EMB standard (purity ≥ 97%, CAS number: 155569-91-8) was purchased from Dr. Ehrenstorfer (Augsburg, Germany). SLICE^® (EMB; 0.2% aquaculture premix) was supplied by Merck Animal Health (New Jersey, American). HPLC grade acetonitrile, ethyl acetate, and n-hexane used for analysis were all obtained from J. T. Baker (Deventer, Holland). LC-MS grade water was bought from Merck (Darmstadt, Germany). Dimethyl sulfoxide (DMSO) was attained from Sigma-Aldrich (Shanghai, China). Ammonium hydroxide (AR, 25–28%) was ordered from Macklin (Shanghai, China). N-methyl imidazole was purchased from Hwrk Chem (Beijing, China). Magnesium sulfate anhydrous, sodium chloride, heparin sodium, and trifluoroacetic anhydride were purchased from Sinopharm Chemical Reagent Company (Shanghai, China). The centrifugal tubes and 0.22 μm nylon syringe filters were acquired from Shanghai CNW Technologies (Shanghai, China).

Standard stock solution was prepared by dissolving 10 mg of EMB standard in acetonitrile, and then diluted to a final concentration of 100 mg/L. Then, working standard solutions were prepared by diluting the standard stock solution with acetonitrile. All standard solutions were contained in screw thread amber glass bottles and stored at −20°C.

For extractant, 0.2% (v/v) ammonium acetonitrile solution was made by the addition of 0.2 mL ammonium hydroxide in 100 mL acetonitrile and mixed well. 0.1% DMSO (v/v) solution was made by the addition of 0.1 mL DMSO in 100 mL distilled water. Heparin sodium water solution (1%, w/v) was prepared by dissolving 0.1 g of heparin sodium in 10 mL of distilled water.

The experimental protocols and care of animals were approved by the Fish Ethics Committee of Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Wuhan, China (ID 2021-Liu Yongtao-03).

Healthy crucian carp (mean body weight 180 ± 20 g, mixed genders) used in our study were supplied by the aquaculture base of the Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences (Wuhan, China). Before starting the experiment, the fish were acclimated for 1 week. Then, they were raised in tanks supplied with dechlorinated tap water (pH 7.5–8.0). The fish were supplied with oxygen by an inflation pump to ensure the dissolved oxygen levels were kept at 6.0-8.0 mg/L in the water. The nitrite and nitrates were detected every 3 days to keep them at 0.1–0.2 mg/L. Then, the un-ionized ammonia values were kept at < 0.02 mg/L. Otherwise, the water temperature was maintained at 22 ± 2°C by means of an aquarium heater.

A total of 375 fish were placed in fiberglass tanks (10 fish in each tank) supplied with 300 L dechlorinated tap water. Fish were divided into five groups: the first to third groups for oral administration (PO) study, the fourth group served as the intravenous administration (IV) group, and the fifth group for the vehicle control group. The fish were medicated with a drug-free normal feed for 7 days, then starved for another day before the beginning of the drug administration.

The EMB, as Slice^® (Merck Animal Health, New Jersey, American), is administered at 50 μg/kg biomass/day for 7 consecutive days as an in-feed therapeutant against fish ectoparasites. The drug used in the study contains 2 mg of EMB in each gram. In brief, the EMB solutions for oral administration were prepared by dissolving with 0.1% DMSO and adjusting to the final concentrations of 50, 200, and 500 μg/kg bw.

The fish were single orally administered EMB solution with 50, 200, and 500 μg/kg bw by a preloaded syringe down the esophagus (28). As for IV treatment, a single 50 μg/kg bw dose of EMB was injected into the caudal vein (29, 30).

After treatment, each fish was observed, if the EMB solution was regurgitated, the fish was excluded and replaced. The fish were first anesthetized with tricaine methanesulfonate (MS-222, 200 mg/L) (30), and then approximately 2 mL of blood from each of 5 fish at each time point was drawn from the caudal vein using 2 mL heparinized syringe with a 26 G × 1 inch needle on 0.5, 1, 2, 4, 6, 8, 10, 12, 24, 48, 72, 96, 120, 144, and 168 h. After collecting blood, the fish were immediately euthanized by immersion in an overdose of 250 mg/L MS-222 solution (31). Then, muscle plus skin, liver, kidney, and gill were collected at each time point described above. Plasma was isolated from the blood by centrifugation at 3,000 r/min for 5 min and stored at −80°C until analysis. Placed blood stored at 4°C and kept overnight, and then, take the upper layer of serum. Tissues were homogenized by a homogenizer and kept at −80°C until determination.

The preparation procedure of samples was based on our previous method with some modifications (32).

All samples were analyzed by an Agilent UPLC (Milford, MA, USA) equipped with quart solvent manager with a quart solvent pump, a sampler manager with an autosampler, and a fluorescence detector (FLD). An Infinity Lab Poroshell 120 EC-C18 column (2.7 μm, 100 × 4.6 mm) (Agilent, USA) was used for separation. The column temperature was set at 40°C, and the injection volume was 20 μL. The detector was operated at an excitation wavelength of 365 nm and an emission wavelength of 465 nm, respectively. The mobile phase consisted of water and acetonitrile and was applied at a flow rate of 1 mL/min. The gradient elution procedure is shown in Table 1.

According to (33) guidelines for the chromatographic method, recovery rate was determined by comparing the measured concentration of blank sample spiked before and after preparation. Samples were treated as description of 2.4, and each concentration was assayed in six replicates. Intra-day and inter-day precision were evaluated by calculating the relative error (RE) and relative standard deviation (RSD).

Standard calibration curve for EMB was constructed by plotting EMB peak areas vs. seven EMB concentrations (5, 10, 50, 100, 200, 500, and 1,000 μg/L) in acetonitrile.

Recovery was determined by analyzing samples spiked at four levels (5, 10, 50, and 200 μg/L or μg/kg for EMB in plasma and various tissues) in five replicates.

As for intra-day precision of the assay method was determined in a single day, for inter-day precision, five replicates of four different concentration levels were evaluated within 3 days.

The pharmacokinetic parameters were calculated by the Phoenix WinNolin 8.1 Software (Certara Corporation, USA), and the concentration-time profiles of EMB were plotted by Prism 8.0 Software (GraphPad Software, USA). The following parameters were estimated: λz: (elimination rate constant), T_1/2z: (terminal elimination half-life = 0.693/Zeta), T_max: (time to attained maximal concentration), C_max: (observed maximal concentration), AUC: (area under the concentration-time curve), MRT_last: (mean flow time of drug in body from zero to last time point) (34).

The absolute bioavailability was calculated by comparing the area under the curve (AUC) of EMB after PO and IV following the below equation: F (%)= AUC oral administration×Dose intravenous administrationAUC intravenous administration×Dose oral administration×100 Dose _{oraladministration} and dose _{intravenousadministration} correspond to the actual doses by oral and intravenous administration, respectively (35).

Serum contents of glucose (GLU), total cholesterol (T-CHO), blood urea nitrogen (BUN), albumin(ALB), total protein (TP), alkaline phosphatase (ALP), and creatinine (CRE) as well as the activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were assayed by a Chemix-800 automatic biochemical analyzer (Japan Sysmex Corporation, Japan) (36, 37). Significant differences were analyzed by SPSS Statistics 25.0 Software (IBM, USA).

The relevant data of recoveries, intra-day and inter-day precisions in plasma and tissues of crucian carp are listed in Table 2.

EMB concentrations and corresponding peak areas in the range of 5–1,000 μg/L showed good linearity with R² ≥ 0.999. The recovery rates varied from 85.74 to 117.63%. The intra-day and inter-day precisions (CV) ranged between 1.23 to 11.82%.

In plasma, the limit of detection (LOD) for EMB was 1 μg/kg (S/N > 3), and the limit of quantitation (LOQ) was 5 μg/kg (S/N > 10). In muscle plus skin, liver, kidney, and gill, the value of LOD, defined by (S/N > 3), was 2 μg/kg and the value of LOQ, defined by (S/N > 10), was 5 μg/kg. The chromatograms of EMB in tissues and plasma are listed in Figure 1.

Consequently, this validated method was reliable for the determination of EMB in plasma and various tissues of crucian carp.

Plasma concentration-time profiles of the target compound in crucian carp following PO treatment with 50, 200, and 500 μg/kg bw were presented in Figure 2 and the plasma concentration-time profile with IV treatment with 50 μg/kg bw was shown in Figure 4. And the relative pharmacokinetic parameters of EMB in crucian carp plasma were documented in Table 3.

After PO treatment with 50, 200, and 500 μg/kg bw, in plasma, values of C_max for EMB were 9.96, 21.77, and 53.86 μg/L, respectively, and the corresponding T_max were all 8 h. Values of λz were calculated to be 0.019, 0.007, and 0.008 1/h, and T_1/2z were found to be 14.99, 39.03, and 64.87 h, meanwhile, AUC₀₋₁₆₈ were estimated to be 382.01, 1,833.94, and 6,310.65 h·μg /L while MRT_last were 51.88, 67.04, 78.00 h, respectively. For the IV route, pharmacokinetic parameters of EMB in crucian carp plasma were determined. The value of λz for EMB was calculated to be 0.038 1/h, meanwhile, T_1/2z was found to be 18.04 h. The MRT_last was 14.71 h while the value of AUC₀₋₁₆₈ was estimated to be 724.90 h·μg/L.

Concentration-time profiles of EMB in muscle plus skin, liver, kidney, and gill after PO treatment with 50, 200, and 500 μg/kg bw and IV treatment with 50 μg/kg bw were shown in Figures 3, 4. Pharmacokinetic parameters of EMB in crucian carp tissues were listed in Tables 4–7.

Seen from the tissues concentration data, following PO treatment, C_max in various tissues after PO treatment with 50, 200, and 500 μg/kg bw were found to be 71.03, 93.95, and 226.78 μg/kg at 8 h (skinny muscle), 54.69 and 199.81 and 365.10 μg/kg at 8 h (liver), 99.16, 194.13, and 375.91 μg/kg at 4 h (kidney) and 104.38, 250.13, and 506.71 μg/kg at 8 h (gill), respectively. Moreover, C_max in various tissues of crucian carp after IV treatment were found to be 41.71 μg/kg at 0.5 h (skinny muscle), 46.59 μg/kg at 4 h (liver), 358.22 μg/kg at 0.5 h (kidney), and 102.54 μg/kg at 8 h (gill), respectively.

For PO route, the parameters were described. Values of AUC₀₋₁₆₈ for crucian carp after administration with 50, 200, and 500 μg/kg bw were 5,698.62, 11,367.16, and 22,258.31 h·μg/L in skinny muscle, 2,870.68, 13,342.86, and 33,285.94 h·μg/L in liver, 8,042.34, 15,709.71 and 30,902.62 h·μg/L in kidney and 9,546.20, 23,888.25 and 61,236.51 h·μg/L in gill. Meanwhile, MRT_last were 66.27, 81.02, and 71.77 h in skinny muscle, 69.84, 62.61, and 70.41 h in liver, 75.03, 59.93, and 60.79 h in kidney, and 70.03, 70.78, and 76.49 h in gill, respectively. For IV treatment with 50 μg/kg bw, in skinny muscle, liver, kidney, and gill, values of λz were calculated to be 0.007, 0.031, 0.006, and 0.009 1/h, respectively, meanwhile, T_1/2z were found to be 103.40, 22.22, 116.93, and 78.13 h. MRT_last were 60.64, 49.94, 60.34, and 61.66 h and AUC₀₋₁₆₈ were estimated to be 1,923.50, 2,871.20, 14,675.57, and 5,416.48 h·μg/L.

According to the AUC values and doses of EMB used in crucian carp plasma following PO and IV routes of administration, the absolute oral bioavailability of EMB (F%) in crucian carp was calculated to be 52.70% at doses of 50 μg/kg bw.

The serum biochemical analysis results are shown in Figure 5.

The serum biochemical indexes including TP, T-CHO, ALB, ALP, BUN, CRE, and GLU were not significantly different (P > 0.05) or regular between the administered group and the control group. After EMB treatment, AST and ALT showed significant differences (P < 0.05) at days 1, 3, 5, and 7 compared to the control group (day 0).

Our study revealed that in single oral doses at 50, 200, and 500 μg/kg bw for crucian carp, concentrations of EMB in plasma constantly raised while the concentration of administration increased. Meanwhile, concentrations ranged from 4.41 to 53.86 μg/L and the maximum concentration was reached at 8 h after drug administration. This phenomenon indicated that EMB was absorbed quickly, and reached the highest concentration in a short time after completion of administration. Previous studies indicated that fed with EMB 50 μg/kg bw for 7 days, concentrations of EMB in the plasma of salmon were ranging from 6 to 440 μg/L (38) and reached maximum levels of 128 μg/kg on the last day of treatment (day 7) (20). The difference in this concentration change is due to the difference in the number of administrations.

Regarding the plasma pharmacokinetic parameters, our study found that AUC₀₋₁₆₈, T_1/2z, and MRT_lastwere found to be dose-dependent, rising with increased dose following PO treatment. AUC represents the extent of drug absorption and determines the degree of bioavailability, larger AUC values indicate a slower metabolism (39). By comparing two administration routes, AUC₀₋₁₆₈ of EMB in crucian carp plasma by IV route were greater than that following PO treatment with 50 μg/kg bw. Our experimental results showed that the T_1/2z for PO varied from 14.99 to 64.87 h in plasma following administration of 50, 200, and 500 μg/kg bw, respectively. The result also demonstrated that the value of T_1/2z for PO treatment was shorter than that following IV treatment at the same dose. Meanwhile, MRT_lastvaried from 51.88 to 78 h in plasma following PO administration, while it was only 14.71 h following IV administration. These results indicated that the metabolism of EMB is slow, and the increased administration concentration may affect the normal function of the liver and kidney of crucian carp. Furthermore, T_1/2zin the plasma of crucian carp in this study was shorter than T_1/2zin the plasma of Atlantic salmon, which was determined to be 10.0 days. Combined with the MRT_last, the conclusion indicated that EMB remains in crucian carp plasma for a long time, which can play a better treatment effect. Such a conclusion is also recognized in other literature. For example, a conclusion about the validity of EMB for salmon longer than 21 days was presented (14), and similar conclusions have emerged in other studies (13, 40, 41).

The absorption of EMB following a single oral administration was quick, as the maximum concentration in the tissues was detected after the medicated completed 4–8 h, the EMB levels in tissues of crucian carp were liver > gill > kidney > muscle plus skin > plasma. However, when the concentration of EMB was detected 7 days after treatment, the content in gill is highest. We observed a higher level of EMB in gill than in skinny muscle sample likely because the mucous is known to concentrate EMB (20). And EMB can be distributed to many tissues throughout the body, initially easy to be concentrated in the liver, kidney, fat, and muscle tissues (42). After oral administration of different concentrations of EMB, values of AUC₀₋₁₆₈in muscle plus skin, liver, kidney, and gill were increased with increasing administered concentrations. Subsequently, the largest λz was found in liver by two routes of administration. MRT_last in muscle and gill was longer than that in liver and kidney, moreover, at 50 μg/kg bw concentration, values of MRT_lastin tissues by PO treatment were longer than IV route. In addition, in muscle plus skin and gill, the values of T_1/2z increased with increasing administered concentration. The calculated T_1/2z of EMB in the skinny muscle after IV treatment was 103.40 h as well as after PO treatment were 29.75, 56.78, and 91.62 h, respectively. The result is far less than the studies with 9.2 days in the study of Sevatdal et al. (20), and in another pharmacokinetic study, the T_1/2z in muscle was calculated as 11.1 days, and a clear linear relationship between dose and residual concentration was observed (43).

The study demonstrated that when used in the range of 50–500 μg/kg bw, the metabolism of EMB is relatively slow while providing an important therapeutic effect.

Bioavailability was proposed by Oser in 1945, referring to the degree and rate of the absorption of the drug (44). Drug content is not the only criterion for determining the curative effect. In addition to drug content, bioavailability should be considered. The bioavailability of EMB was only 38% in cod by determined concentrations of EMB in plasma collected from treated fish after IV (50 μg/kg bw) and PO (50 μg/kg bw) (24). However, the bioavailability of EMB in crucian carp was 52.70%. It showed that EMB has a high bioavailability in crucian carp, and the crucial factor that causes bioavailability to vary is the different fish species.

The serum biochemical indexes including TP, T-CHO, ALB, ALP, BUN, CRE, and GLU were not significantly different (P > 0.05) from the control group. In contrast, AST and ALT in experimental groups exhibited significant differences (P < 0.05) compared with the control group. In summary, the results of our study indicated that EMB would induce altered serum parameters.

AST and ALT are important indicator enzymes of the liver (45). The contents of AST and ALT in tilapia were related to hepatic injury (46). In comparison with the control group, ALT and AST levels were increased after administration. In brief, the results of our study indicated that EMB would induce serum parameters and EMB has an effect on liver when metabolized in crucian carp. However, the single PO treatment showed a significant increase in AST and ALT levels on day 1 (P < 0.05), with the content gradually decreasing after day 1, and it still showed a downward trend until day 7, which was still significantly higher than those of the control group.

The effect of EMB on the biochemical parameters of rainbow trout was studied. It was found that the fish was administered with EMB of 50 μg/kg bw daily for 21 consecutive days, and the result showed that AST content was increased (22). Increased ALT and AST levels obviously projected EMB as an inducer of hepatic dysfunction in Nile tilapia possibly due to damage of hepatic cells, disrupted Kreb's cycle, and other hepato-cellular damage (46, 47).

The levels of ALT and AST content proved that in the range of 50–500 μg/kg bw EMB stimulated the crucian carp, but the content became normal over time, indicating that there was no substantial damage caused to the body, meanwhile, it was also proved in previous studies (12, 19, 48).

The results of the present study suggested that the effects of EMB on serum biochemical indices are revocable. Nevertheless, it did not reach normalcy after 7 days of dosing. Those data would provide a valid reference for researchers and aquaculturists in assessing the health condition of cultured crucian carp after EMB administration in tropical conditions.