The in vitro assay demonstrated the effectiveness of NaCl against pirarucu monogenoids, allowing for a better understanding of the responses observed in the in vivo efficacy tests, as NaCl effects in the target organism indicate greater safety and reduces the number of fish required for the in vivo test (Park et al. 2014; Zorin et al. 2019. Queiroz et al. 2020). Direct NaCl exposure on pirarucu parasites led to a dose-dependent relationship, with increasing salt concentrations resulting in lower exposure times to reach 100% mortality rates. A 100% effectiveness in the 11 g.L− 1 in vitro assay was observed at 1 HPT, corroborating previous reports for Poecilia reticulata (Schelkle et al. 2010) and Bidyanus bidyanus (Forwood et al. 2013) displaying the same parasitosis and undergoing similar salt concentration treatments.
The pharmacological effects of NaCl on aquaculture health are associated to osmotic concentration gradient, osmoregulation and compensatory mechanisms changes, resulting in cellular dehydration and, consequently, parasite death (Kim. 2012). Sodium chloride can be absorbed by monogenoid teguments, which displays a membrane associated to the internal parasite environment, and by the parasite excretory system, which contains specialized cells named flame cells comprising collecting ducts and capillaries capable of transporting chloride to the internal parasite environment (Cohen et al. 2004; Woo. 2006). In the present study, the dose-dependent effect of NaCl on pirarucu monogenioids reflects the probable parasite mortality cause.
The LC50 − 90= 11.64 g.L− 1 calculated for juvenile pirarucu in the present study is higher than those reported for other species at the same growth stage. According to Zucker (1985), NaCl is considered lightly toxic for pirarucu (> 10 < 100 ppm), ranking 2 in a 1 to 5 toxicity scale. Our findings, however, suggest that pirarucu are highly tolerant to this compound (LC50 − 96h = 11.64 g.L− 1) compared to other freshwater fish.
The low toxicity of NaCl to pirarucu indicated by the calculated LC50 − 96h allowed for in vivo NaCl effectiveness determinations. At the applied NaCl concentrations, a low efficacy (20.5% − 11 g.L− 1) against monogenoids was noted compared to the in vitro test (100% − 11 g.L− 1) following the same exposure period.
The effectiveness difference noted in the in vivo and in vitro tests can be attributed to the presence of biological barriers, such as mucus, teguments and the operculum in the in vivo tests, making it difficult for NaCl to reach the parasites, interfering with product absorption. In addition, abiotic water factors can also decrease NaCl effects and interfere with its effectiveness.
Our findings and other literature reports suggest that NaCl results in differential responses in the host and parasite. Furthermore, its effectiveness is directly related to NaCl concentrations, exposure times and/or parasite and host species sensitivities. Despite being a cheap alternative for ectoparasite control with no residual effects, NaCl should be used cautiously, respecting hist tolerance limits (Tavares-Dias., 2021).
Hematology, biochemistry, and histology assessments are commonly employed to assess fish health conditions and the effect of xenobiotics (Andrade-Porto et al. 2017; Rodrigues et al. 2020; Queiroz et al. 2020; Ventura et al. 2020). Herein, no significant blood changes were observed following NaCl exposure. However, decreased hemoglobin and HCM values were noted following exposure to the highest NaCl concentration (11g.L− 1). Under adverse conditions, the organism attempts to adapt by employing compensatory mechanisms, in this case increasing water cell absorption and decreasing hemoglobin concentrations. These results corroborate reports for Cyprinus carpio under prolonged exposure (40 days) to 3.0 g.L− 1 NaCl, resulting in altered erythrogram values, including decreased hemoglobin and CHCM values and increased mortality rates (Mubarik et al. 2018). Prolonged exposure in Labeo rohita to 8.0 g.L− 1 NaCl increased total erythrocytes and thrombocytes and hematocrit percentages and decreased hemoglobin values (Murmu et al. 2020). In another study, however, a short 15-min NaCl bath (15 g.L− 1) did not significantly alter Peckoltia oligospila blood parameters (Santos et al. 2020), while Sander lucioperca exposed to short baths (30 min.) at 10 and 20 g.L− 1 NaCl exhibited significant increases in hemoglobin concentrations, hematocrit percentages and number of erythrocytes (Demska-Zakęs et al. 2021). These results suggest that NaCl alters fish oxygen transport capacity in a species specific and dose dependent manner. Pirarucu survival was not compromised in the present study, indicating that the fish were able to adapt to the evaluated conditions.
The different NaCl concentrations evaluated herein resulted in altered mean glucose and cortisol values, albeit not statistically different (p > 0.05), suggesting that NaCl does not reverse the stress situation induced by the parasitosis in all groups. In addition, higher NaCl concentrations than those tested herein induce hypercotisolemia and hyperglycemia in other species, such as Brycon amazonicum (Urbinati and Carneiro, 2006) and Colossoma macropomum (Chagas et al., 2012).
Fish gills are responsible for gas exchanges, osmoregulation, acid-base regulation and the excretion of nitrogenous wastes (Evans et al. 2005). Their structures are sensitive and may present different responses to environmental stressors (Nilsson. 2017; Rodrigues et al. 2017; Ugurlu et al. 2019). In the present study, histological changes were observed with increasing NaCl concentrations. The increased HAI values and MAV observed at 8 and 10 g.L− 1 NaCl exposure were mainly associated to marginal gill canal dilation and lamellar epithelium hyperplasia, respectively. These changes may indicate pirarucu sensitivity to NaCl, although they were reversible, returning to normal when the stressor agent was removed from the environment (Sayed et al. 2020).
Some changes were also observed in the gills of fish belonging to the control group, probably due to the presence of monogeneoid parasites. Other cases of reversible lesions caused by monogenoids in fish gills have been reported previously by Jerônimo et al. (2014) and Costa et al. (2017). Thus, despite the observed changes, therapeutic NaCl baths in juvenile pirarucu at the concentrations evaluated herein do not interfere with fish homeostasis.
In the present study, 100% of the monogenoids present in the gills of the fish were identified as D. cycloancistrium, probably because this is the most common species in pirarucu, corroborating Andrade-Porto et al. (2017) and Queiroz et al. (2020). Our results suggest that NaCl exhibits anthelmintic activity against D. cycloancistrium in pirarucu influenced by exposure time and concentration. Pirarucu exhibit a certain tolerance to NaCl (11 g.L− 1), displaying a 100% survival rate during 2 h of exposure, without significant hematological, hormonal, and histopathological alterations. However, the concentrations employed herein exhibit low efficacy (< 36) against D. cycloancistrium, indicating that other studies evaluating the influence of long and/or sequenced baths employing higher NaCl concentrations are required.