The present study examined the potential use of eugenol and EOLA as anesthetic substances for application in juveniles of an Amazonian freshwater stingray species, Potamotrygon wallacei. To date, the use of both anesthetics is unprecedented for representatives of Potamotrygonidae family, and only a few investigations have assessed the effects of anesthetic substances obtained from herbal products on blood and plasma biochemistry (Frick at al. 2009); no studies have focused on use of EOLA in any elasmobranch fish. This also applies for the evaluation of potential histological alterations on the gills of these fish when they are in contact with these substances in the water. This limitation is, in part, due to complicated logistics in acclimatization and handling procedures to keep elasmobranchs in captivity, which makes the collection of physiological data after anesthesia a non-trivial task.
The similarity of the physical-chemical characteristics of the water observed during the acclimatization of the stingrays, as well as in the induction and recovery times in the experimental aquariums, are in line with the amplitude of these parameters documented in the areas of natural occurrence of the cururu stingray (Oliveira et al. 2017). This indicates that the behavioral (swimming capacity and reflex activity) and physiological responses (SF and blood parameters), as well as the histological changes observed in the gills of P. wallacei during anesthesia and recovery are attributed only to the effects of tested anesthetics, and not due to changes in the water quality used in the experiments.
As well as in teleosts, depression of ventilatory frequency and cardiac rate are expected responses in elasmobranch species under anesthesia (Hove and Moss 1997). For instance, a transitory decrease in ventilatory frequency (measured trough spiracular opening and closing movements) in the freshwater stingray P. motoro was observed after oral administration of 50 mg kg− 1 of ketamine (Raines and Clancy 2009). Conversely, in the spotted bamboo shark (Chiloscyllium plagiosum), the intravenous administration of propofol (2.5 mg kg− 1) resulted in stable respiratory and heart rates during anesthesia (Miller et al. 2005). These results indicate that cardiorespiratory responses may diverge in different species and according to the dosage and anesthetic type. In our study, the SF values obtained for the basal group of juveniles of P. wallacei before exposure to eugenol and EOLA are in line with those recorded for the same species (Pastório 2014) under normoxic conditions (40 to 60 beats min− 1). On the other hand, it was observed that the first contact with eugenol resulted in a hyperactivity response, which was reflected in the maintenance of a high SF (> 60 beats min− 1) during the onset of the induction phase in almost all tested concentrations. This pattern, associated with an excessively long excitatory phase (Stage II), may have resulted in stress and physical exhaustion, making the anesthetic induction with this herbal compound aversive for stingrays. These ventilatory responses are in line with the behavioral manifestations of hyperactivity and intense excitability observed in these eugenol-exposed animals, which were not attenuated during the phase of deep anesthesia. In this line of evidence, Barbas et al. (2021) demonstrated for juveniles of tambaqui (Colossoma macropomum), an Amazonian teleost species, that 65 mg L− 1 eugenol induced the fish to immobilization (with loss of muscular tonus and reflex) in a time-dependent manner. However, electroencephalographic studies revealed that eugenol failed to depress the central nervous system (CNS), resulting in an intense neuronal excitatory condition. Thus, the evidence of the behavioral responses observed in juveniles of P. wallacei when exposed to eugenol corroborates the above cited results (Barbas et al. 2021), suggesting that stingrays had been immobilized (with a loss of muscle tonus) by the action of eugenol, without depressing their CNS function. These features led us to carefully consider the use of this herbal product as a safe anesthetic and draws our attention to its possible toxic action for animals when submitted to anesthetic practices. In a comparative perspective, the sequence of behavioral patterns shown by juveniles of P. wallacei under exposure to eugenol and EOLA was like those observed by Grusha (2005) for the marine stingray R. bonasus submitted to two distinct eugenol concentrations. On the other hand, the sharks T. semifasciata and M. californicus treated with an isoeugenol-based anesthetic (AQUI-S 20E®), a eugenol isomer, showed a prolonged induction time and more than one excitatory phase (Silbernagel and Yochem 2016). In this study, the stingrays anesthetized with eugenol showed long recovery times, a response that differs from that observed for viola ray, Z. brevirostris, anesthetized with 85 mg L− 1 eugenol (Takatsuka et al. 2019). Conversely, juveniles of P. wallacei showed considerably shorter recovery times (4.2 min) after anesthesia, which are coincident to those observed in teleost fish (Marking and Meyer 1985; Keene et al. 1998), suggesting that eugenol can act differently in freshwater stingrays when compared to marine elasmobranchs.
The exposure of P. wallacei juveniles to 50 mg L− 1 eugenol revealed that this concentration was not suited to deep anesthesia due to the long time to reach light or deep anesthesia (> 10 min). The overexposure of P. wallacei to eugenol probably resulted in excessive absorption of this substance, culminating in 50% mortality during the recovery phase. On the other hand, the abrupt entry into the anesthesia phase and deaths recorded at concentrations above 75 mg L− 1 eugenol increase the risk of anesthesia, cause deep respiratory depression and the anesthetic induction process must be stopped immediately as soon as deep anesthesia is observed (phase III).
When juveniles of P. wallacei were exposed to EOLA at concentrations of 50 and 100 µL L− 1 they did not reach mild or deep anesthesia after 10 min; therefore, these concentrations are not recommended to the practice of anesthesia for these stingrays. On the other hand, at concentrations over 300 µL L− 1 of this essential oil, neurological sequelae and subsequent deaths were observed after exposure, which may be related to a decrease in blood flow at the cerebral level during anesthesia. In bamboo sharks, Miller et al. (2005) observed signs of neurological alterations associated to intravenous administration of propofol. In the other concentrations of EOLA evaluated in this study, a uniform decline in the respiratory pattern was observed during anesthesia, with no obvious stressing effects, such as the initial high SF and sudden falls in ventilation observed with eugenol. This fact resulted in a better condition for physiological recovery after anesthesia, which was evidenced by the faster return to regular feeding and routine swimming after recovery from the anesthesia.
Considering that eugenol has a higher density (1.06) than that of EOLA (0.80), this physical property could be a relevant aspect when referring to bottom-dwelling elasmobranchs, like P. wallacei. Even when previously diluted with ethyl alcohol for its use (Neiffer and Stamper 2009), eugenol may be easily left at the bottom of the experimental aquarium, exacerbating its irritating effects when coming into direct contact with the eyes (causing evident eye retraction), skin (burns on the edges of the disc, and areas of focal necrosis on the body surface) and gills. The overall features of gill histology assessed in the present study confirms the detrimental effects of this anesthetic on the architecture of this tissue. The necrotizing characteristics promoted by eugenol in the gills led to impaired protection responses in the primary epithelium, promoting irreversible damage, such as ionocyte (chloride cell) apoptosis and lamellar necrosis. In contrast, despite promoting tissue irritation, EOLA triggered less irreversible damage and greater reversible changes in the protective response of the epithelium, such as hyperplasia and cellular hypertrophy. This increase in the proliferation and size of cells can lead to the fusion of lamellae, a response mechanism to protect the lamellar epithelium from direct contact with toxic agents; this increases the distance from the gill surface used for the diffusion of blood to inhibit the intake of substance from the surrounding environment (Heath 1987; Karan et al. 1998).
Another feasible explanation could be related to the inflammatory chemical mediators released from the injured tissue responsible for vasodilation and increased local vascular permeability. In this process, the increase in blood flow through capillary dilation, while allowing the arrival of defense cells, might predispose to edema and the formation of aneurysms (Pober and Sessa 2015). In our study, we observed the incidence of melanomacrophages in the blood vessels of branchial filaments of juveniles of P. wallacei exposed to both anesthetics. Melanomacrophages are cells with phagocytic activity having a considerable number of dark pigments inside, like melanin, lipofuscin and hemosiderin. These cells are commonly found in the stroma of lymphoid tissues of teleost fishes, such as the kidney and liver, and are associated with the immune response in organs like the gills, liver, kidney and spleen (Agius and Roberts 2003; Steinel and Bolnick 2017) and related inflammatory processes (Manrique et al. 2014). However, other functions associated with the natural physiological mechanisms of cell renewal, waste cleaning and collagen recycling (Gutierre et al. 2017), lipid deposition during fasting (Neyrão et al. 2019), and iron recycling from hemoglobin molecules (Wolke 1992; Passantino et al. 2005; Sales et al. 2017) have also been documented. Studies on elasmobranch species have been sporadic (Borucinska et al. 2009; Moraes et al. 2016) and inconclusive. In Potamotrygon motoro, the presence of these cells was recognized and associated with the immune response, acting as the site of antigen processing (Moraes et al. 2016), and with environmental pollution triggered by gold and oil exploitation in the occurrence area of this species in Peruvian territory (Ramos-Espinoza et al. 2017). However, in the present study, the arrival of melanomacrophages in the gills of P. wallacei exposed to concentrations of 100 µL L− 1 eugenol and 175 µL L− 1 EOLA cannot be attributed entirely to the toxic effect of these anesthetics. The presence of this cell type surrounding the blood vessels of the gill filaments seems to be more associated with natural physiological processes, because they were only observed at intermediate concentrations, and since despite the most expressive changes evidenced in the epithelium at the highest concentrations of both anesthetics, this cell type was not found. Thus, further investigations are required to better understand the role of melanomacrophages in routine physiological and immunological responses in the gills and other tissues of freshwater stingrays.
The hematocrit values obtained in juveniles of P. wallacei were within the range recorded for this species under different environmental and experimental conditions (Brinn et al. 2012; Pastório 2014; Oliveira et al. 2016) and similar to those observed in marine stingray species, such as D. americana (Cain et al. 2004), A. rostrata (Routley et al. 2002; Speers-Roesch et al. 2012), Hypanus sabinus (Lambert et al. 2018) and Zearaja maugeana (Morash et al. 2020). In all these studies, a commonly observed characteristic was the lack of changes in this parameter in response to different types of stressors. Brinn et al. (2012) mentioned that the variables related to erythrogram did not function as suitable physiological indicators in the assessment of stress responses in juveniles of P. wallacei (= Potamotrygon cf. histrix) submitted to river transport. Our results corroborate this hypothesis and indicate that, at least for the tested concentrations, both eugenol and L. alba essential oil did not promote noticeable changes in this blood parameter in cururu stingrays, even after 48 h of recovery. In addition, the absence of statistical differences in the observed values of the plasma metabolite profile analyzed immediately after the anesthetic action and in the recovery 48 h later, especially glucose levels, is clearly indicative that the extent of contact with the different anesthetic concentrations of both eugenol and EOLA was not enough to promote changes in these blood physiological parameters, and hence do not interfere in the overall homeostasis of P. wallacei juveniles. These findings are in line with previous results for the same species exposed to anesthesia (Frick et al. 2012) or different types of stressors, like transport (Brinn et al. 2012; Ariotti et al. 2021) and progressive hypoxia (Pastório 2014).
On the other hand, the inability of these anesthetics to produce changes in the plasma lactate levels contrast with previous studies conducted with other elasmobranch species anesthetized with eugenol or its isomer, the isoeugenol. In Australian tiger sharks Cephaloscyllium laticeps, sedated with an anesthetic derived from clove oil (AQUI-S® isoeugenol, New Zealand Ltd.), the mean plasma lactate levels were also significantly higher than those of untreated sharks (Frick et al. 2012), and like P. wallacei, no significant differences in plasma glucose were observed between the treatment groups. Usually, sharks alter blood parameters (like hematocrit, pH, lactate, and glucose levels) only when submitted to vigorous and intense physical exercise (Skomal 2007; Frick et al. 2010; Brooks et al. 2012). In juveniles of Carcharhinus taurus, lactate levels remained significantly higher up to 12 h after capture stress (Kneebone et al. 2013). In a similar approach, Fuller et al. (2020) found that individuals of the Atlantic sharpnose shark (Rhizoprionodon terraenovae) showing higher lactate levels in blood after capture stress also exhibited lower clinical patterns of behavior required for safe release.