a promising route towards more ethical and environmentally responsible aquaculture practices. Overall, increasing the implementation of natural anesthetics into aquaculture represents a favorable shift toward systems that balance productivity with welfare and sustainability. Moreover, by combining pharmacological efficacy, biological safety, and ecological compatibility, monoterpenes provide a scientifically grounded, welfare-enhancing alternative that supports the evolution of responsible aquaculture.