Anthelmintic efficacy of dillapiole and the crude extract of Piper aduncum in the control of monogenoids in Colossoma macropomum

Anthelmintic efficacy of dillapiole and the crude extract of Piper aduncum in the control of monogenoids in Colossoma macropomum | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Anthelmintic efficacy of dillapiole and the crude extract of Piper aduncum in the control of monogenoids in Colossoma macropomum Marieta Nascimento Queiroz, Paulo Adelino Medeiros, Zelina Estevam os Santos Torres, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7567560/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 11 You are reading this latest preprint version Abstract Piper aduncum , a medicinal plant, was evaluated for control of monogenoids and effects on the welfare of Colossoma macropomum using a crude ethanolic extract and its major constituent dillapiole. Treatments of 40 and 60 mg·L⁻¹ of the ethanolic extract were tested in in vitro and in vivo experiments with water and methanol controls. Dillapiole was evaluated at 28 and 33 mg·L⁻¹ with water and Tween-20 controls and a basal acclimated group. Parasitological, immunophysiological, biochemical and histological analyses were performed on 50 g fish. Both extract concentrations significantly reduced parasite load versus controls, yielding antiparasitic efficacies of 85.89% and 99.21% for 40 and 60 mg·L⁻¹, respectively. Control fish showed increases in hematocrit and hemoglobin, whereas fish treated with 60 mg·L⁻¹ extract exhibited hyperglycemia, elevated cortisol and reductions in total protein and Ca²⁺ and K⁺ ion levels, indicating physiological stress at higher extract concentration. In water controls, thrombocyte and leukocyte elevations correlated with heavy parasitism; these responses were attenuated in extract-treated groups. Dillapiole at 28 and 33 mg·L⁻¹ produced significant parasite reductions with efficacies of 52.44% and 80.24%, respectively. Dillapiole treatments induced increased thrombocytes, leukocytes and differential counts, suggesting stimulation of immune responses that favored recovery after therapeutic baths. Histological assessment showed milder tissue alterations in dillapiole-treated fish compared to controls. Results indicate that both P. aduncum extract and dillapiole effectively control monogenoid infestations in C. macropomum , combining antiparasitic action with moderate, concentration-dependent physiological impacts. These findings support further development of P. aduncum-based therapies for sustainable monogenoid management in tropical aquaculture globally. Anthelmintic aquaculture prophylaxis herbal medicine tambaqui Figures Figure 1 Figure 2 Figure 3 Figure 4 1. INTRODUCTION The intensification of aquaculture production has boosted the growth of the sector, but it has also predisposed fish to a greater susceptibility to pathogens, especially parasitosis. Furthermore, the supply of products and effective protocols remains insufficient to combat these diseases, which cause high rates of mortality and significant economic losses (Moraes et al. 2023 ; FAO 2024 ; Madsen & Stauffer 2024). In this context, Colossoma macropomum , an endemic species of the Amazon and the main fish produced in the region, with increasing relevance in South America, Central America, the Caribbean and Asia (Valladão et al. 2018; Woynarovich & Van Anrooy 2019), depends on technological innovations and new therapeutic alternatives to sustain intensive cultivation systems. Several studies have indicated that the number of authorized drugs that can be used to control the main parasitoses in this species is very low, especially those caused by myxozoans (Vine et al. 2016), acanthocephalans (Andrade-Porto et al. 2021; Morais et al. 2023) and monogenoids (Affonso et al. 2023; Dias et al. 2015 ; Maciel & Affonso 2021 ; De Queiroz et al. 2022 ), which constitutses a major obstacle for increased production and productive expansion. In particular, monogenoids, ectoparasites of the skin and gills, are among the most common, and induce excessive mucus production, hyperplasia, lamella fusion and branchial necrosis, in addition to favoring secondary infections (Jerônimo et al. 2014 ; Noga 2010 ). Due to the negative impacts of these parasites on the productive sector, some chemotherapeutic products (hydrogen peroxide, formalin, mebendazole and praziquantel) have been evaluated for their therapeutic potential in C. macropomum (Andrade-Porto et al. 2017 ; Chagas et al. 2016; Hirazawa et al. 2016 ; Maciel & Affonso 2021 ). However, there is still a considerable range of variation in the results regarding their efficiency in reducing infestations; in addition, the prolonged use of some of these products can favor the development of resistant microorganisms, and serious risks of contamination of the host, the environment and consumers (FAO 2024 ; Ng et al. 2024; Huys et al. 2007). To reduce the farmers dependence on conventional chemicals, the use of extracts, essential oils and substances isolated from plants has shown promise in the control of parasites in aquaculture (De Queiroz et al. 2022 ; Ng et al. 2024). For example, an extract from Euphorbia fischeriana controled Dactylogyrus vastator in Carassius auratus (Zhang et al. 2014 ); Acetone from Bixa orellana and oil from Lipia alba were effective against monogenoids in C. macropomum (Andrade et al. 2016 ; Soares et al. 2017); and oils from Melaleuca alternifolia , Mentha piperita and Copaifera ducke showed effectiveness against Anacanthorus penilabiatus and Mymarothecium viatorum in Piaractus mesopotamicus (Costa et al. 2017 ). Piper aduncum , or monkey pepper, which is widely distributed in the tropics, is highlighted in studies due to its leaf extracts (yield > 17%, with ~ 41% dillapiole) and its essential oil (2.5–3.5%, up to 98% dillapiole), which are rich in prenylated compounds, flavonoids and sesquiterpenes (Corral et al. 2018 ; De Queiroz et al. 2022 ; Efdi et al. 2023 ). Its extracts have shown efficacy against monogenoids in Arapaima gigas and C. macropomum (Queiroz et al. 2022 ) and Hysterothylacium sp. in A. gigas (Corral et al. 2018 ), while the dillapiole isolate exhibits parasiticidal, bactericidal, fungicidal and insecticidal actions (Flores et al. 2009 ; Almeida et al. 2009 ; Dal Picolo et al. 2014 ;), thus evidencing its potential for sustainable disease management. Thus, with the aim of offering a sustainable phytopharmacological alternative, this study evaluated the anthelmintic efficacy of dillapiole and the ethanolic extract of Piper aduncum against monogenoids in juvenile Colossoma macropomum , in addition to investigating their effects on the welfare of the fish. 2. MATERIAL AND METHODS 2.1. Study site and Ethics Committee The experiments were carried out at the Experimental Aquaculture Station of the Laboratory of Physiology Applied to Fish Farming (LAFAP), at the National Institute for Amazonian Research (INPA), Amazonas, Brazil. This work was carried out in accordance with the guidelines published by the National Council for Control of Animal Experimentation (CONCEA 2015 ) and was approved by the Ethics Committee on the Use of Animals (CEUA) of the National Institute for Amazonian Research (INPA) (Process No. 030/2016). 2.2 Acclimation of the fish and monitoring of water quality Juvenile C. macropomum (10 ± 1.0 g) were obtained from a commercial fish farm, and then placed in six 500-L polyethylene tanks of (100 fish/tank), with a low water-renewal rate, constant aeration, temperature of 26.0 ± 0.5 ºC, dissolved oxygen of 6.5 ± 1.0 mg·L ⁻1 and a pH of 6.5 ± 0.5. The fish were fed twice a day for 30 days with a 36% crude protein commercial feed until apparent satiety. At the end of this period, three fish from each tank were removed for parasitological screening before the execution of the experiments. The following physical and chemical variables of the water were determined: dissolved oxygen (DO), temperature and electrical conductivity, with the aid of a digital oximeter (YSI, 85/10); pH, with a digital pH meter (YSI, 60/10); total ammonia concentration (NH₃ + NH₄⁺), according to Verdouw et al. ( 1978 ); nitrite (NO₂⁻) and carbon dioxide (CO₂), according to Boyd and Tucker ( 1992 ). The mean values (± SD) of the water quality variables were temperature 27.70 ± 0.07 ºC; pH 6.38 ± 0.04 (log H⁺); dissolved oxygen 7.11 ± 0.20 mg·L⁻¹; total ammonia 0.676 ± 0.066 mg·L ⁻1 ; CO₂ 17.92 ± 2.22 mg·L ⁻1 ; nitrite 0.005 ± 0.001 mg·L ⁻1 ; and electrical conductivity 51.60 ± 1.60 µS·cm − 1 . During the therapeutic baths, these variables did not present statistical differences between the treatments and were within the recommended limits for C. macropomum (Barroso et al. 2020). 2.3 Screening and determination of in vitro concentrations Initially, to determine the concentrations of crude ethanolic extract and dillapiole, a phytochemical (screening) experiment was performed. Collections, characterization, chemical analysis and toxicity tests of both compounds were performed as described by Queiroz et al. ( 2022 ). In the experiment, the anti-monogenoid activity of P. aduncum was evaluated in the juvenile C. macropomum after five-hour baths, in which four fish (45 ± 10 g) were euthanized by cerebral concussion (CONCEA 2018) and immediately immersed in two concentrations of crude ethanolic extract (20 and 200 mg·L ⁻1 , solubilized in methanol 0.1% v/v) and dillapiole (10 and 100 mg·L ⁻1 , solubilized in Tween-20 0.4% v/v). For each product, two control groups were paired: pure water and vehicle (methanol 0.1% or Tween-20 0.0004% v/v). Every 30 min, the gills were examined under a stereomicroscope (Zeiss, Stemi 2000-C) to record lethargy, detachment of the gills and parasite mortality (Fajer-Ávila et al. 2003), with the percentage of efficacy being calculated according to Martins et al. ( 2001 ) and Onaka et al. ( 2003 ). With the results of this screening, four final concentrations for each product were selected for a quantitative in vitro assay: 40, 60, 80 and 100 mg·L ⁻1 of the ethanolic extract (controls in water and methanol 0.1% v/v) and 15, 30, 60 and 90 mg·L ⁻1 of dillapiole (controls in water and Tween-20 0.0004% v/v). For the assay, ten fish (45 ± 10 g) were euthanized (CONCEA 2018) and subjected to baths of the aforementioned concentrations, repeating the microscopic observation of lethargy, gill detachment and mortality at intervals of 30 min over five hours. The evaluation method remained identical, differing only in the amplitude of the doses tested in order to characterize the efficacy response curve of the ethanolic extract and the dillapiole. 2.4 In vivo therapeutic assays of the ethanolic extract and dillapiole in juvenile C. macropomum 2.4.1 Therapeutic baths For the in vivo assay, we used the acute toxicity test (LC₅₀- 96h ) in non-parasitized C. macropomum , as described by Queiroz et al. ( 2022 a), to ensure that the predicted doses would be sublethal). Based on the results obtained in vitro , 40 and 60 mg·L ⁻1 for the crude ethanolic extract and 28 and 33 mg·L ⁻1 for dillapiole were used in the in vivo therapeutic baths. For this, two tests were performed, each involving twelve 60-L aquariums (total capacity 80 L), containing ten juvenile C. macropomum (48.6 ± 5.0 g). The fish were submitted to two consecutive therapeutic baths of four hours, interspersed by three hours of recovery between baths, in 500-L tanks with continuous aeration and a photoperiod of 12 h/12 h, and a basal group (not parasitized and untreated) was used as a negative control. At the end of the second bath, five fish from each experimental unit were collected for parasitological analysis of the gills and five for histological examination (Marinho-Neto et al. 2019 ; Affonso et al. 2023). The remaining subjects remained in the 500-L fiber tanks for seven days, during which they were monitored daily for mortality, alterations in behavior and clinical signs of abnormal health. 2.4.2 Analysis of antiparasitic activity and identification of monogenoids After the euthanasia of the fish, the gill arches were removed and individualized in Petri dishes for analysis under a stereomicroscope (Zeiss, Stemi 2000-C). After counting the parasites, the gills were fixed in 5% formalin (Eiras et al. 2010 ) for further counting and identification of the species of monogeneoids. The estimate of the percentage of efficacy of the treatments was calculated according to Martins et al. ( 2001 ) and Onaka et al. ( 2003 ). Parasitic indices followed the definitions of Bush et al. ( 1997 ). The monogenoid species were identified according to De Kritsky et al. ( 1979 ), Thatcher and Krytsky (1983), Cohen and Kohn ( 2005 ), Belmont-Jégu et al. ( 2004 ) and Kritsky et al. ( 1996 ). For the study of the sclerotized structures (bars, hooks, anchors of the haptor, copulatory complex), the parasites were mounted on slides with a coverslip, using Grey & Wass for clarification (Thatcher 2006). 2.4.3 Clinical laboratory analysis After the two therapeutic baths of 4 h each, blood samples were collected from nine fish per treatment via caudal vein puncture, using syringes with 10% EDTA, and then stored under refrigeration at 4°C. An aliquot of whole blood was used to determine hematocrit, hemoglobin concentration and hematimetric indices: mean corpuscular volume (MCV = Ht × 10/RBC); mean corpuscular hemoglobin (MCH = Hb × 10/RBC); total blood concentration (TBC); mean corpuscular hemoglobin concentration (MCHC = Hb × 100/Ht) and the erythrocyte count via the indirect method (Costa et al. 2021 ). Leukograms and thrombograms were obtained by blood extensions considering left shift when the proportion of band neutrophils exceeded 3% of the total leukocytes (Claudiano et al. 2019 ). For the biochemical analyses, the determination of total proteins, albumin, magnesium and alanine aminotransferase (ALT) was performed in a semi-automatic analyzer, with the calculation of globulin and the albumin/globulin ratio (Claudiano et al. 2019 ), and glycemia according to Castro et al. ( 2014 ). For plasma ions (Ca 2 ⁺, Na⁺, K⁺ and Mg 2 ⁺), the samples were diluted to 1:1000 (v/v) and quantified using flame spectrophotometry (Perkin-Elmer, AAS 1100B). 2.4.4 Histological analysis of the gills After the baths with the crude extract or dillapiole, the gill structures of 36 fish from each experiment and nine from the basal sample were fixed in 5% buffered formalin for 24 h, thn transferred to 70% alcohol for 48 h. From each gill, the second gill arch was removed and embedded in paraffin (Sigma-Aldrich, Paraplast Plus) in the sagittal position, and serial cuts of 2–5 µm were obtained with the aid of a rotary microtome (Leica, RM 2245). To describe the histopathologies and obtain the histological alteration indices, three histochemical protocols were used: 1) eosin-hematoxylin 2) alcian blue and 3) Schiff's periodic acid. With the results, a descriptive and qualitative analysis was performed to classify the histopathological alterations in the gills of the animals: the mean alteration value (MAV), according to Schwaiger et al. ( 1997 ) and the histological alteration index (HAI), according to Poleksić and Mitrović-Tutundžić ( 1994 ). 2.5 Statistical analysis The results of the parasitological, immunophysiological, histological and water quality analyses were submitted to statistical analysis. Results that presented a normal distribution (Shapiro-Wilk test) and homogeneity between deviations (Levene’s test) were submitted to analysis of variance (ANOVA), followed by Tukey’s test at 5% significance. Those that did not meet the parametric assumptions were submitted to the Kruskal-Wallis test, using the statistical program BioEstat 5.0. 3. RESULTS 3.1 In vitro anti-monogenoid activity of P. aduncum The results of the phytochemical screening demonstrated that there was no mortality of monogenoids in the control groups (water and solvents) during the tests, nor any visible deterioration of the gill tissue of the fish exposed to the products. In the experiment with the crude ethanolic extract of P. aduncum , there was a dose-and time-dependent effect on the antiparasitic efficacy. For the ethanolic extract, at the lowest concentration tested (40 mg·L ⁻1 ), the efficacy gradually increased, reaching about 70% at the end of 5 h of exposure (Fig. 1 a). At concentrations of 60 and 80 mg·L ⁻1 , efficacy was greater than 70% after 3 h, reaching values close to 100% with 5 h of exposure. The highest concentration evaluated (100 mg·L ⁻1 ), with 4 h of exposure, showed an efficacy of 100%, evidencing faster action compared to the other doses (Fig. 1 a). In the test with dillapiole alone, mortality was greater than 90% in the first 2 h at concentrations of 60 and 90 mg·L ⁻1 , reaching 100% after 3 h of exposure. At 30 mg·L ⁻1 , there was intermediate efficacy, with approximately 60% between 4 and 5 h of exposure (Fig. 1 b). The lowest concentration (15 mg·L ⁻1 ) showed low initial efficacy, with a progressive increase up to about 30% at the end of the experimental period (Fig. 1 b). The number of parasites adhered to the gill arches varied significantly between treatments (p < 0.05), with a significant reduction for the highest concentrations of both products (Fig. 1 c and 1 d). In the samples exposed to the crude ethanolic extract (Fig. 1 c), the concentrations 40 and 60 mg·L ⁻1 significantly reduced the mean monogenoid count when compared to the controls (water and methanol), with a decrease that was proportional to the increase in dose. For dillapiole, the concentrations 28 and 33 mg·L -1 also promoted a significant reduction in the number of parasites adhering to the gill arches, confirming the efficacy of the compound, even at intermediate doses (Fig. 1 d). The detailed count of the behavioral alterations caused by the crude ethanolic extract and dillapiole are represented in Tables 1 and 2 , respectively. For the extract, a dose- and time-dependent effect is observed, with intense lethargy occurring in the first 30 min at concentrations of 80 and 100 mg·L ⁻1 , with mortality greater than 80% after 3 h and 100% at the end of 4 h of exposure. At 60 mg·L -1 , some of the parasites remained alive until the third hour and, after 5 h, there was almost total death or detachment from the tissue. At 40 mg·L -1 , the effect was more gradual, with a progressive increase in lethargy and partial mortality, with a significant number of parasites still alive at the end of the assay (Table 1 ). Table 1 Number of gill monogenoids in Colossoma macropomum classified as alive, dead or exhibiting behavioral alterations during the in vitro test with the crude extract of Piper aduncum and different exposure periods. Period Crude extract mg.L − 1 Alive Behavioral alterations Dead Lethargic Detached 0 0 (Water) 37 0 0 0 0 (Methanol) 54 0 0 0 40 70 0 0 0 60 34 0 0 0 80 37 0 0 0 100 35 0 0 0 30 min 0 (Water) 37 0 0 0 0 (Methanol) 54 0 0 0 40 70 25 0 0 60 34 10 0 0 80 37 15 0 0 100 33 20 0 2 1 h 0 (Water) 37 0 0 0 0 (Methanol) 54 0 0 0 40 70 25 0 0 60 22 10 0 12 80 25 15 11 12 100 20 20 10 15 2 h 0 (Water) 37 0 0 0 0 (Methanol) 54 0 0 0 40 70 25 0 0 60 22 13 9 12 80 20 10 11 17 100 20 20 10 15 3 h 0 (Water) 37 0 0 0 0 (Methanol) 54 0 0 0 40 60 40 10 10 60 11 13 9 23 80 8 3 20 29 100 4 4 15 31 4 h 0 (Water) 37 0 0 0 0 (Methanol) 54 0 0 0 40 40 40 30 30 60 11 0 20 23 80 0 0 20 37 100 0 0 0 35 5 h 0 (Water) 37 0 0 0 0 (Methanol) 54 0 0 0 40 40 20 50 30 60 11 0 20 23 80 0 0 20 37 100 0 0 0 35 Values are expressed as absolute counts per observation interval. Controls: water and Tween-20 (0.0004%). Table 2 Number of gill monogenoids in Colossoma macropomum classified as alive, dead or exhibiting behavioral alterations during the in vitro exposure to dillapiole isolated from Piper aduncum and different time intervals. Period Dillapiole (mg/L) Alive Behavioral alterations Dead Lethargic Detached 0 0 (Water) 46 0 0 0 0 (Water + Tween) 50 0 0 0 15 65 0 0 0 30 48 0 0 0 60 40 0 0 0 90 36 0 0 0 30 min 0 (Water) 46 0 0 0 0 (Water + Tween) 50 0 0 0 15 65 0 0 0 30 48 0 0 0 60 40 0 0 0 90 4 4 0 32 1 h 0 (Water) 46 0 0 0 0 (Water + Tween) 50 0 0 0 15 65 0 0 0 30 46 0 0 2 60 35 20 5 5 90 0 0 0 36 2 h 0 (Water) 46 0 0 0 0 (Water + Tween) 50 0 0 0 15 65 0 0 0 30 46 20 5 2 60 20 15 15 20 90 0 0 0 36 3 h 0 (Water) 46 0 0 0 0 (Water + Tween) 50 0 0 0 15 65 0 0 0 30 20 20 10 28 60 0 0 0 40 90 0 0 0 36 4 h 0 (Water) 46 0 0 0 0 (Water + Tween) 50 0 0 0 15 65 0 0 0 30 20 30 20 28 60 0 0 0 40 90 0 0 0 36 5 h 0 (Water) 46 0 0 0 0 (Water + Tween) 50 0 0 0 15 60 30 30 5 30 20 20 20 28 60 0 0 0 40 90 0 0 0 36 Controls: water and water + Tween-20 (0.0004% v/v). In the test with dillapiole (Table 2 ), there was a faster and more intense action on the parasite when compared to the extract. At 90 mg·L -1 , 89% of parasites were dead immediately after 30 min and 100% died at the end of 1 h of exposure. At 60 and 90 mg·L ⁻1 , severe lethargy was observed in the first hour and total mortality up to 3 h. At intermediate concentrations (15 and 30 mg·L -1 ), the effect was more limited and progressive, with part of the parasites becoming lethargic or detached and others still alive at the end of 5 h of exposure (Table 2 ). As shown in Tables 1 and 2 , the remaining specimens showed reduced movement and lethargy from the first evaluation intervals. 3.2 In vivo effect of dillapiole and the crude extract of P. aduncum in C. macropomum In the in vivo tests, three monogenoid species parasitizing C. macropomum were identified: Anacanthorus spathulatus , Notozothecium janauachensi and Mymarothecium boegeri (Table 3 ). In almost all the treatments with the crude ethanolic extract, A. spathulatus presented 100% prevalence, except in 60 mg·L ⁻1 which showed a significant reduction in prevalence (66.6%) and lower mean abundance when compared to the controls (p 0.05; Table 3 ). Table 3. Prevalence and mean abundance of monogenean species ( Anacanthorus spathulatus , Notozothecium janauachensi and Mymarothecium boegeri ) recovered from the gill arches of Colossoma macropomum after in vivo therapeutic baths with the crude extract of and dillapiole from Piper aduncum . Parasitic species Crude extract Control Concentrations (mg.L -1 ) “p” 1 Water Methanol 40 60 P (%) MA P (%) MA P (%) MA P (%) MA A. spathulatus 100 36.55±7.5 b 100 34.11±7.5 b 100 22.22±6.4 b 66.6 3.55±2.49 a * M. boegeri 66.66 1.77±1.35 55.55 1.00±0.88 55.55 0.77±0.86 33.3 0.22±0.34 NS N. janauachensis 77.00 1.22±0.74 66.66 1.00±0.66 55.55 0.55±0.61 33.3 0.33±0.44 NS Parasitic species Dillapiole Control Concentrations (mg.L -1 ) “p” 1 Water Tween-20 28 33 P (%) MA P (%) MA P (%) MA P (%) MA A. spathulatus 100 43.33±5.18 c 100 43.88±5.9 bc 100 22.22±3.58 b 100 13.88±4.32 a *** M. boegeri 88.9 1.88±0.61 b 100 1.77±0.51 b 100 1.55±0.49 ab 55.6 0.44±0.49 a * N. janauachensis 77.8 1.44±1.037 100 1.77±0.69 100 1.22±0.30 77.8 0.88±0.39 NS 1 Different letters on the same rows indicate statistically significant differences between treatments according to Tukey’s test. * indicates p < 0.05; *** indicates p < 0.01; NS indicates no significant differences. P = prevalence; MA = mean abundance; N = 9 fish per treatment. Values are presented as mean ± standard deviation. In the baths with dillapiole, the prevalence of A. spathulatus was 100% in all the treatments, while the mean abundance progressively decreased with increasing concentrations of the product (Table 3 ). A statistically significant difference (p < 0.05) between the controls and the 33 mg·L ⁻1 treatment was also observed in this parasite species. For M. boegeri , prevalence and abundance were also reduced in the test with the highest concentration of dillapiole, presenting significant differences in relation to controls (p < 0.05. Table 3 )). The behavioral analysis indicated that the fish of the control treatments (water, methanol 0.1% and Tween-20 0.0004%) showed normal swimming and respiratory rate during the baths. In contrast, fish exposed to the highest concentration of the extract (60 mg·L ⁻1 ) and the highest concentration of dillapiole (33 mg·L ⁻1 ) showed behavioral alterations, including an increase in the frequency of opercular beats and a reduction in swimming activity. After the seven-day recovery period, no mortality was observed. The fish returned to normal feeding after the second day of recovery, presenting coloration and integrity of the fins similar to the basal group. However, individuals in the control groups exhibited clinical signs such as erosions on the fins and whitish spots. In Table 4 , the hemato-biochemical results of the fish exposed to the crude extract at 60 mg·L⁻¹ demonstrated hyperglycemia (p < 0.05) in relation to the other treatments and to the basal group, as well as significant decreases in total protein and plasma calcium levels at the highest concentrations (p < 0.05). Cortisol levels were significantly higher in fish treated with the extract, especially at a dose of 60 mg·L ⁻1 , presenting statistical differences when compared to the controls and the basal group (Table 4 ). The mean values of thrombocytes and leukocytes were higher in the water control in comparison to the other treatments, except the basal one; there was eosinophilia in all the groups, which was not observed in the basal group, without differences for lymphocytes and monocytes (p < 0.05; Table 4 ). Table 4 Blood parameters of Colossoma macropomum submitted to two four-hour baths with the crude extract of Piper aduncum . Blood variables Controls Crude extract (mg.L − 1 ) “p-value” 1 Basal Water Methanol 40 60 Hematological parameters Hematocrit (%) 23.68 ± 1.85 24.5 ± 1.33 24.11 ± 1.01 22.72 ± 1.25 21.44 ± 0.5 NS Hemoglobin (g/dL − 1 ) 5.59 ± 0.60 6.72 ± 0.59 6.30 ± 0.53 6.13 ± 0.53 5.68 ± 0.29 NS RBC (x10 6 .µL − 1 ) 1.50 ± 0.22 1.47 ± 0.20 1.50 ± 0.28 1.52 ± 0.12 1.47 ± 0.20 NS MCV(fL) 161.36 ± 19.1 169.69 ± 17.24 166.19 ± 25.93 150.11 ± 15.30 148.75 ± 20.17 NS MCHC (g.dL − 1 ) 23.78 ± 2.63 27.62 ± 2.89 26.10 ± 1.60 27.01 ± 2.02 26.55 ± 1.54 NS MCH (pg) 37.92 ± 4.79 46.62 ± 5.5 43.54 ± 7.67 40.84 ± 6.96 39.22 ± 4.50 NS Biochemical parameters Glucose (mg.dL − 1 ) 89.44 ± 11.64 b 55.40 ± 5.66 d 64.25 ± 5.9 cd 71.75 ± 6.67 bc 121.69 ± 26.39 a *** TP (g.dL − 1 ) 2.54 ± 0.44 a 2.23 ± 0.20 ab 2.32 ± 0.14 a 2.23 ± 0.11 ab 1.88 ± 0.15 b * Ca 2+ (mg/L) 1,215.55 ± 21.72 a 997.77 ± 124.69 b 862.22 ± 81.97 b 728.88 ± 72.34 b 550 ± 62.22 c *** Mg 2+ (mg/L) 86.66 ± 8.14 88.88 ± 10.37 84.44 ± 7.16 78.88 ± 5.92 80 ± 6.66 NS Na + (mg/L) 4,012.47 ± 408.13 a 3,618.915 ± 204.06 ab 3,662.644 ± 369.26 ab 3,400.27 ± 310.96 b 3,986.84 ± 430.92 b * K + (mg/L) 376.00 ± 102 423.33 ± 177.7 447.77 ± 118.51 232.22 ± 98.51 267.77 ± 131.35 NS Cortisol (µdL) 67.53 ± 18.05 ab 47.63 ± 17.04 ab 42.93 ± 7.17 b 68.46 ± 6.99 a 69.24 ± 5.3 a * Innate immunity cell count Thrombocytes (x10³.µL − 1 ) 93.00 ± 20.20 ab 155.66 ± 24.86 a 72.00 ± 11.6 b 66.55 ± 10.44 b 90.88 ± 30.06 b * Leukocytes (x10³.µL − 1 ) 5.47 ± 2.73 b 10.59 ± 1.84 a 8.86 ± 1.62 ab 7.89 ± 1.84 b 8.52 ± 2.09 ab * Lymphocytes (x10³.µL − 1 ) 1.76 ± 0.90 ab 2.79 ± 0.81 a 2.47 ± 0.76 ab 1.48 ± 0.56 b 1.82 ± 0.55 ab * Monocytes (x10³.µL − 1 ) 0.97 ± 0.42 ab 1.30 ± 0.30 ab 1.52 ± 0.54 a 0.92 ± 0.22 b 1.23 ± 0.63 ab * Neutrophils (x10³.µL − 1 ) 1.80 ± 0.96 b 3.43 ± 0.96 a 2.72 ± 0.74 ab 2.90 ± 1.02 ab 3.06 ± 1.29 a * Eosinophils (x10³.µL − 1 ) 0.81 ± 0.59 b 2.92 ± 0.70 a 2.09 ± 0.59 a 2.55 ± 0.57 a 2.33 ± 1.64 a *** SGC (x10³.µL − 1 ) 0.10 ± 0.08 0.08 ± 0.05 0.04 ± 0.03 0.03 ± 0.45 0.07 ± 0.05 NS 1 Different letters between the lines indicate statistical differences between treatments on Tukey's test (N = 9): * represents p < 0.05; *** represents p < 0.01; NS represents no statistical differences. Basal = acclimation. RBC = red blood cell count. MCV = mean corpuscular volume. MCHC = mean corpuscular hemoglobin concentration. MCH = mean corpuscular hemoglobin. TP = total proteins; Ca 2 + = calcium, Mg² + = magnesium, Na + = sodium, K + = potassium, SGC = special granulocytic cells. Means ± standard deviation. In Table 5 , two 4-h baths with dillapiole (33 mg·L ⁻1 ) reduced hematocrit, hemoglobin and MCH in comparison with the control (p < 0.05). There was also a reduction in glycemia, a decrease in calcium and potassium and an increase in sodium (p < 0.05), as well as lower plasma cortisol in the treated groups when compared to the controls (p < 0.05). In the leukogram, there was an increase in thrombocytes, total leukocytes and lymphocytes at the two highest concentrations, as well as neutrophilia and eosinophilia in the treated groups, which was not observed in the controls (p < 0.05; Table 5 ). Table 5 Blood parameters of Colossoma macropomum submitted to two four-hour baths with dillapiole. Blood variables Basal Control Dillapiole (mg.L − 1 ) p-value 1 Water Tween 20 28 33 Hematological parameters Hematocrit (%) 23.68 ± 1.85 b 25.55 ± 2.38 a 23.94 ± 1.53 ab 21.72 ± 1.08 bc 20.22 ± 1.62 c *** Hemoglobin (g/dL − 1 ) 5.59 ± 0.60 ab 6.85 ± 0.97 a 6.17 ± 0.87 ab 5.59 ± 0.85 ab 4.95 ± 0.54 b * RBC (x10 6 .µL − 1 ) 1.50 ± 0.22 1.51 ± 0.14 1.71 ± 0.21 1.60 ± 0.17 1.58 ± 0.14 NS MCV(fL) 161.36 ± 19.11 170.71 ± 16.83 142.70 ± 15.98 137.87 ± 13.99 129.19 ± 15.70 NS MCHC (g.dL − 1 ) 23.78 ± 2.63 24.83 ± 2.99 25.11 ± 4.28 25.82 ± 4.36 24.54 ± 2.37 NS MCH (pg) 37.92 ± 4.79 ab 42.31 ± 6.62 a 38.36 ± 6.84 ab 35.53 ± 6.39 ab 31.76 ± 5.12 b * Biochemical parameters Glucose (mg.dL − 1 ) 89.44 ± 11.64 a 49.58 ± 8.57 c 50.23 ± 10.87 c 66.36 ± 13.20 bc 81.70 ± 14.04 bc * TP (g.dL − 1 ) 2.54 ± 0.44 2.35 ± 0.16 2.36 ± 0.14 2.50 ± 0.19 2.51 ± 0.16 NS Ca 2+ (mg/L) 1,215.55 ± 21.72 a 1,215.55 ± 69.38 a 1,166.66 ± 97.77 ab 1,013.33 ± 53.33 b 1045.55 ± 151.85 b * Mg 2+ (mg/L) 86.66 ± 8.14 91.11 ± 8.41 91.11 ± 15.06 92.22 ± 10.86 83.33 ± 17.77 NS Na + (mg/L) 4,012.47 ± 408.13 ab 3,658.177 ± 263.20 b 3,561.662 ± 353.20 b 3,984.90 ± 330.8 ab 4,198.169 ± 108.15 a * K + (mg/L) 376.66 ± 102.22 ab 554.44 ± 128.39 a 487.77 ± 183.70 a 236.66 ± 77.77 b 186.66 ± 106.66 b * Cortisol (µdL) 67.53 ± 18.05 a 59.54 ± 4.26 b 55.93 ± 10.30 b 67.22 ± 3.73 a 46.14 ± 14.47 b * Innate immunity cell count Thrombocytes (x10³.µL − 1 ) 89.66 ± 25.40 c 195.00 ± 47.33 ab 161.55 ± 25.62 bc 178.66 ± 59.40 ab 259 ± 58.22 a * Leukocytes (x10³.µL − 1 ) 5.47 ± 2.73 c 13.68 ± 2.48 a 11.55 ± 1.1.70 bc 15.81 ± 2.28 a 14.361 ± 2.044 ab *** Lymphocytes (x10³.µL − 1 ) 1.76 ± 0.70 b 3.00 ± 0.79 ab 2.33 ± 0.60 b 3.57 ± 0.96 a 3.17 ± 0.91 a * Monocytes (x10³.µL − 1 ) 0.97 ± 0.42 b 1.39 ± 0.28 ab 2.07 ± 0.54 a 2.72 ± 0.71 a 1.49 ± 0.43 ab * Neutrophils (x10³.µL − 1 ) 1.80 ± 0.96 b 5.28 ± 1.27 a 3.56 ± 1.01 ab 5.76 ± 0.76 a 7.09 ± 1.70 a * Eosinophils (x10³.µL − 1 ) 0.81 ± 0.29 c 3.90 ± 0.99 a 3.26 ± 1.08 ab 3.68 ± 0.94 a 2.57 ± 0.77 b *** SGC (x10³.µL − 1 ) 0.005 ± 0.006 0.006 ± 0.005 0.013 ± 0.009 0.006 ± 0.007 0.002 ± 0.004 NS 1 Different letters between the lines indicate statistical differences between treatments on Tukey's test (N = 9): * represents p < 0.05; *** represents p < 0.01; NS represents no statistical differences. Basal = acclimation. RBC = red blood cell count. MCV = mean corpuscular volume. MCHC = mean corpuscular hemoglobin concentration. MCH = mean corpuscular hemoglobin. TP = total proteins; Ca 2 + = calcium, Mg² + = magnesium, Na + = sodium, K + = potassium, SGC = special granulocytic cells. Means ± standard deviation. The histopathological alterations index (HAI) showed significant differences (p < 0.05) between the treatments with 40 and 60 mg·L ⁻1 of the extract and the control with methanol (Fig. 2 a). In the dillapiole experiments, the HAI was higher in the control groups, but did not differ statistically in relation to the treatments and the basal group (Fig. 2 b). Quantitative evaluation of histological parameters indicated variations in the index/mean of the lesions between the crude extract and dillapiole treatments (Fig. 2 c–d). In the crude extract, the mean values ranged from 1.33 to 1.96, with lower means in the treated groups (40–60 mg·L ⁻1 ) compared to the controls with water/methanol; there was no dose-dependent effect nor statistically significant differences with the controls (p > 0.05; Fig. 2 c). Similarly, the mean values of the histological alterations in the gills of fish treated with dillapiole remained similar between the concentrations tested and did not show significant differences when compared to basal group (Fig. 2 d). Histopathological evaluation of C. macropomum gills exposed to the ethanolic extract revealed the classification of lesions with different frequency patterns between the treatments (Fig. 3 ). In the basal group, it was possible to observe branchial lamellae with preserved morphology, without evident alterations (Fig. 3 a), or presenting mild and discrete alterations, such as small areas of hypertrophy and focal hyperplasia (Fig. 3 b). In the controls with water and methanol, the frequency and intensity of lesions increased, with characteristic alterations, including hyperplasia of the lamellar epithelium with partial fusion of secondary lamellae (Fig. 3 c and 3 d), abundant presence of monogenoids adhered to the branchial surface (Fig. 3 c–d), dilatation of the marginal canal (Fig. 3 e) and a marked proliferation of mucosal cells, evidencing the impact of the parasitic infestation. At concentrations of 40 and 60 mg·L ⁻1 of crude extract, there was a reduction in the frequency and severity of these alterations, with decreased hyperplasia and better lamellar organization compared to the controls (Fig. 3 f–h). Some samples showed areas with reduced epithelial thickness and a slight presence of aneurysmal lamellae (Fig. 3 h) and a smaller number of mucous cells and few residual parasites adhered to the arches (Fig. 3 i). In general, the presence of parasites was high in the controls, while in the treatments with 40 and 60 mg·L ⁻1 of the extract and in the basal group the occurrence was low or sporadic (Fig. 3 a, 3 b, 3 g–i). In the histopathological evaluation of the fish of the dillapiole assay, the fish of the control groups (water and Tween-20) showed a higher frequency of epithelial hyperplasia and hypertrophy, associated with partial fusion of the secondary lamellae and an abundant presence of parasites adhered to the gill filaments (Fig. 4 c–d). These alterations also included dilatation of the marginal canal and intense proliferation of mucous cells in the controls. The therapeutic baths reduced the frequency and intensity of these lesions, especially in the bath with 33 mg·L ⁻1 , which showed more preserved lamellar architecture and fewer mucocytes (Fig. 4 f). At the concentration of 28 mg·L⁻¹, the pattern was intermediate, there were still areas of hyperplasia, but these were less extensive than in the controls (Fig. 4 e). The presence of parasites was high in the control groups and practically absent in the fish treated with the highest concentration of dillapiole. In the basal group, gills with preserved morphology or mild and focal alterations were observed (Fig. 4 a-b). 4. DISCUSSION The results of the present study unequivocally demonstrate that the crude ethanolic extract of P. aduncum and its major compound, dillapiole, have a highly effective anti-monogenoid activity, evidenced by the dose- and time-dependent action in the in vitro assays and by the substantial reduction of the parasitic load in the in vivo assays. These findings, gathered in a pioneering way, provide consistent data on the potential of these substances as herbal alternatives in the control of branchial parasites in C. macropomum . Evaluating the toxic effects of drugs on animals, as well as testing their effect directly on parasites, in vitro , are fundamental steps that allow us to determine the therapeutic concentrations to be used, as well as enable us to understand their mechanism of action on parasites when applied in vivo (Affonso et al. 2022; de Queiroz et al. 2022 ). The in vitro tests showed that both the crude extract and dillapiole showed a progressive lethality that was proportional to the increase in concentration and exposure time. Specifically, the extract demonstrated 100% efficacy at a concentration of 100 mg·L ⁻1 after 4 h, and dillapiole, in turn, reached the same level of efficacy in the first hour at a concentration of 90 mg·L ⁻1 , confirming the faster and more intense action of the isolated compound. These results corroborate previous observations by de Queiroz et al. ( 2022 ), who also identified the dose-dependent action of P. aduncum extract against monogenoids in A. gigas and C. macropomum , although previous studies have not directly compared the extract to the dillapiole isolate under controlled laboratory conditions. The comparison between the two products suggests that dillapiole plays a central role in anthelmintic action, a hypothesis supported by the phytochemical analyses that indicate its predominance (up to 98%) in the essential oil (Almeida et al. 2009 ; de Queiroz et al. 2022 ). However, it cannot be ruled out that other secondary metabolites present in the crude extract, including chromenes, flavonoids, alkaloids and monoterpenes (Efdi et al. 2023 ), exert synergistic or potentiating effects on the parasites, increasing the biological activity of the product, as already proposed by Corral et al. ( 2018 ) and Andrade et al. ( 2016 ). The mechanism of action of dillapiole on monogenoids remains unclear. However, studies into phenylpropanoids demonstrate that these molecules have the ability to interact with the lipids of cell and mitochondrial membranes, compromising the integrity of the lipid bilayer, increasing ion permeability and leading to cell death (Carson et al. 2002 ). In addition, dillapiole is recognized as a potent inhibitor of cytochrome P450 complex enzymes (Omura and Sato, 1964 ; Mukerjee et al. 1979 ), and interferes with oxidative and biotransformative processes of the organisms. This metabolic inhibition may have contributed to the lethal efficacy on the parasites, which was observed even at low concentrations and during reduced exposure times, a desirable characteristic for the rapid control of infestations in an intensive farming environment. The relevance of these findings becomes even more evident when one considers that alternatives for monogenoid control have limitations that are associated with environmental toxicity, residue formation and potential induction of resistance (Guardone et al. 2022). In Brazilian aquaculture, chemotherapeutic treatments, such as formalin and albendazole, have legal restrictions and prolonged withdrawal periods (Cordeiro et al. 2021 ). In this context, plant products, such as dillapiole and the crude extract of P. aduncum , represent a strategic alternative for safer and more effective disease management and are aligned with the principles of sustainability. In the in vivo test, the applications of therapeutic baths in successive doses resulted in a significant reduction in the prevalence and mean abundance of A. spathulatus , the main parasite species identified in the fish studied, in addition to lower occurrences of N. janauachensi and M. boegeri . This pattern confirms that chronic infestation in C. macropomum can be efficiently controlled with 4-h bathing protocols at moderate concentrations, a strategy already proposed by Andrade et al. ( 2016 ) for plant extracts. The positive effects on the parasitic load were accompanied by the recovery of feeding behavior and integrity of the fins, especially noticeable in the groups treated with dillapiole at 33 mg·L − 1 and with the crude extract at 60 mg·L − 1 , demonstrating a positive effect on animal welfare. The hematological and biochemical analyses provided important information about the tolerance of fish to the treatments. While in the crude extract hyperglycemia and increased cortisol levels occurred at higher concentrations, dillapiole showed lower glycemic elevation and a more controlled response to stress (Affonso et al. 2009 ). This finding may be associated with the anesthetic effect described for concentrated dillapiole, which reduces oxygen consumption, basal metabolism and, consequently, the release of catecholamines in the face of stressful stimuli (Lemos et al. 2023). Furthermore, treatment with 33 mg·L ⁻1 of dillapiole resulted in decreased erythrocyte parameters, including a drop in mean corpuscular hemoglobin and hematocrit, suggesting a picture of iron deficiency anemia, potentially resulting from interference in the absorption of iron and essential ions (Marshall 2002 ; Evans et al. 2005 ). The concomitant drop in calcium and potassium levels reinforces this hypothesis, since the active transport of these ions by the mitochondrial cells of the gills is fundamental for ionic balance and the physiological integrity of the fish (Perry et al. 2003 ; Baldisserotto 2025 ). The leukocyte parameters revealed distinct modulations: in the fish exposed to baths with the crude extract. There was an improvement in response and, in both treatments ( P. aduncum extract and dillapiole), there was an inflammatory response that is characteristic of parasitosis, with the elevation of neutrophils and eosinophils – cells associated with defense against parasites and inflammatory processes (Claudiano et al. 2019 ). These findings suggest that both phytochemicals can exert immunomodulatory action, thus expanding their applicability as prophylactic agents (Mansoori et al. 2024). The histological findings corroborate the effectiveness of the treatments. In the controls, the presence of hypertrophy, hyperplasia, lamellar fusion and necrosis, and the abundance of parasites are alterations that are compatible with the response to intense infestations (Vanhove et al. 2024 ). In fish treated with the crude extract and dillapiole, especially in the highest concentrations, the frequency and severity of the lesions were reduced, as well as the histopathological alterations index (HAI) when compared to the controls. This confirms that the reduction in the parasitic load is associated with tissue preservation and lower respiratory impairment, unlike what was observed in treatments with synthetic agents such as glyphosate, which, although effective in eliminating parasites, caused extensive lesions in the gills. As a result, this work advances previous studies by demonstrating that the use of dillapiole and the crude extract of P. aduncum not only promotes the effective removal of monogenoids, but also favors homeostasis and animal welfare. This is evidenced by: (i) an organized inflammatory response that is typical of parasitoses (neutrophilia/eosinophilia), (ii) a serum biochemical profile maintained within physiological ranges, (iii) a hormonal modulation (cortisol) that is compatible with the maintenance of homeostasis, (iv) branchial histopathological findings with reduced scores/no relevant differences when compared to the controls and (v) adequate behavior during and after baths, even after successive therapeutic cycles. Together, this confirms that the extract and dillapiole are viable and promising herbal alternatives, able to integrate biosecurity and disease control programs for C. macropomum in intensive systems, while also being aligned with the demand for sustainable aquaculture and the reduction in the use of conventional chemotherapeutics (Ali et al. 2021; Norbury et al. 2022; Freitas et al. 2023). 5. CONCLUSION The results suggest that both products, dillapiole and the crude ethanolic extract of P. aduncum , have an effective and safe anthelmintic effect in the control of the monogenoids A. spathulatus , N. janauachensis and M. boegeri in juvenile C. macropomum , with up to 99% efficacy when using 60 mg·L ⁻1 of the extract and 85% when using 33 mg·L ⁻1 of dillapiole. In addition to the significant reduction of the parasitic load, the treatments preserved the physiological homeostasis of the fish, which showed behavioral recovery and integrity of the gill tissues after recovery. Taken together, the findings support the practical applicability of these herbal medicines as biosecurity components in intensive farming systems, with the potential to reduce the use of conventional chemotherapies. Declarations Data availability statement: No data are available for this study. Funding statement: Fundação de Amparo à Pesquisa do Estado do Amazonas, PROCAD AMAZONIA/CAPES (88881.200614/2018-01) e INCT-ADAPTA III/Conselho Nacional de Desenvolvimento Científico e Tecnológico (Process 409202/2024-0). Conflict of interest disclosure: The authors declare that there are no conflicts of interest. Ethics approval statement: This study was approved by the Ethics Committee on Animal Use, INPA, No. 030/2016. Patient consent statement: Not applicable. Permission to reproduce material from other sources: Not applicable. Acknowledgments The authors would like to thank Universidade Nilton Lins (UNL) and Instituto Nacional de Pesquisas da Amazônia (INPA). M.N. Queiroz also thanks Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for the grant. Funding This study was partially funded by Fundação de Amparo à Pesquisa do Estado do Amazonas via PROCAD AMAZONIA/CAPES (88881.200614/2018-01) and INCT-ADAPTA III/Conselho Nacional de Desenvolvimento Científico e Tecnológico (Process 409202/2024-0). The authors also thank Dr. Francisco Celio Maia Chave (Embrapa Amazônia Ocidental, Amazonas, Brazil) for supplying the raw material for the preparation of the plant extract. Author contributions : Marieta Nascimento de Queiroz: Conceptualization; Methodology; Investigation; Formal analysis; Writing – original draft. Paulo Adelino de Medeiros: Methodology; Investigation; Resources; Data curation; Writing – review & editing. Zelina Estevam dos Santos Torres: Investigation; Data curation; Validation; Writing – review & editing. Adrian Martin Pohlit: Formal analysis; Visualization; Writing – review & editing. Valdelira Lia Araújo Fernandes: Investigation; Resources; Writing – review & editing. 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J Aquat Ecosyst Stress Recover 6(1):75–86 Soares BV, Neves LR, Ferreira DO, Oliveira MSB, Chaves FCM, Chagas EC, Gonçalves RA, Tavares-Dias M (2016) Antiparasitic activity, histopathology and physiology of Colossoma macropomum (tambaqui) exposed to the essential oil of Lippia sidoides (Verbenaceae). Vet Parasitol 234:49–56 Thatcher VE, Kritsky DC (1983) Neotropical Monogenoidea. 4. Linguadactyloides brinkmanni gen. et sp. n. (Dactylogyridae: Linguadactyloidinae subfam. nov.) with observations on the pathology in Colossoma macropomum. Proc. Helminthol. Soc. Wash. 50:305–311 Vanhove MPM, Pariselle A, Kmentová N (2024) Monogenean parasitic flatworms. Curr Biol 34(22):R1122–R1124. https://doi.org/10.1016/j.cub.2024.10.033 Verdouw H, Van Echted CJA, Dekkers EMJ (1978) Ammonia determination based on indophenol formation with sodium salicylate. Water Res 12:339–402. https://doi.org/10.1016/0043-1354(78)90107-0 Videira M, Velasco M, Malcher CS, Santos P, Matos P, Matos E (2016) An outbreak of myxozoan parasites in farmed freshwater fish Colossoma macropomum in the Amazon region, Brazil. Aquac Rep 3:31–34 Vila R, Tomi F, Mundina M, Santana AI, Solís PN, López Arce JB, Balderrama Iclina JL, Iglesias J, Gupta MP, Casanova J, Cañigueral S (2005) Unusual composition of the essential oils from the leaves of Piper aduncum. Flavour Fragr J 20(1):67–69 Zhang XP, Li WX, Ai TS, Zou H, Wu SG, Wang GT (2014) The efficacy of four common anthelmintic drugs and traditional Chinese medicinal plant extracts to control Dactylogyrus vastator (Monogenea). Aquaculture 420:302–307. https://doi.org/10.1016/j.aquaculture.2013.09.022 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 08 Oct, 2025 Reviews received at journal 07 Oct, 2025 Reviews received at journal 01 Oct, 2025 Reviews received at journal 27 Sep, 2025 Reviewers agreed at journal 16 Sep, 2025 Reviewers agreed at journal 12 Sep, 2025 Reviewers agreed at journal 12 Sep, 2025 Reviewers invited by journal 12 Sep, 2025 Editor assigned by journal 12 Sep, 2025 Submission checks completed at journal 12 Sep, 2025 First submitted to journal 08 Sep, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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1","display":"","copyAsset":false,"role":"figure","size":95059,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eIn vitro\u003c/em\u003e efficacy of \u003cem\u003ePiper aduncum\u003c/em\u003e-based products against gill monogenoids \u0026nbsp;of \u003cem\u003eColossoma macropomum\u003c/em\u003e. (A) Percentage of parasite mortality over time after exposure to the crude ethanolic extract at concentrations of 40, 60, 80, and 100 mg·L⁻¹ compared to controls (water and methanol 0.1%). (B) Percentage of parasite mortality over time after exposure to dillapiole at concentrations of 15, 30, 60, and 90 mg·L⁻¹ compared to controls (water and Tween-20 0.0004%). (C) Mean number of monogenoids adhered to gill arches after exposure to crude extract. (D) Mean number of monogenoids adhered to gill arches after exposure to dillapiole. Different letters above the bars indicate statistically significant differences between treatments (p \u0026lt; 0.05). Values represent means ± standard error.\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7567560/v1/91cfa58c0a48450d7639194f.jpg"},{"id":91867686,"identity":"ba95a56d-f8e9-49ee-9ef1-5d3317c7170c","added_by":"auto","created_at":"2025-09-22 13:27:08","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":90710,"visible":true,"origin":"","legend":"\u003cp\u003eMean histological alterations and histopathological alteration index (HAI) in the gills of \u003cem\u003eColossoma macropomum\u003c/em\u003e after \u003cem\u003ein vivo\u003c/em\u003e baths with \u003cem\u003ePiper aduncum\u003c/em\u003e-based treatments. (A) Histopathological alteration index in treatments with crude extract. (B) Histopathological alteration index in treatments with dillapiole. (C) Mean values of histological alterations in treatments with crude extract. (D) Mean values of histological alterations in treatments with dillapiole. Bars represent mean ± standard deviation. Different letters above the columns indicate statistically significant differences between treatments, according to ANOVA, followed by Tukey’s test (p \u0026lt; 0.05). HAI — Histopathological alteration index.\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7567560/v1/c61f5232dfb0f4e29ca68102.jpg"},{"id":91868111,"identity":"3c93f2ba-59be-4a36-9974-efe52b6e6046","added_by":"auto","created_at":"2025-09-22 13:35:08","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":403397,"visible":true,"origin":"","legend":"\u003cp\u003eHistopathological alterations in the gills of \u003cem\u003eColossoma macropomum\u003c/em\u003e exposed to the ethanolic extract of \u003cem\u003ePiper aduncum\u003c/em\u003e(paraffin sections, 20 μm thick, objective lens ×40).\u003c/p\u003e\n\u003cp\u003ea) Basal group showing preserved gill architecture without lesions.\u003c/p\u003e\n\u003cp\u003eb) Basal group with mild focal epithelial alterations.\u003c/p\u003e\n\u003cp\u003ec) Control (water): hyperplasia with partial fusion of secondary lamellae and presence of parasites.\u003c/p\u003e\n\u003cp\u003ed) Control (water): hyperplasia with lamellar fusion and abundant gill parasites.\u003c/p\u003e\n\u003cp\u003ee) Control (water): hyperplasia with lamellar fusion and dilation of the marginal channel.\u003c/p\u003e\n\u003cp\u003ef) Control (methanol): hyperplasia with lamellar fusion and parasite adhesion.\u003c/p\u003e\n\u003cp\u003eg) 40 mg·L⁻¹: reduction of hyperplasia.\u003c/p\u003e\n\u003cp\u003eh) 40 mg·L⁻¹: reduction of hyperplasia and presence of lamellar aneurysm.\u003c/p\u003e\n\u003cp\u003ei) 60 mg·L⁻¹: reduction of hyperplasia and increased mucous cell proliferation.\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7567560/v1/4308c9dce6ed1f625c27a895.jpg"},{"id":91868113,"identity":"76715528-1df0-4103-95b2-9b2b01bbf2b4","added_by":"auto","created_at":"2025-09-22 13:35:08","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":304243,"visible":true,"origin":"","legend":"\u003cp\u003eHistopathological alterations in the gills of \u003cem\u003eColossoma macropomum\u003c/em\u003e exposed to dillapiole (paraffin sections, 20 μm thick).\u003c/p\u003e\n\u003cp\u003ea) Basal group showing preserved gill architecture without lesions.\u003c/p\u003e\n\u003cp\u003eb) Basal group with mild epithelial alterations.\u003c/p\u003e\n\u003cp\u003ec) Control (water): hyperplasia with partial fusion of secondary lamellae and presence of parasites.\u003c/p\u003e\n\u003cp\u003ed) Control (Tween-20): hyperplasia with partial lamellar fusion and widening of the marginal channel.\u003c/p\u003e\n\u003cp\u003ee) 28 mg·L⁻¹: reduction of lamellar hyperplasia compared to controls.\u003c/p\u003e\n\u003cp\u003ef) 33 mg·L⁻¹: reduction of hyperplasia and increased mucous cell proliferation.\u003c/p\u003e","description":"","filename":"Picture4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7567560/v1/1b93d0b3afb446c1a8950e5a.jpg"},{"id":91963746,"identity":"8e31806d-549a-4927-bebd-a080ce00b717","added_by":"auto","created_at":"2025-09-23 08:10:31","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2619794,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7567560/v1/6f1a4566-e885-42c6-a43f-bf7baae6cb4d.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Anthelmintic efficacy of dillapiole and the crude extract of Piper aduncum in the control of monogenoids in Colossoma macropomum","fulltext":[{"header":"1. INTRODUCTION","content":"\u003cp\u003eThe intensification of aquaculture production has boosted the growth of the sector, but it has also predisposed fish to a greater susceptibility to pathogens, especially parasitosis. Furthermore, the supply of products and effective protocols remains insufficient to combat these diseases, which cause high rates of mortality and significant economic losses (Moraes et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; FAO \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Madsen \u0026amp; Stauffer 2024).\u003c/p\u003e\u003cp\u003eIn this context, \u003cem\u003eColossoma macropomum\u003c/em\u003e, an endemic species of the Amazon and the main fish produced in the region, with increasing relevance in South America, Central America, the Caribbean and Asia (Vallad\u0026atilde;o et al. 2018; Woynarovich \u0026amp; Van Anrooy 2019), depends on technological innovations and new therapeutic alternatives to sustain intensive cultivation systems. Several studies have indicated that the number of authorized drugs that can be used to control the main parasitoses in this species is very low, especially those caused by myxozoans (Vine et al. 2016), acanthocephalans (Andrade-Porto et al. 2021; Morais et al. 2023) and monogenoids (Affonso et al. 2023; Dias et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Maciel \u0026amp; Affonso \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; De Queiroz et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), which constitutses a major obstacle for increased production and productive expansion. In particular, monogenoids, ectoparasites of the skin and gills, are among the most common, and induce excessive mucus production, hyperplasia, lamella fusion and branchial necrosis, in addition to favoring secondary infections (Jer\u0026ocirc;nimo et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Noga \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Due to the negative impacts of these parasites on the productive sector, some chemotherapeutic products (hydrogen peroxide, formalin, mebendazole and praziquantel) have been evaluated for their therapeutic potential in \u003cem\u003eC. macropomum\u003c/em\u003e (Andrade-Porto et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Chagas et al. 2016; Hirazawa et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Maciel \u0026amp; Affonso \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). However, there is still a considerable range of variation in the results regarding their efficiency in reducing infestations; in addition, the prolonged use of some of these products can favor the development of resistant microorganisms, and serious risks of contamination of the host, the environment and consumers (FAO \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Ng et al. 2024; Huys et al. 2007).\u003c/p\u003e\u003cp\u003eTo reduce the farmers dependence on conventional chemicals, the use of extracts, essential oils and substances isolated from plants has shown promise in the control of parasites in aquaculture (De Queiroz et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Ng et al. 2024). For example, an extract from \u003cem\u003eEuphorbia fischeriana\u003c/em\u003e controled \u003cem\u003eDactylogyrus vastator\u003c/em\u003e in \u003cem\u003eCarassius auratus\u003c/em\u003e (Zhang et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2014\u003c/span\u003e); Acetone from \u003cem\u003eBixa orellana\u003c/em\u003e and oil from \u003cem\u003eLipia alba\u003c/em\u003e were effective against monogenoids in \u003cem\u003eC. macropomum\u003c/em\u003e (Andrade et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Soares et al. 2017); and oils from \u003cem\u003eMelaleuca alternifolia\u003c/em\u003e, \u003cem\u003eMentha piperita\u003c/em\u003e and \u003cem\u003eCopaifera ducke\u003c/em\u003e showed effectiveness against \u003cem\u003eAnacanthorus penilabiatus\u003c/em\u003e and \u003cem\u003eMymarothecium viatorum\u003c/em\u003e in \u003cem\u003ePiaractus mesopotamicus\u003c/em\u003e (Costa et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cem\u003ePiper aduncum\u003c/em\u003e, or monkey pepper, which is widely distributed in the tropics, is highlighted in studies due to its leaf extracts (yield\u0026thinsp;\u0026gt;\u0026thinsp;17%, with ~\u0026thinsp;41% dillapiole) and its essential oil (2.5\u0026ndash;3.5%, up to 98% dillapiole), which are rich in prenylated compounds, flavonoids and sesquiterpenes (Corral et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; De Queiroz et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Efdi et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Its extracts have shown efficacy against monogenoids in \u003cem\u003eArapaima gigas\u003c/em\u003e and \u003cem\u003eC. macropomum\u003c/em\u003e (Queiroz et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) and \u003cem\u003eHysterothylacium\u003c/em\u003e sp. in \u003cem\u003eA. gigas\u003c/em\u003e (Corral et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), while the dillapiole isolate exhibits parasiticidal, bactericidal, fungicidal and insecticidal actions (Flores et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Almeida et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Dal Picolo et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2014\u003c/span\u003e;), thus evidencing its potential for sustainable disease management.\u003c/p\u003e\u003cp\u003eThus, with the aim of offering a sustainable phytopharmacological alternative, this study evaluated the anthelmintic efficacy of dillapiole and the ethanolic extract of \u003cem\u003ePiper aduncum\u003c/em\u003e against monogenoids in juvenile \u003cem\u003eColossoma macropomum\u003c/em\u003e, in addition to investigating their effects on the welfare of the fish.\u003c/p\u003e"},{"header":"2. MATERIAL AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Study site and Ethics Committee\u003c/h2\u003e\u003cp\u003eThe experiments were carried out at the Experimental Aquaculture Station of the Laboratory of Physiology Applied to Fish Farming (LAFAP), at the National Institute for Amazonian Research (INPA), Amazonas, Brazil. This work was carried out in accordance with the guidelines published by the National Council for Control of Animal Experimentation (CONCEA \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) and was approved by the Ethics Committee on the Use of Animals (CEUA) of the National Institute for Amazonian Research (INPA) (Process No. 030/2016).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Acclimation of the fish and monitoring of water quality\u003c/h2\u003e\u003cp\u003eJuvenile \u003cem\u003eC. macropomum\u003c/em\u003e (10\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0 g) were obtained from a commercial fish farm, and then placed in six 500-L polyethylene tanks of (100 fish/tank), with a low water-renewal rate, constant aeration, temperature of 26.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 \u0026ordm;C, dissolved oxygen of 6.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0 mg\u0026middot;L\u003csup\u003e⁻1\u003c/sup\u003e and a pH of 6.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5. The fish were fed twice a day for 30 days with a 36% crude protein commercial feed until apparent satiety. At the end of this period, three fish from each tank were removed for parasitological screening before the execution of the experiments.\u003c/p\u003e\u003cp\u003eThe following physical and chemical variables of the water were determined: dissolved oxygen (DO), temperature and electrical conductivity, with the aid of a digital oximeter (YSI, 85/10); pH, with a digital pH meter (YSI, 60/10); total ammonia concentration (NH₃ + NH₄⁺), according to Verdouw et al. (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e1978\u003c/span\u003e); nitrite (NO₂⁻) and carbon dioxide (CO₂), according to Boyd and Tucker (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1992\u003c/span\u003e). The mean values (\u0026plusmn;\u0026thinsp;SD) of the water quality variables were temperature 27.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07 \u0026ordm;C; pH 6.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 (log H⁺); dissolved oxygen 7.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20 mg\u0026middot;L⁻\u0026sup1;; total ammonia 0.676\u0026thinsp;\u0026plusmn;\u0026thinsp;0.066 mg\u0026middot;L\u003csup\u003e⁻1\u003c/sup\u003e; CO₂ 17.92\u0026thinsp;\u0026plusmn;\u0026thinsp;2.22 mg\u0026middot;L\u003csup\u003e⁻1\u003c/sup\u003e; nitrite 0.005\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001 mg\u0026middot;L\u003csup\u003e⁻1\u003c/sup\u003e; and electrical conductivity 51.60\u0026thinsp;\u0026plusmn;\u0026thinsp;1.60 \u0026micro;S\u0026middot;cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. During the therapeutic baths, these variables did not present statistical differences between the treatments and were within the recommended limits for \u003cem\u003eC. macropomum\u003c/em\u003e (Barroso et al. 2020).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Screening and determination of in vitro concentrations\u003c/h2\u003e\u003cp\u003eInitially, to determine the concentrations of crude ethanolic extract and dillapiole, a phytochemical (screening) experiment was performed. Collections, characterization, chemical analysis and toxicity tests of both compounds were performed as described by Queiroz et al. (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In the experiment, the anti-monogenoid activity of \u003cem\u003eP. aduncum\u003c/em\u003e was evaluated in the juvenile \u003cem\u003eC. macropomum\u003c/em\u003e after five-hour baths, in which four fish (45\u0026thinsp;\u0026plusmn;\u0026thinsp;10 g) were euthanized by cerebral concussion (CONCEA 2018) and immediately immersed in two concentrations of crude ethanolic extract (20 and 200 mg\u0026middot;L\u003csup\u003e⁻1\u003c/sup\u003e, solubilized in methanol 0.1% v/v) and dillapiole (10 and 100 mg\u0026middot;L\u003csup\u003e⁻1\u003c/sup\u003e, solubilized in Tween-20 0.4% v/v). For each product, two control groups were paired: pure water and vehicle (methanol 0.1% or Tween-20 0.0004% v/v). Every 30 min, the gills were examined under a stereomicroscope (Zeiss, Stemi 2000-C) to record lethargy, detachment of the gills and parasite mortality (Fajer-\u0026Aacute;vila et al. 2003), with the percentage of efficacy being calculated according to Martins et al. (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2001\u003c/span\u003e) and Onaka et al. (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2003\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eWith the results of this screening, four final concentrations for each product were selected for a quantitative \u003cem\u003ein vitro\u003c/em\u003e assay: 40, 60, 80 and 100 mg\u0026middot;L\u003csup\u003e⁻1\u003c/sup\u003e of the ethanolic extract (controls in water and methanol 0.1% v/v) and 15, 30, 60 and 90 mg\u0026middot;L\u003csup\u003e⁻1\u003c/sup\u003e of dillapiole (controls in water and Tween-20 0.0004% v/v). For the assay, ten fish (45\u0026thinsp;\u0026plusmn;\u0026thinsp;10 g) were euthanized (CONCEA 2018) and subjected to baths of the aforementioned concentrations, repeating the microscopic observation of lethargy, gill detachment and mortality at intervals of 30 min over five hours. The evaluation method remained identical, differing only in the amplitude of the doses tested in order to characterize the efficacy response curve of the ethanolic extract and the dillapiole.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 In vivo therapeutic assays of the ethanolic extract and dillapiole in juvenile C. macropomum\u003c/h2\u003e\u003cdiv id=\"Sec7\" class=\"Section3\"\u003e\u003ch2\u003e2.4.1 Therapeutic baths\u003c/h2\u003e\u003cp\u003eFor the \u003cem\u003ein vivo\u003c/em\u003e assay, we used the acute toxicity test (LC₅₀-\u003csub\u003e96h\u003c/sub\u003e) in non-parasitized \u003cem\u003eC. macropomum\u003c/em\u003e, as described by Queiroz et al. (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2022\u003c/span\u003ea), to ensure that the predicted doses would be sublethal). Based on the results obtained \u003cem\u003ein vitro\u003c/em\u003e, 40 and 60 mg\u0026middot;L\u003csup\u003e⁻1\u003c/sup\u003e for the crude ethanolic extract and 28 and 33 mg\u0026middot;L\u003csup\u003e⁻1\u003c/sup\u003e for dillapiole were used in the \u003cem\u003ein vivo\u003c/em\u003e therapeutic baths. For this, two tests were performed, each involving twelve 60-L aquariums (total capacity 80 L), containing ten juvenile \u003cem\u003eC. macropomum\u003c/em\u003e (48.6\u0026thinsp;\u0026plusmn;\u0026thinsp;5.0 g). The fish were submitted to two consecutive therapeutic baths of four hours, interspersed by three hours of recovery between baths, in 500-L tanks with continuous aeration and a photoperiod of 12 h/12 h, and a basal group (not parasitized and untreated) was used as a negative control. At the end of the second bath, five fish from each experimental unit were collected for parasitological analysis of the gills and five for histological examination (Marinho-Neto et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Affonso et al. 2023). The remaining subjects remained in the 500-L fiber tanks for seven days, during which they were monitored daily for mortality, alterations in behavior and clinical signs of abnormal health.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section3\"\u003e\u003ch2\u003e2.4.2 Analysis of antiparasitic activity and identification of monogenoids\u003c/h2\u003e\u003cp\u003eAfter the euthanasia of the fish, the gill arches were removed and individualized in Petri dishes for analysis under a stereomicroscope (Zeiss, Stemi 2000-C). After counting the parasites, the gills were fixed in 5% formalin (Eiras et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) for further counting and identification of the species of monogeneoids. The estimate of the percentage of efficacy of the treatments was calculated according to Martins et al. (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2001\u003c/span\u003e) and Onaka et al. (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). Parasitic indices followed the definitions of Bush et al. (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e1997\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe monogenoid species were identified according to De Kritsky et al. (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e1979\u003c/span\u003e), Thatcher and Krytsky (1983), Cohen and Kohn (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2005\u003c/span\u003e), Belmont-J\u0026eacute;gu et al. (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2004\u003c/span\u003e) and Kritsky et al. (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e1996\u003c/span\u003e). For the study of the sclerotized structures (bars, hooks, anchors of the haptor, copulatory complex), the parasites were mounted on slides with a coverslip, using Grey \u0026amp; Wass for clarification (Thatcher 2006).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section3\"\u003e\u003ch2\u003e2.4.3 Clinical laboratory analysis\u003c/h2\u003e\u003cp\u003eAfter the two therapeutic baths of 4 h each, blood samples were collected from nine fish per treatment via caudal vein puncture, using syringes with 10% EDTA, and then stored under refrigeration at 4\u0026deg;C. An aliquot of whole blood was used to determine hematocrit, hemoglobin concentration and hematimetric indices: mean corpuscular volume (MCV\u0026thinsp;=\u0026thinsp;Ht \u0026times; 10/RBC); mean corpuscular hemoglobin (MCH\u0026thinsp;=\u0026thinsp;Hb \u0026times; 10/RBC); total blood concentration (TBC); mean corpuscular hemoglobin concentration (MCHC\u0026thinsp;=\u0026thinsp;Hb \u0026times; 100/Ht) and the erythrocyte count via the indirect method (Costa et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Leukograms and thrombograms were obtained by blood extensions considering left shift when the proportion of band neutrophils exceeded 3% of the total leukocytes (Claudiano et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eFor the biochemical analyses, the determination of total proteins, albumin, magnesium and alanine aminotransferase (ALT) was performed in a semi-automatic analyzer, with the calculation of globulin and the albumin/globulin ratio (Claudiano et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), and glycemia according to Castro et al. (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). For plasma ions (Ca\u003csup\u003e2\u003c/sup\u003e⁺, Na⁺, K⁺ and Mg\u003csup\u003e2\u003c/sup\u003e⁺), the samples were diluted to 1:1000 (v/v) and quantified using flame spectrophotometry (Perkin-Elmer, AAS 1100B).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section3\"\u003e\u003ch2\u003e2.4.4 Histological analysis of the gills\u003c/h2\u003e\u003cp\u003eAfter the baths with the crude extract or dillapiole, the gill structures of 36 fish from each experiment and nine from the basal sample were fixed in 5% buffered formalin for 24 h, thn transferred to 70% alcohol for 48 h. From each gill, the second gill arch was removed and embedded in paraffin (Sigma-Aldrich, Paraplast Plus) in the sagittal position, and serial cuts of 2\u0026ndash;5 \u0026micro;m were obtained with the aid of a rotary microtome (Leica, RM 2245). To describe the histopathologies and obtain the histological alteration indices, three histochemical protocols were used: 1) eosin-hematoxylin 2) alcian blue and 3) Schiff's periodic acid. With the results, a descriptive and qualitative analysis was performed to classify the histopathological alterations in the gills of the animals: the mean alteration value (MAV), according to Schwaiger et al. (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e1997\u003c/span\u003e) and the histological alteration index (HAI), according to Poleksić and Mitrović-Tutundžić (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e1994\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Statistical analysis\u003c/h2\u003e\u003cp\u003eThe results of the parasitological, immunophysiological, histological and water quality analyses were submitted to statistical analysis. Results that presented a normal distribution (Shapiro-Wilk test) and homogeneity between deviations (Levene\u0026rsquo;s test) were submitted to analysis of variance (ANOVA), followed by Tukey\u0026rsquo;s test at 5% significance. Those that did not meet the parametric assumptions were submitted to the Kruskal-Wallis test, using the statistical program BioEstat 5.0.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. RESULTS","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003e3.1 In vitro anti-monogenoid activity of P. aduncum\u003c/h2\u003e\n \u003cp\u003eThe results of the phytochemical screening demonstrated that there was no mortality of monogenoids in the control groups (water and solvents) during the tests, nor any visible deterioration of the gill tissue of the fish exposed to the products.\u003c/p\u003e\n \u003cp\u003eIn the experiment with the crude ethanolic extract of \u003cem\u003eP. aduncum\u003c/em\u003e, there was a dose-and time-dependent effect on the antiparasitic efficacy. For the ethanolic extract, at the lowest concentration tested (40 mg\u0026middot;L\u003csup\u003e⁻1\u003c/sup\u003e), the efficacy gradually increased, reaching about 70% at the end of 5 h of exposure (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ea). At concentrations of 60 and 80 mg\u0026middot;L\u003csup\u003e⁻1\u003c/sup\u003e, efficacy was greater than 70% after 3 h, reaching values close to 100% with 5 h of exposure. The highest concentration evaluated (100 mg\u0026middot;L\u003csup\u003e⁻1\u003c/sup\u003e), with 4 h of exposure, showed an efficacy of 100%, evidencing faster action compared to the other doses (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ea). In the test with dillapiole alone, mortality was greater than 90% in the first 2 h at concentrations of 60 and 90 mg\u0026middot;L\u003csup\u003e⁻1\u003c/sup\u003e, reaching 100% after 3 h of exposure. At 30 mg\u0026middot;L\u003csup\u003e⁻1\u003c/sup\u003e, there was intermediate efficacy, with approximately 60% between 4 and 5 h of exposure (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eb). The lowest concentration (15 mg\u0026middot;L\u003csup\u003e⁻1\u003c/sup\u003e) showed low initial efficacy, with a progressive increase up to about 30% at the end of the experimental period (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eb).\u003c/p\u003e\n \u003cp\u003eThe number of parasites adhered to the gill arches varied significantly between treatments (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), with a significant reduction for the highest concentrations of both products (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ec and \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ed). In the samples exposed to the crude ethanolic extract (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ec), the concentrations 40 and 60 mg\u0026middot;L\u003csup\u003e⁻1\u003c/sup\u003e significantly reduced the mean monogenoid count when compared to the controls (water and methanol), with a decrease that was proportional to the increase in dose. For dillapiole, the concentrations 28 and 33 mg\u0026middot;L\u003csup\u003e-1\u003c/sup\u003e also promoted a significant reduction in the number of parasites adhering to the gill arches, confirming the efficacy of the compound, even at intermediate doses (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ed).\u003c/p\u003e\n \u003cp\u003eThe detailed count of the behavioral alterations caused by the crude ethanolic extract and dillapiole are represented in Tables \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e, respectively. For the extract, a dose- and time-dependent effect is observed, with intense lethargy occurring in the first 30 min at concentrations of 80 and 100 mg\u0026middot;L\u003csup\u003e⁻1\u003c/sup\u003e, with mortality greater than 80% after 3 h and 100% at the end of 4 h of exposure. At 60 mg\u0026middot;L\u003csup\u003e-1\u003c/sup\u003e, some of the parasites remained alive until the third hour and, after 5 h, there was almost total death or detachment from the tissue. At 40 mg\u0026middot;L\u003csup\u003e-1\u003c/sup\u003e, the effect was more gradual, with a progressive increase in lethargy and partial mortality, with a significant number of parasites still alive at the end of the assay (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eNumber of gill monogenoids in \u003cem\u003eColossoma macropomum\u003c/em\u003e classified as alive, dead or exhibiting behavioral alterations during the \u003cem\u003ein vitro\u003c/em\u003e test with the crude extract of \u003cem\u003ePiper aduncum\u003c/em\u003e and different exposure periods.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003ePeriod\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eCrude extract\u003c/p\u003e\n \u003cp\u003emg.L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\" rowspan=\"2\"\u003e\n \u003cp\u003eAlive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"4\"\u003e\n \u003cp\u003eBehavioral alterations\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eDead\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eLethargic\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eDetached\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"6\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0 (Water)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0 (Methanol)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e54\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"6\"\u003e\n \u003cp\u003e30 min\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0 (Water)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0 (Methanol)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e54\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"6\"\u003e\n \u003cp\u003e1 h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0 (Water)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0 (Methanol)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e54\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"6\"\u003e\n \u003cp\u003e2 h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0 (Water)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0 (Methanol)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e54\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e17\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"6\"\u003e\n \u003cp\u003e3 h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0 (Water)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0 (Methanol)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e54\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e23\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e29\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e31\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"6\"\u003e\n \u003cp\u003e4 h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0 (Water)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0 (Methanol)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e54\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e23\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e37\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e35\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"6\"\u003e\n \u003cp\u003e5 h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0 (Water)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0 (Methanol)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e54\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e23\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e37\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e35\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"9\"\u003eValues are expressed as absolute counts per observation interval. Controls: water and Tween-20 (0.0004%).\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003cdiv align=\"left\" class=\"colspec\"\u003e\u003cbr\u003e\u003c/div\u003e\n \u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eNumber of gill monogenoids in \u003cem\u003eColossoma macropomum\u003c/em\u003e classified as alive, dead or exhibiting behavioral alterations during the \u003cem\u003ein vitro\u003c/em\u003e exposure to dillapiole isolated from \u003cem\u003ePiper aduncum\u003c/em\u003e and different time intervals.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003ePeriod\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eDillapiole (mg/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eAlive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003eBehavioral alterations\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eDead\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eLethargic\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDetached\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"6\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0 (Water)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e46\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0 (Water\u0026thinsp;+\u0026thinsp;Tween)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e48\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e90\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"6\"\u003e\n \u003cp\u003e30 min\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0 (Water)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e46\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0 (Water\u0026thinsp;+\u0026thinsp;Tween)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e48\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e90\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e32\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"6\"\u003e\n \u003cp\u003e1 h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0 (Water)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e46\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0 (Water\u0026thinsp;+\u0026thinsp;Tween)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e46\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e90\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e36\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"6\"\u003e\n \u003cp\u003e2 h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0 (Water)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e46\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0 (Water\u0026thinsp;+\u0026thinsp;Tween)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e46\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e90\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e36\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"6\"\u003e\n \u003cp\u003e3 h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0 (Water)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e46\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0 (Water\u0026thinsp;+\u0026thinsp;Tween)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e28\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e40\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e90\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e36\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"6\"\u003e\n \u003cp\u003e4 h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0 (Water)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e46\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0 (Water\u0026thinsp;+\u0026thinsp;Tween)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e28\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e40\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e90\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e36\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"6\"\u003e\n \u003cp\u003e5 h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0 (Water)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e46\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0 (Water\u0026thinsp;+\u0026thinsp;Tween)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e28\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e40\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e90\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e36\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"7\"\u003eControls: water and water\u0026thinsp;+\u0026thinsp;Tween-20 (0.0004% v/v).\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003eIn the test with dillapiole (Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e), there was a faster and more intense action on the parasite when compared to the extract. At 90 mg\u0026middot;L\u003csup\u003e-1\u003c/sup\u003e, 89% of parasites were dead immediately after 30 min and 100% died at the end of 1 h of exposure. At 60 and 90 mg\u0026middot;L\u003csup\u003e⁻1\u003c/sup\u003e, severe lethargy was observed in the first hour and total mortality up to 3 h. At intermediate concentrations (15 and 30 mg\u0026middot;L\u003csup\u003e-1\u003c/sup\u003e), the effect was more limited and progressive, with part of the parasites becoming lethargic or detached and others still alive at the end of 5 h of exposure (Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). As shown in Tables \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e, the remaining specimens showed reduced movement and lethargy from the first evaluation intervals.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003e3.2 \u003cem\u003eIn vivo effect of dillapiole and the crude extract of P. aduncum in C. macropomum\u003c/em\u003e\u003c/h2\u003e\n \u003cp\u003eIn the \u003cem\u003ein vivo\u003c/em\u003e tests, three monogenoid species parasitizing \u003cem\u003eC. macropomum\u003c/em\u003e were identified: \u003cem\u003eAnacanthorus spathulatus\u003c/em\u003e, \u003cem\u003eNotozothecium janauachensi\u003c/em\u003e and \u003cem\u003eMymarothecium boegeri\u003c/em\u003e (Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). In almost all the treatments with the crude ethanolic extract, \u003cem\u003eA. spathulatus\u003c/em\u003e presented 100% prevalence, except in 60 mg\u0026middot;L\u003csup\u003e⁻1\u003c/sup\u003e which showed a significant reduction in prevalence (66.6%) and lower mean abundance when compared to the controls (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The species \u003cem\u003eN. janauachensi\u003c/em\u003e and \u003cem\u003eM. boegeri\u003c/em\u003e occurred less frequently and did not present statistically significant differences in abundance between treatments (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05; Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eTable 3.\u003c/strong\u003e Prevalence and mean abundance of monogenean species (\u003cem\u003eAnacanthorus spathulatus\u003c/em\u003e, \u003cem\u003eNotozothecium janauachensi\u003c/em\u003e and \u003cem\u003eMymarothecium boegeri\u003c/em\u003e) recovered from the gill arches of \u003cem\u003eColossoma macropomum\u003c/em\u003e after \u003cem\u003ein vivo\u003c/em\u003e therapeutic baths with the crude extract of and dillapiole from \u003cem\u003ePiper aduncum\u003c/em\u003e.\u003c/p\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"105%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"4\" style=\"width: 18px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eParasitic species\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"14\" style=\"width: 74px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCrude extract\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"7\" style=\"width: 37px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eControl\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"7\" style=\"width: 37px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eConcentrations (mg.L\u003csup\u003e-1\u003c/sup\u003e)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" style=\"width: 6px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026ldquo;p\u0026rdquo;\u003csup\u003e1\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"4\" style=\"width: 18px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eWater\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 18px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMethanol\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"4\" style=\"width: 20px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e40\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 17px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e60\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"3\" style=\"width: 7px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eP\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMA\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 6px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eP (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMA\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 6px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eP\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 13px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMA\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 6px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eP\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMA\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 18px;\"\u003e\n \u003cp\u003e\u003cem\u003eA. spathulatus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 6px;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 11px;\"\u003e\n \u003cp\u003e36.55\u0026plusmn;7.5\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 6px;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e34.11\u0026plusmn;7.5\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 6px;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 13px;\"\u003e\n \u003cp\u003e22.22\u0026plusmn;6.4\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 6px;\"\u003e\n \u003cp\u003e66.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e3.55\u0026plusmn;2.49\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 18px;\"\u003e\n \u003cp\u003e\u003cem\u003eM. boegeri\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 6px;\"\u003e\n \u003cp\u003e66.66\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 11px;\"\u003e\n \u003cp\u003e1.77\u0026plusmn;1.35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 6px;\"\u003e\n \u003cp\u003e55.55\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e1.00\u0026plusmn;0.88\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 6px;\"\u003e\n \u003cp\u003e55.55\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 13px;\"\u003e\n \u003cp\u003e0.77\u0026plusmn;0.86\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 6px;\"\u003e\n \u003cp\u003e33.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e0.22\u0026plusmn;0.34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 18px;\"\u003e\n \u003cp\u003e\u003cem\u003eN. janauachensis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 6px;\"\u003e\n \u003cp\u003e77.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 11px;\"\u003e\n \u003cp\u003e1.22\u0026plusmn;0.74\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 6px;\"\u003e\n \u003cp\u003e66.66\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e1.00\u0026plusmn;0.66\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 6px;\"\u003e\n \u003cp\u003e55.55\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 13px;\"\u003e\n \u003cp\u003e0.55\u0026plusmn;0.61\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 6px;\"\u003e\n \u003cp\u003e33.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e0.33\u0026plusmn;0.44\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"4\" style=\"width: 18px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eParasitic species\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"14\" style=\"width: 74px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDillapiole\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"7\" style=\"width: 37px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eControl\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"7\" style=\"width: 37px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eConcentrations (mg.L\u003csup\u003e-1\u003c/sup\u003e)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" style=\"width: 6px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026ldquo;p\u0026rdquo;\u003csup\u003e1\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"4\" style=\"width: 18px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eWater\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 18px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTween-20\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 19px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e28\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"4\" style=\"width: 18px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e33\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eP\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 12px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMA\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eP (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 12px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMA\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eP\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 13px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMA\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 5px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eP\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 12px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMA\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 18px;\"\u003e\n \u003cp\u003e\u003cem\u003eA. spathulatus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 12px;\"\u003e\n \u003cp\u003e43.33\u0026plusmn;5.18\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 12px;\"\u003e\n \u003cp\u003e43.88\u0026plusmn;5.9\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 13px;\"\u003e\n \u003cp\u003e22.22\u0026plusmn;3.58\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 5px;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 12px;\"\u003e\n \u003cp\u003e13.88\u0026plusmn;4.32\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e***\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 18px;\"\u003e\n \u003cp\u003e\u003cem\u003eM. boegeri\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e88.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 12px;\"\u003e\n \u003cp\u003e1.88\u0026plusmn;0.61\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 12px;\"\u003e\n \u003cp\u003e1.77\u0026plusmn;0.51\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 13px;\"\u003e\n \u003cp\u003e1.55\u0026plusmn;0.49\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 5px;\"\u003e\n \u003cp\u003e55.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 12px;\"\u003e\n \u003cp\u003e0.44\u0026plusmn;0.49\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 18px;\"\u003e\n \u003cp\u003e\u003cem\u003eN. janauachensis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e77.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 12px;\"\u003e\n \u003cp\u003e1.44\u0026plusmn;1.037\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 12px;\"\u003e\n \u003cp\u003e1.77\u0026plusmn;0.69\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 13px;\"\u003e\n \u003cp\u003e1.22\u0026plusmn;0.30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 5px;\"\u003e\n \u003cp\u003e77.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 12px;\"\u003e\n \u003cp\u003e0.88\u0026plusmn;0.39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003csup\u003e1\u003c/sup\u003eDifferent letters on the same rows indicate statistically significant differences between treatments according to Tukey\u0026rsquo;s test. * indicates p \u0026lt; 0.05; *** indicates p \u0026lt; 0.01; NS indicates no significant differences. P = prevalence; MA = mean abundance; N = 9 fish per treatment. Values are presented as mean \u0026plusmn; standard deviation.\u003c/p\u003e\n \u003cp\u003eIn the baths with dillapiole, the prevalence of \u003cem\u003eA. spathulatus\u003c/em\u003e was 100% in all the treatments, while the mean abundance progressively decreased with increasing concentrations of the product (Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). A statistically significant difference (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) between the controls and the 33 mg\u0026middot;L\u003csup\u003e⁻1\u003c/sup\u003e treatment was also observed in this parasite species. For \u003cem\u003eM. boegeri\u003c/em\u003e, prevalence and abundance were also reduced in the test with the highest concentration of dillapiole, presenting significant differences in relation to controls (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e)).\u003c/p\u003e\n \u003cp\u003eThe behavioral analysis indicated that the fish of the control treatments (water, methanol 0.1% and Tween-20 0.0004%) showed normal swimming and respiratory rate during the baths. In contrast, fish exposed to the highest concentration of the extract (60 mg\u0026middot;L\u003csup\u003e⁻1\u003c/sup\u003e) and the highest concentration of dillapiole (33 mg\u0026middot;L\u003csup\u003e⁻1\u003c/sup\u003e) showed behavioral alterations, including an increase in the frequency of opercular beats and a reduction in swimming activity. After the seven-day recovery period, no mortality was observed. The fish returned to normal feeding after the second day of recovery, presenting coloration and integrity of the fins similar to the basal group. However, individuals in the control groups exhibited clinical signs such as erosions on the fins and whitish spots.\u003c/p\u003e\n \u003cp\u003eIn Table \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e, the hemato-biochemical results of the fish exposed to the crude extract at 60 mg\u0026middot;L⁻\u0026sup1; demonstrated hyperglycemia (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in relation to the other treatments and to the basal group, as well as significant decreases in total protein and plasma calcium levels at the highest concentrations (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Cortisol levels were significantly higher in fish treated with the extract, especially at a dose of 60 mg\u0026middot;L\u003csup\u003e⁻1\u003c/sup\u003e, presenting statistical differences when compared to the controls and the basal group (Table \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). The mean values of thrombocytes and leukocytes were higher in the water control in comparison to the other treatments, except the basal one; there was eosinophilia in all the groups, which was not observed in the basal group, without differences for lymphocytes and monocytes (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab4\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eBlood parameters of \u003cem\u003eColossoma macropomum\u003c/em\u003e submitted to two four-hour baths with the crude extract of \u003cem\u003ePiper aduncum\u003c/em\u003e.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eBlood variables\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003eControls\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eCrude extract (mg.L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e\u0026ldquo;p-value\u0026rdquo;\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eBasal\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eWater\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMethanol\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e40\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"7\"\u003e\n \u003cp\u003e\u003cem\u003eHematological parameters\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHematocrit (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e23.68\u0026thinsp;\u0026plusmn;\u0026thinsp;1.85\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e24.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e24.11\u0026thinsp;\u0026plusmn;\u0026thinsp;1.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e22.72\u0026thinsp;\u0026plusmn;\u0026thinsp;1.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e21.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHemoglobin (g/dL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.59\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.53\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.53\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.68\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRBC (x10\u003csup\u003e6\u003c/sup\u003e.\u0026micro;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMCV(fL)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e161.36\u0026thinsp;\u0026plusmn;\u0026thinsp;19.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e169.69\u0026thinsp;\u0026plusmn;\u0026thinsp;17.24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e166.19\u0026thinsp;\u0026plusmn;\u0026thinsp;25.93\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e150.11\u0026thinsp;\u0026plusmn;\u0026thinsp;15.30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e148.75\u0026thinsp;\u0026plusmn;\u0026thinsp;20.17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMCHC (g.dL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e23.78\u0026thinsp;\u0026plusmn;\u0026thinsp;2.63\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e27.62\u0026thinsp;\u0026plusmn;\u0026thinsp;2.89\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e26.10\u0026thinsp;\u0026plusmn;\u0026thinsp;1.60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e27.01\u0026thinsp;\u0026plusmn;\u0026thinsp;2.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e26.55\u0026thinsp;\u0026plusmn;\u0026thinsp;1.54\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMCH (pg)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e37.92\u0026thinsp;\u0026plusmn;\u0026thinsp;4.79\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e46.62\u0026thinsp;\u0026plusmn;\u0026thinsp;5.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e43.54\u0026thinsp;\u0026plusmn;\u0026thinsp;7.67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e40.84\u0026thinsp;\u0026plusmn;\u0026thinsp;6.96\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e39.22\u0026thinsp;\u0026plusmn;\u0026thinsp;4.50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eBiochemical parameters\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGlucose (mg.dL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e89.44\u0026thinsp;\u0026plusmn;\u0026thinsp;11.64\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e55.40\u0026thinsp;\u0026plusmn;\u0026thinsp;5.66\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e64.25\u0026thinsp;\u0026plusmn;\u0026thinsp;5.9\u003csup\u003ecd\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e71.75\u0026thinsp;\u0026plusmn;\u0026thinsp;6.67\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e121.69\u0026thinsp;\u0026plusmn;\u0026thinsp;26.39\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e***\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTP (g.dL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.54\u0026thinsp;\u0026plusmn;\u0026thinsp;0.44\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.88\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCa\u003csup\u003e2+\u003c/sup\u003e (mg/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1,215.55\u0026thinsp;\u0026plusmn;\u0026thinsp;21.72\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e997.77\u0026thinsp;\u0026plusmn;\u0026thinsp;124.69\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e862.22\u0026thinsp;\u0026plusmn;\u0026thinsp;81.97\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e728.88\u0026thinsp;\u0026plusmn;\u0026thinsp;72.34\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e550\u0026thinsp;\u0026plusmn;\u0026thinsp;62.22\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e***\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMg\u003csup\u003e2+\u003c/sup\u003e (mg/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e86.66\u0026thinsp;\u0026plusmn;\u0026thinsp;8.14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e88.88\u0026thinsp;\u0026plusmn;\u0026thinsp;10.37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e84.44\u0026thinsp;\u0026plusmn;\u0026thinsp;7.16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e78.88\u0026thinsp;\u0026plusmn;\u0026thinsp;5.92\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e80\u0026thinsp;\u0026plusmn;\u0026thinsp;6.66\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNa\u003csup\u003e+\u003c/sup\u003e (mg/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4,012.47\u0026thinsp;\u0026plusmn;\u0026thinsp;408.13\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3,618.915\u0026thinsp;\u0026plusmn;\u0026thinsp;204.06\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3,662.644\u0026thinsp;\u0026plusmn;\u0026thinsp;369.26\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3,400.27\u0026thinsp;\u0026plusmn;\u0026thinsp;310.96\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3,986.84\u0026thinsp;\u0026plusmn;\u0026thinsp;430.92\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eK\u003csup\u003e+\u003c/sup\u003e (mg/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e376.00\u0026thinsp;\u0026plusmn;\u0026thinsp;102\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e423.33\u0026thinsp;\u0026plusmn;\u0026thinsp;177.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e447.77\u0026thinsp;\u0026plusmn;\u0026thinsp;118.51\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e232.22\u0026thinsp;\u0026plusmn;\u0026thinsp;98.51\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e267.77\u0026thinsp;\u0026plusmn;\u0026thinsp;131.35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCortisol (\u0026micro;dL)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e67.53\u0026thinsp;\u0026plusmn;\u0026thinsp;18.05\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e47.63\u0026thinsp;\u0026plusmn;\u0026thinsp;17.04\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e42.93\u0026thinsp;\u0026plusmn;\u0026thinsp;7.17\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e68.46\u0026thinsp;\u0026plusmn;\u0026thinsp;6.99\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e69.24\u0026thinsp;\u0026plusmn;\u0026thinsp;5.3\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eInnate immunity cell count\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eThrombocytes (x10\u0026sup3;.\u0026micro;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e93.00\u0026thinsp;\u0026plusmn;\u0026thinsp;20.20\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e155.66\u0026thinsp;\u0026plusmn;\u0026thinsp;24.86\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e72.00\u0026thinsp;\u0026plusmn;\u0026thinsp;11.6\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e66.55\u0026thinsp;\u0026plusmn;\u0026thinsp;10.44\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e90.88\u0026thinsp;\u0026plusmn;\u0026thinsp;30.06\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLeukocytes (x10\u0026sup3;.\u0026micro;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.47\u0026thinsp;\u0026plusmn;\u0026thinsp;2.73\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10.59\u0026thinsp;\u0026plusmn;\u0026thinsp;1.84\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8.86\u0026thinsp;\u0026plusmn;\u0026thinsp;1.62\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.89\u0026thinsp;\u0026plusmn;\u0026thinsp;1.84\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8.52\u0026thinsp;\u0026plusmn;\u0026thinsp;2.09\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLymphocytes (x10\u0026sup3;.\u0026micro;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.76\u0026thinsp;\u0026plusmn;\u0026thinsp;0.90\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.79\u0026thinsp;\u0026plusmn;\u0026thinsp;0.81\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.76\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.56\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.82\u0026thinsp;\u0026plusmn;\u0026thinsp;0.55\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMonocytes (x10\u0026sup3;.\u0026micro;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.97\u0026thinsp;\u0026plusmn;\u0026thinsp;0.42\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.54\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.92\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.63\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNeutrophils (x10\u0026sup3;.\u0026micro;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.96\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.96\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.74\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.90\u0026thinsp;\u0026plusmn;\u0026thinsp;1.02\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.06\u0026thinsp;\u0026plusmn;\u0026thinsp;1.29\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eEosinophils (x10\u0026sup3;.\u0026micro;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.81\u0026thinsp;\u0026plusmn;\u0026thinsp;0.59\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.92\u0026thinsp;\u0026plusmn;\u0026thinsp;0.70\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.59\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.57\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.33\u0026thinsp;\u0026plusmn;\u0026thinsp;1.64\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e***\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSGC (x10\u0026sup3;.\u0026micro;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"7\"\u003e\u003csup\u003e1\u003c/sup\u003eDifferent letters between the lines indicate statistical differences between treatments on Tukey\u0026apos;s test (N\u0026thinsp;=\u0026thinsp;9): * represents p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; *** represents p\u0026thinsp;\u0026lt;\u0026thinsp;0.01; NS represents no statistical differences. Basal\u0026thinsp;=\u0026thinsp;acclimation. RBC\u0026thinsp;=\u0026thinsp;red blood cell count. MCV\u0026thinsp;=\u0026thinsp;mean corpuscular volume. MCHC\u0026thinsp;=\u0026thinsp;mean corpuscular hemoglobin concentration. MCH\u0026thinsp;=\u0026thinsp;mean corpuscular hemoglobin. TP\u0026thinsp;=\u0026thinsp;total proteins; Ca\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;+\u0026thinsp;=\u0026thinsp;calcium, Mg\u0026sup2;\u003csup\u003e+\u003c/sup\u003e= magnesium, Na\u003csup\u003e+\u003c/sup\u003e= sodium, K\u003csup\u003e+\u003c/sup\u003e= potassium, SGC\u0026thinsp;=\u0026thinsp;special granulocytic cells. Means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation.\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003eIn Table \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e, two 4-h baths with dillapiole (33 mg\u0026middot;L\u003csup\u003e⁻1\u003c/sup\u003e) reduced hematocrit, hemoglobin and MCH in comparison with the control (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). There was also a reduction in glycemia, a decrease in calcium and potassium and an increase in sodium (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), as well as lower plasma cortisol in the treated groups when compared to the controls (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In the leukogram, there was an increase in thrombocytes, total leukocytes and lymphocytes at the two highest concentrations, as well as neutrophilia and eosinophilia in the treated groups, which was not observed in the controls (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab5\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eBlood parameters of \u003cem\u003eColossoma macropomum\u003c/em\u003e submitted to two four-hour baths with dillapiole.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eBlood variables\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eBasal\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eControl\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eDillapiole (mg.L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003ep-value\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eWater\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTween 20\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e28\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e33\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"7\"\u003e\n \u003cp\u003e\u003cem\u003eHematological parameters\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHematocrit (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e23.68\u0026thinsp;\u0026plusmn;\u0026thinsp;1.85\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e25.55\u0026thinsp;\u0026plusmn;\u0026thinsp;2.38\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e23.94\u0026thinsp;\u0026plusmn;\u0026thinsp;1.53\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e21.72\u0026thinsp;\u0026plusmn;\u0026thinsp;1.08\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20.22\u0026thinsp;\u0026plusmn;\u0026thinsp;1.62\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e***\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHemoglobin (g/dL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.60\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.97\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.87\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.85\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.95\u0026thinsp;\u0026plusmn;\u0026thinsp;0.54\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRBC (x10\u003csup\u003e6\u003c/sup\u003e.\u0026micro;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.60\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.58\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMCV(fL)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e161.36\u0026thinsp;\u0026plusmn;\u0026thinsp;19.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e170.71\u0026thinsp;\u0026plusmn;\u0026thinsp;16.83\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e142.70\u0026thinsp;\u0026plusmn;\u0026thinsp;15.98\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e137.87\u0026thinsp;\u0026plusmn;\u0026thinsp;13.99\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e129.19\u0026thinsp;\u0026plusmn;\u0026thinsp;15.70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMCHC (g.dL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e23.78\u0026thinsp;\u0026plusmn;\u0026thinsp;2.63\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e24.83\u0026thinsp;\u0026plusmn;\u0026thinsp;2.99\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e25.11\u0026thinsp;\u0026plusmn;\u0026thinsp;4.28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e25.82\u0026thinsp;\u0026plusmn;\u0026thinsp;4.36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e24.54\u0026thinsp;\u0026plusmn;\u0026thinsp;2.37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMCH (pg)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e37.92\u0026thinsp;\u0026plusmn;\u0026thinsp;4.79\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e42.31\u0026thinsp;\u0026plusmn;\u0026thinsp;6.62\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e38.36\u0026thinsp;\u0026plusmn;\u0026thinsp;6.84\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e35.53\u0026thinsp;\u0026plusmn;\u0026thinsp;6.39\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e31.76\u0026thinsp;\u0026plusmn;\u0026thinsp;5.12\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"7\"\u003e\n \u003cp\u003e\u003cem\u003eBiochemical parameters\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGlucose (mg.dL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e89.44\u0026thinsp;\u0026plusmn;\u0026thinsp;11.64\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e49.58\u0026thinsp;\u0026plusmn;\u0026thinsp;8.57\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e50.23\u0026thinsp;\u0026plusmn;\u0026thinsp;10.87\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e66.36\u0026thinsp;\u0026plusmn;\u0026thinsp;13.20\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e81.70\u0026thinsp;\u0026plusmn;\u0026thinsp;14.04\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTP (g.dL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.54\u0026thinsp;\u0026plusmn;\u0026thinsp;0.44\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCa\u003csup\u003e2+\u003c/sup\u003e (mg/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1,215.55\u0026thinsp;\u0026plusmn;\u0026thinsp;21.72\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1,215.55\u0026thinsp;\u0026plusmn;\u0026thinsp;69.38\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1,166.66\u0026thinsp;\u0026plusmn;\u0026thinsp;97.77\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1,013.33\u0026thinsp;\u0026plusmn;\u0026thinsp;53.33\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1045.55\u0026thinsp;\u0026plusmn;\u0026thinsp;151.85\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMg\u003csup\u003e2+\u003c/sup\u003e (mg/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e86.66\u0026thinsp;\u0026plusmn;\u0026thinsp;8.14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e91.11\u0026thinsp;\u0026plusmn;\u0026thinsp;8.41\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e91.11\u0026thinsp;\u0026plusmn;\u0026thinsp;15.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e92.22\u0026thinsp;\u0026plusmn;\u0026thinsp;10.86\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e83.33\u0026thinsp;\u0026plusmn;\u0026thinsp;17.77\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNa\u003csup\u003e+\u003c/sup\u003e (mg/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4,012.47\u0026thinsp;\u0026plusmn;\u0026thinsp;408.13\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3,658.177\u0026thinsp;\u0026plusmn;\u0026thinsp;263.20\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3,561.662\u0026thinsp;\u0026plusmn;\u0026thinsp;353.20\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3,984.90\u0026thinsp;\u0026plusmn;\u0026thinsp;330.8\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4,198.169\u0026thinsp;\u0026plusmn;\u0026thinsp;108.15\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eK\u003csup\u003e+\u003c/sup\u003e (mg/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e376.66\u0026thinsp;\u0026plusmn;\u0026thinsp;102.22\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e554.44\u0026thinsp;\u0026plusmn;\u0026thinsp;128.39\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e487.77\u0026thinsp;\u0026plusmn;\u0026thinsp;183.70\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e236.66\u0026thinsp;\u0026plusmn;\u0026thinsp;77.77\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e186.66\u0026thinsp;\u0026plusmn;\u0026thinsp;106.66\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCortisol (\u0026micro;dL)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e67.53\u0026thinsp;\u0026plusmn;\u0026thinsp;18.05\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e59.54\u0026thinsp;\u0026plusmn;\u0026thinsp;4.26\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e55.93\u0026thinsp;\u0026plusmn;\u0026thinsp;10.30\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e67.22\u0026thinsp;\u0026plusmn;\u0026thinsp;3.73\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e46.14\u0026thinsp;\u0026plusmn;\u0026thinsp;14.47\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"7\"\u003e\n \u003cp\u003e\u003cem\u003eInnate immunity cell count\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eThrombocytes (x10\u0026sup3;.\u0026micro;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e89.66\u0026thinsp;\u0026plusmn;\u0026thinsp;25.40\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e195.00\u0026thinsp;\u0026plusmn;\u0026thinsp;47.33\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e161.55\u0026thinsp;\u0026plusmn;\u0026thinsp;25.62\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e178.66\u0026thinsp;\u0026plusmn;\u0026thinsp;59.40\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e259\u0026thinsp;\u0026plusmn;\u0026thinsp;58.22\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLeukocytes (x10\u0026sup3;.\u0026micro;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.47\u0026thinsp;\u0026plusmn;\u0026thinsp;2.73\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13.68\u0026thinsp;\u0026plusmn;\u0026thinsp;2.48\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11.55\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1.70\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e15.81\u0026thinsp;\u0026plusmn;\u0026thinsp;2.28\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14.361\u0026thinsp;\u0026plusmn;\u0026thinsp;2.044\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e***\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLymphocytes (x10\u0026sup3;.\u0026micro;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.76\u0026thinsp;\u0026plusmn;\u0026thinsp;0.70\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.79\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.60\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.96\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.91\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMonocytes (x10\u0026sup3;.\u0026micro;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.97\u0026thinsp;\u0026plusmn;\u0026thinsp;0.42\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.39\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.54\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.71\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.49\u0026thinsp;\u0026plusmn;\u0026thinsp;0.43\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNeutrophils (x10\u0026sup3;.\u0026micro;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.96\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.28\u0026thinsp;\u0026plusmn;\u0026thinsp;1.27\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.56\u0026thinsp;\u0026plusmn;\u0026thinsp;1.01\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.76\u0026thinsp;\u0026plusmn;\u0026thinsp;0.76\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.09\u0026thinsp;\u0026plusmn;\u0026thinsp;1.70\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eEosinophils (x10\u0026sup3;.\u0026micro;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.81\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.99\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.26\u0026thinsp;\u0026plusmn;\u0026thinsp;1.08\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.68\u0026thinsp;\u0026plusmn;\u0026thinsp;0.94\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.77\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e***\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSGC (x10\u0026sup3;.\u0026micro;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.005\u0026thinsp;\u0026plusmn;\u0026thinsp;0.006\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.006\u0026thinsp;\u0026plusmn;\u0026thinsp;0.005\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.013\u0026thinsp;\u0026plusmn;\u0026thinsp;0.009\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.006\u0026thinsp;\u0026plusmn;\u0026thinsp;0.007\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.002\u0026thinsp;\u0026plusmn;\u0026thinsp;0.004\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"7\"\u003e\u003csup\u003e1\u003c/sup\u003eDifferent letters between the lines indicate statistical differences between treatments on Tukey\u0026apos;s test (N\u0026thinsp;=\u0026thinsp;9): * represents p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; *** represents p\u0026thinsp;\u0026lt;\u0026thinsp;0.01; NS represents no statistical differences. Basal\u0026thinsp;=\u0026thinsp;acclimation. RBC\u0026thinsp;=\u0026thinsp;red blood cell count. MCV\u0026thinsp;=\u0026thinsp;mean corpuscular volume. MCHC\u0026thinsp;=\u0026thinsp;mean corpuscular hemoglobin concentration. MCH\u0026thinsp;=\u0026thinsp;mean corpuscular hemoglobin. TP\u0026thinsp;=\u0026thinsp;total proteins; Ca\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;+\u0026thinsp;=\u0026thinsp;calcium, Mg\u0026sup2;\u003csup\u003e+\u003c/sup\u003e= magnesium, Na\u003csup\u003e+\u003c/sup\u003e= sodium, K\u003csup\u003e+\u003c/sup\u003e= potassium, SGC\u0026thinsp;=\u0026thinsp;special granulocytic cells. Means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation.\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003eThe histopathological alterations index (HAI) showed significant differences (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) between the treatments with 40 and 60 mg\u0026middot;L\u003csup\u003e⁻1\u003c/sup\u003e of the extract and the control with methanol (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ea). In the dillapiole experiments, the HAI was higher in the control groups, but did not differ statistically in relation to the treatments and the basal group (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eb).\u003c/p\u003e\n \u003cp\u003eQuantitative evaluation of histological parameters indicated variations in the index/mean of the lesions between the crude extract and dillapiole treatments (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ec\u0026ndash;d). In the crude extract, the mean values ranged from 1.33 to 1.96, with lower means in the treated groups (40\u0026ndash;60 mg\u0026middot;L\u003csup\u003e⁻1\u003c/sup\u003e) compared to the controls with water/methanol; there was no dose-dependent effect nor statistically significant differences with the controls (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05; Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ec). Similarly, the mean values of the histological alterations in the gills of fish treated with dillapiole remained similar between the concentrations tested and did not show significant differences when compared to basal group (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ed).\u003c/p\u003e\n \u003cp\u003eHistopathological evaluation of \u003cem\u003eC. macropomum\u003c/em\u003e gills exposed to the ethanolic extract revealed the classification of lesions with different frequency patterns between the treatments (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). In the basal group, it was possible to observe branchial lamellae with preserved morphology, without evident alterations (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ea), or presenting mild and discrete alterations, such as small areas of hypertrophy and focal hyperplasia (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eb). In the controls with water and methanol, the frequency and intensity of lesions increased, with characteristic alterations, including hyperplasia of the lamellar epithelium with partial fusion of secondary lamellae (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ec and \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ed), abundant presence of monogenoids adhered to the branchial surface (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ec\u0026ndash;d), dilatation of the marginal canal (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ee) and a marked proliferation of mucosal cells, evidencing the impact of the parasitic infestation. At concentrations of 40 and 60 mg\u0026middot;L\u003csup\u003e⁻1\u003c/sup\u003e of crude extract, there was a reduction in the frequency and severity of these alterations, with decreased hyperplasia and better lamellar organization compared to the controls (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ef\u0026ndash;h). Some samples showed areas with reduced epithelial thickness and a slight presence of aneurysmal lamellae (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eh) and a smaller number of mucous cells and few residual parasites adhered to the arches (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ei). In general, the presence of parasites was high in the controls, while in the treatments with 40 and 60 mg\u0026middot;L\u003csup\u003e⁻1\u003c/sup\u003e of the extract and in the basal group the occurrence was low or sporadic (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ea, \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eb, \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eg\u0026ndash;i).\u003c/p\u003e\n \u003cp\u003eIn the histopathological evaluation of the fish of the dillapiole assay, the fish of the control groups (water and Tween-20) showed a higher frequency of epithelial hyperplasia and hypertrophy, associated with partial fusion of the secondary lamellae and an abundant presence of parasites adhered to the gill filaments (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ec\u0026ndash;d). These alterations also included dilatation of the marginal canal and intense proliferation of mucous cells in the controls. The therapeutic baths reduced the frequency and intensity of these lesions, especially in the bath with 33 mg\u0026middot;L\u003csup\u003e⁻1\u003c/sup\u003e, which showed more preserved lamellar architecture and fewer mucocytes (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ef). At the concentration of 28 mg\u0026middot;L⁻\u0026sup1;, the pattern was intermediate, there were still areas of hyperplasia, but these were less extensive than in the controls (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ee). The presence of parasites was high in the control groups and practically absent in the fish treated with the highest concentration of dillapiole. In the basal group, gills with preserved morphology or mild and focal alterations were observed (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ea-b).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"4. DISCUSSION","content":"\u003cp\u003eThe results of the present study unequivocally demonstrate that the crude ethanolic extract of \u003cem\u003eP. aduncum\u003c/em\u003e and its major compound, dillapiole, have a highly effective anti-monogenoid activity, evidenced by the dose- and time-dependent action in the \u003cem\u003ein vitro\u003c/em\u003e assays and by the substantial reduction of the parasitic load in the \u003cem\u003ein vivo\u003c/em\u003e assays. These findings, gathered in a pioneering way, provide consistent data on the potential of these substances as herbal alternatives in the control of branchial parasites in \u003cem\u003eC. macropomum\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eEvaluating the toxic effects of drugs on animals, as well as testing their effect directly on parasites, \u003cem\u003ein vitro\u003c/em\u003e, are fundamental steps that allow us to determine the therapeutic concentrations to be used, as well as enable us to understand their mechanism of action on parasites when applied \u003cem\u003ein vivo\u003c/em\u003e (Affonso et al. 2022; de Queiroz et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The \u003cem\u003ein vitro\u003c/em\u003e tests showed that both the crude extract and dillapiole showed a progressive lethality that was proportional to the increase in concentration and exposure time. Specifically, the extract demonstrated 100% efficacy at a concentration of 100 mg\u0026middot;L\u003csup\u003e⁻1\u003c/sup\u003e after 4 h, and dillapiole, in turn, reached the same level of efficacy in the first hour at a concentration of 90 mg\u0026middot;L\u003csup\u003e⁻1\u003c/sup\u003e, confirming the faster and more intense action of the isolated compound. These results corroborate previous observations by de Queiroz et al. (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), who also identified the dose-dependent action of \u003cem\u003eP. aduncum\u003c/em\u003e extract against monogenoids in \u003cem\u003eA. gigas\u003c/em\u003e and \u003cem\u003eC. macropomum\u003c/em\u003e, although previous studies have not directly compared the extract to the dillapiole isolate under controlled laboratory conditions.\u003c/p\u003e\u003cp\u003eThe comparison between the two products suggests that dillapiole plays a central role in anthelmintic action, a hypothesis supported by the phytochemical analyses that indicate its predominance (up to 98%) in the essential oil (Almeida et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; de Queiroz et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). However, it cannot be ruled out that other secondary metabolites present in the crude extract, including chromenes, flavonoids, alkaloids and monoterpenes (Efdi et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), exert synergistic or potentiating effects on the parasites, increasing the biological activity of the product, as already proposed by Corral et al. (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) and Andrade et al. (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe mechanism of action of dillapiole on monogenoids remains unclear. However, studies into phenylpropanoids demonstrate that these molecules have the ability to interact with the lipids of cell and mitochondrial membranes, compromising the integrity of the lipid bilayer, increasing ion permeability and leading to cell death (Carson et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). In addition, dillapiole is recognized as a potent inhibitor of cytochrome P450 complex enzymes (Omura and Sato, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e1964\u003c/span\u003e; Mukerjee et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e1979\u003c/span\u003e), and interferes with oxidative and biotransformative processes of the organisms. This metabolic inhibition may have contributed to the lethal efficacy on the parasites, which was observed even at low concentrations and during reduced exposure times, a desirable characteristic for the rapid control of infestations in an intensive farming environment.\u003c/p\u003e\u003cp\u003eThe relevance of these findings becomes even more evident when one considers that alternatives for monogenoid control have limitations that are associated with environmental toxicity, residue formation and potential induction of resistance (Guardone et al. 2022). In Brazilian aquaculture, chemotherapeutic treatments, such as formalin and albendazole, have legal restrictions and prolonged withdrawal periods (Cordeiro et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In this context, plant products, such as dillapiole and the crude extract of \u003cem\u003eP. aduncum\u003c/em\u003e, represent a strategic alternative for safer and more effective disease management and are aligned with the principles of sustainability.\u003c/p\u003e\u003cp\u003eIn the \u003cem\u003ein vivo\u003c/em\u003e test, the applications of therapeutic baths in successive doses resulted in a significant reduction in the prevalence and mean abundance of \u003cem\u003eA. spathulatus\u003c/em\u003e, the main parasite species identified in the fish studied, in addition to lower occurrences of \u003cem\u003eN. janauachensi\u003c/em\u003e and \u003cem\u003eM. boegeri\u003c/em\u003e. This pattern confirms that chronic infestation in \u003cem\u003eC. macropomum\u003c/em\u003e can be efficiently controlled with 4-h bathing protocols at moderate concentrations, a strategy already proposed by Andrade et al. (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) for plant extracts. The positive effects on the parasitic load were accompanied by the recovery of feeding behavior and integrity of the fins, especially noticeable in the groups treated with dillapiole at 33 mg\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and with the crude extract at 60 mg\u0026middot;L \u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, demonstrating a positive effect on animal welfare.\u003c/p\u003e\u003cp\u003eThe hematological and biochemical analyses provided important information about the tolerance of fish to the treatments. While in the crude extract hyperglycemia and increased cortisol levels occurred at higher concentrations, dillapiole showed lower glycemic elevation and a more controlled response to stress (Affonso et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). This finding may be associated with the anesthetic effect described for concentrated dillapiole, which reduces oxygen consumption, basal metabolism and, consequently, the release of catecholamines in the face of stressful stimuli (Lemos et al. 2023). Furthermore, treatment with 33 mg\u0026middot;L\u003csup\u003e⁻1\u003c/sup\u003e of dillapiole resulted in decreased erythrocyte parameters, including a drop in mean corpuscular hemoglobin and hematocrit, suggesting a picture of iron deficiency anemia, potentially resulting from interference in the absorption of iron and essential ions (Marshall \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Evans et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). The concomitant drop in calcium and potassium levels reinforces this hypothesis, since the active transport of these ions by the mitochondrial cells of the gills is fundamental for ionic balance and the physiological integrity of the fish (Perry et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Baldisserotto \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe leukocyte parameters revealed distinct modulations: in the fish exposed to baths with the crude extract. There was an improvement in response and, in both treatments (\u003cem\u003eP. aduncum\u003c/em\u003e extract and dillapiole), there was an inflammatory response that is characteristic of parasitosis, with the elevation of neutrophils and eosinophils \u0026ndash; cells associated with defense against parasites and inflammatory processes (Claudiano et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). These findings suggest that both phytochemicals can exert immunomodulatory action, thus expanding their applicability as prophylactic agents (Mansoori et al. 2024).\u003c/p\u003e\u003cp\u003eThe histological findings corroborate the effectiveness of the treatments. In the controls, the presence of hypertrophy, hyperplasia, lamellar fusion and necrosis, and the abundance of parasites are alterations that are compatible with the response to intense infestations (Vanhove et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). In fish treated with the crude extract and dillapiole, especially in the highest concentrations, the frequency and severity of the lesions were reduced, as well as the histopathological alterations index (HAI) when compared to the controls. This confirms that the reduction in the parasitic load is associated with tissue preservation and lower respiratory impairment, unlike what was observed in treatments with synthetic agents such as glyphosate, which, although effective in eliminating parasites, caused extensive lesions in the gills.\u003c/p\u003e\u003cp\u003eAs a result, this work advances previous studies by demonstrating that the use of dillapiole and the crude extract of \u003cem\u003eP. aduncum\u003c/em\u003e not only promotes the effective removal of monogenoids, but also favors homeostasis and animal welfare. This is evidenced by: (i) an organized inflammatory response that is typical of parasitoses (neutrophilia/eosinophilia), (ii) a serum biochemical profile maintained within physiological ranges, (iii) a hormonal modulation (cortisol) that is compatible with the maintenance of homeostasis, (iv) branchial histopathological findings with reduced scores/no relevant differences when compared to the controls and (v) adequate behavior during and after baths, even after successive therapeutic cycles. Together, this confirms that the extract and dillapiole are viable and promising herbal alternatives, able to integrate biosecurity and disease control programs for \u003cem\u003eC. macropomum\u003c/em\u003e in intensive systems, while also being aligned with the demand for sustainable aquaculture and the reduction in the use of conventional chemotherapeutics (Ali et al. 2021; Norbury et al. 2022; Freitas et al. 2023).\u003c/p\u003e"},{"header":"5. CONCLUSION","content":"\u003cp\u003eThe results suggest that both products, dillapiole and the crude ethanolic extract of \u003cem\u003eP. aduncum\u003c/em\u003e, have an effective and safe anthelmintic effect in the control of the monogenoids \u003cem\u003eA. spathulatus\u003c/em\u003e, \u003cem\u003eN. janauachensis\u003c/em\u003e and \u003cem\u003eM. boegeri\u003c/em\u003e in juvenile \u003cem\u003eC. macropomum\u003c/em\u003e, with up to 99% efficacy when using 60 mg\u0026middot;L\u003csup\u003e⁻1\u003c/sup\u003e of the extract and 85% when using 33 mg\u0026middot;L\u003csup\u003e⁻1\u003c/sup\u003e of dillapiole. In addition to the significant reduction of the parasitic load, the treatments preserved the physiological homeostasis of the fish, which showed behavioral recovery and integrity of the gill tissues after recovery. Taken together, the findings support the practical applicability of these herbal medicines as biosecurity components in intensive farming systems, with the potential to reduce the use of conventional chemotherapies.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability statement:\u003c/strong\u003e No data are available for this study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding statement:\u0026nbsp;\u003c/strong\u003eFundação de Amparo à Pesquisa do Estado do Amazonas, PROCAD AMAZONIA/CAPES (88881.200614/2018-01)\u0026nbsp;e\u0026nbsp;INCT-ADAPTA III/Conselho Nacional de Desenvolvimento Científico e Tecnológico (Process 409202/2024-0).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest disclosure:\u003c/strong\u003e The authors declare that there are no conflicts of interest.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval statement:\u003c/strong\u003e This study was approved by the Ethics Committee on Animal Use, INPA, No.\u0026nbsp;030/2016.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePatient consent statement:\u003c/strong\u003e Not applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePermission to reproduce material from other sources:\u003c/strong\u003e Not applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank Universidade Nilton Lins (UNL) and Instituto Nacional de Pesquisas da Amazônia (INPA).\u0026nbsp;M.N. Queiroz\u0026nbsp;also thanks Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for the grant.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was partially funded by Fundação de Amparo à Pesquisa do Estado do Amazonas via PROCAD AMAZONIA/CAPES (88881.200614/2018-01)\u0026nbsp;and\u0026nbsp;INCT-ADAPTA III/Conselho Nacional de Desenvolvimento Científico e Tecnológico (Process 409202/2024-0). The authors also thank Dr. Francisco Celio Maia Chave (Embrapa Amazônia Ocidental, Amazonas, Brazil) for supplying the raw material for the preparation of the plant extract.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e:\u003c/p\u003e\n\u003cp\u003eMarieta Nascimento de Queiroz: Conceptualization; Methodology; Investigation; Formal analysis; Writing – original draft.\u003c/p\u003e\n\u003cp\u003ePaulo Adelino de Medeiros: Methodology; Investigation; Resources; Data curation; Writing – review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003eZelina Estevam dos Santos Torres: Investigation; Data curation; Validation; Writing – review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003eAdrian Martin Pohlit: Formal analysis; Visualization; Writing – review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003eValdelira Lia Araújo Fernandes: Investigation; Resources; Writing – review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003eGustavo da Silva Claudiano: Supervision; Funding acquisition; Project administration; Writing – review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003eEduardo Akifumi Ono: Resources; Supervision; Validation; Writing – review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003eElizabeth Gusmão Affonso (corresponding author): Conceptualization; Supervision; Funding acquisition; Project administration; Writing – review \u0026amp; editing; Correspondence.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAffonso EG, Barros FP, Brasil EM, Tavares-Dias M, Ono EA (2009) Indicadores fisiol\u0026oacute;gicos de estresse em peixes expostos ao per\u0026oacute;xido de hidrog\u0026eacute;nio (H2O2). 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Flavour Fragr J 20(1):67\u0026ndash;69\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhang XP, Li WX, Ai TS, Zou H, Wu SG, Wang GT (2014) The efficacy of four common anthelmintic drugs and traditional Chinese medicinal plant extracts to control Dactylogyrus vastator (Monogenea). Aquaculture 420:302\u0026ndash;307. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.aquaculture.2013.09.022\u003c/span\u003e\u003cspan address=\"10.1016/j.aquaculture.2013.09.022\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"","identity":"aquaculture-international","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"10499","submissionUrl":"https://submission.nature.com/new-submission/10499/3","title":"Aquaculture International","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"","reportingPortfolio":"VoR Journals","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Anthelmintic, aquaculture, prophylaxis, herbal medicine, tambaqui","lastPublishedDoi":"10.21203/rs.3.rs-7567560/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7567560/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cem\u003ePiper aduncum\u003c/em\u003e, a medicinal plant, was evaluated for control of monogenoids and effects on the welfare of \u003cem\u003eColossoma macropomum\u003c/em\u003e using a crude ethanolic extract and its major constituent dillapiole. Treatments of 40 and 60 mg\u0026middot;L⁻\u0026sup1; of the ethanolic extract were tested in in vitro and in vivo experiments with water and methanol controls. Dillapiole was evaluated at 28 and 33 mg\u0026middot;L⁻\u0026sup1; with water and Tween-20 controls and a basal acclimated group. Parasitological, immunophysiological, biochemical and histological analyses were performed on 50 g fish. Both extract concentrations significantly reduced parasite load versus controls, yielding antiparasitic efficacies of 85.89% and 99.21% for 40 and 60 mg\u0026middot;L⁻\u0026sup1;, respectively. Control fish showed increases in hematocrit and hemoglobin, whereas fish treated with 60 mg\u0026middot;L⁻\u0026sup1; extract exhibited hyperglycemia, elevated cortisol and reductions in total protein and Ca\u0026sup2;⁺ and K⁺ ion levels, indicating physiological stress at higher extract concentration. In water controls, thrombocyte and leukocyte elevations correlated with heavy parasitism; these responses were attenuated in extract-treated groups. Dillapiole at 28 and 33 mg\u0026middot;L⁻\u0026sup1; produced significant parasite reductions with efficacies of 52.44% and 80.24%, respectively. Dillapiole treatments induced increased thrombocytes, leukocytes and differential counts, suggesting stimulation of immune responses that favored recovery after therapeutic baths. Histological assessment showed milder tissue alterations in dillapiole-treated fish compared to controls. Results indicate that both \u003cem\u003eP. aduncum\u003c/em\u003e extract and dillapiole effectively control monogenoid infestations in \u003cem\u003eC. macropomum\u003c/em\u003e, combining antiparasitic action with moderate, concentration-dependent physiological impacts. These findings support further development of P. aduncum-based therapies for sustainable monogenoid management in tropical aquaculture globally.\u003c/p\u003e","manuscriptTitle":"Anthelmintic efficacy of dillapiole and the crude extract of Piper aduncum in the control of monogenoids in Colossoma macropomum","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-22 13:27:04","doi":"10.21203/rs.3.rs-7567560/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-08T08:18:19+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-07T23:25:57+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-01T21:47:21+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-28T01:31:16+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"35195601496935173739890387040394527181","date":"2025-09-16T22:22:27+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"222039347870668585811584741535840591009","date":"2025-09-12T17:56:56+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"200500770389100797096978369872772797700","date":"2025-09-12T16:25:02+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-12T16:13:45+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-12T12:35:19+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-12T07:10:01+00:00","index":"","fulltext":""},{"type":"submitted","content":"Aquaculture International","date":"2025-09-08T21:15:58+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"","identity":"aquaculture-international","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"10499","submissionUrl":"https://submission.nature.com/new-submission/10499/3","title":"Aquaculture International","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"","reportingPortfolio":"VoR Journals","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"ff04ca1d-c31f-430b-af5a-778c33b98458","owner":[],"postedDate":"September 22nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-10-16T11:08:31+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-22 13:27:04","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7567560","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7567560","identity":"rs-7567560","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}