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Peer-Review Record

Depletion of Albendazole and Its Metabolites and Their Impact on the Gut Microbial Community Following Multiple Oral Dosing in Yellow River Carp (Cyprinus carpio haematopterus)

by Yue Liu, Yan Dai, Yan-Ni Zhang, Wen-Rui Wang, Yu-Xin Chen, Yang-Guang Jin, Long-Ji Sun, Shi-Hao Li, Fang Yang, Xing-Ping Li * and Fan Yang *
Reviewer 1: Anonymous
Reviewer 2: Anonymous
Reviewer 3:
Submission received: 28 June 2025 / Revised: 7 August 2025 / Accepted: 11 August 2025 / Published: 14 August 2025
(This article belongs to the Special Issue Aquaculture Pharmacology)

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript describes the tissue distribution characteristics of albendazole and its metabolites in Yellow River carp as well as its negative effect on the gut microbiota. Overall, the study design was appropriate and the manuscript writing was well-organized. However, I have some questions and comments to the authors as follows.

 

Introduction

  1. Line 67: The reference [11] is inappropriate as this is unrelated to the albendazole use in aquaculture.
  2. Line 82-85: Please provide the reference for the MRL of albendazole according to the Chinese regulation.
  3. Please review previous studies regarding albendazole pharmacokinetic, tissue residue, efficacy, toxicity, and other aspects in aquatic animals. Also, please point out the research gap.

 

Materials and methods

  1. Line 119: Did the authors really use albendazole powder (6 g/100 g)? The drug purity is only 6% which is considered too low for this kind of study. Otherwise, other unidentified ingredients (94%) can interfere the results. In other word, the observed effects on tissue distribution and gut microbiota might be due to other components rather than albendazole per se. Please recheck this information.
  2. Line 155: Why did the authors select the dose of 12 mg/kg? Please provide some references to support this dose selection.
  3. Line 156-157: The method of drug administration was unclear (either via oral gavage or medicated feed?). How could the authors mix the drug with “feed” to prepare a “suspension”?
  4. Line 159-164: Please specify whether the authors used anticoagulant and anesthetics during the blood sampling.
  5. Line 202: Please clarify whether “99%” means “99th percentile tolerance”?

 

Results

  1. Table 5 and 6: Please use 2 significant figures for the elimination rate constant (λz). For instance, use 0.024 instead of 0.02, or use 0.010 instead of 0.01.
  2. Figure 4 (Line 325): Please remove “….in ruminant muscle”.
  3. Figure 5 and 6: Please specify what are “a” and “b” in these figures. For example, “a” in Figure 5 is Phylum, and “b” is genus.

 

Discussion

  1. Line 479-480: Please remove the phrase “…,confirming that the liver and kidney are key organs for ABZ metabolism” because the present data was not enough to support this conclusion. The detection of high drug residue in the kidney does not necessarily imply that the kidney is the major organ for drug metabolism.
  2. The discussion on gut microbiota (Line 515-564) is terrible, with a lot of misinformation. Please consult experts in this field and revise the discussion accordingly. The specific examples are as follows:

13.1 Line 518-532: Please note that the phylum Pseudomonadota (or Proteobacteria) is a very big phylum of Gram-negative bacteria. This phylum is not only include Pseudomonas spp., but also contain several important pathogenic bacteria of fish and human such as Vibrio, Aeromonas, Salmonella, E. coli, Edwardsiella, Franciscella. The authors should not emphasize Pseudomonas too much while ignore other more important bacteria of fish.

13.2 Line 538: Mycobacterium is a bacteria in the phylum Actinomycetota (or Actinobacteria), not belonging to the phylum Pseudomonadota or Bacteroidota.

13.3 Line 553: This sentence is totally wrong! Streptococcus spp. such as S. iniae and S. agalactiae are one of the most significant pathogens of fish.

13.4 Line 562: This is misconception. Co-administration with suitable antibiotics or probiotics is strongly discouraged (not advisable!). Antibiotics or probiotics are mutually antagonistic. Co-administration of antibiotics and probiotics can leading to treatment failure and increasing drug resistance in bacteria.

 

  1. Conclusion is too long. Please make it more concise.

Author Response

Comment: This manuscript describes the tissue distribution characteristics of albendazole and its metabolites in Yellow River carp as well as its negative effect on the gut microbiota. Overall, the study design was appropriate and the manuscript writing was well-organized. However, I have some questions and comments to the authors as follows.

Response: We sincerely thank the reviewer for their careful evaluation of our manuscript and for recognizing the merits of our study design and manuscript structure. We appreciate the constructive comments and suggestions, which have helped us improve the quality and clarity of our work. Below, we provide point-by-point responses to each of the reviewer’s questions and comments. All changes made in response to the reviewer’s suggestions have been highlighted in the revised manuscript.

 

  1. Comment: Line 67: The reference [11] is inappropriate as this is unrelated to the albendazole use in aquaculture.

Response: Thank you for pointing this out. We agree with the reviewer’s observation that Reference [11] was not directly relevant to the use of albendazole in aquaculture. In response, we have replaced the previous reference with the following, which is more pertinent. The new reference is now numbered as [12]. Thank you for your valuable suggestion.

[12] Min, J.K., Ji, Y.K., Dong, W.S., Mi, O.E. Application potential of albendazole as an aquatic animal drug based on its safety, efficacy, and residue profiles. Toxicological Research. 2024, 40, 519–531. https://doi.org/10.1007/s43188-024-00244-1.

 

  1. Comment: Line 82-85: Please provide the reference for the MRL of albendazole according to the Chinese regulation.

Response: Thank you for this important observation. We have now added China’s National Food Safety Standard GB 31650‑2019 as the appropriate reference specifying the MRL for albendazole in edible animal products. This standard sets the MRL for albendazole in fish (skin-on muscle) at 100 µg/kg, with albendazole‑2‑aminosulfone (ABZ‑2‑NH2–SO2) designated as the marker residue. The citation has been added in the revised manuscript accordingly.

New citations:

[19] MARA (Ministry of Agriculture and Rural Affairs of the People’s Republic of China). The National Standard for Food Safety-Maximum Residue Limit of Veterinary Drugs in Food/GB 31650 2019. 2019. https://www.fas.usda.gov/data/china-china-publishes-maximum-residue-limits-veterinary-drugs-food.

 

  1. Comment: Please review previous studies regarding albendazole pharmacokinetic, tissue residue, efficacy, toxicity, and other aspects in aquatic animals. Also, please point out the research gap.

Response: We have carefully reviewed and incorporated relevant literature on the pharmacokinetics, tissue residue, efficacy, and toxicity of albendazole (ABZ) in aquatic species. We have listed the additions and updates below.

Previous studies have demonstrated that ABZ is effective against a variety of endoparasites in aquaculture, such as monogeneans [25,26,27], Loma salmonae [28], and Neoechinorhynchus buttnerae [29], and it is widely used due to its broad-spectrum antiparasitic activity [12]. Pharmacokinetic and residue studies in fish species such as tambaqui [30], rainbow trout [15], tilapia [15], Atlantic salmon [15] and Yellow River carp [31] have shown that ABZ and its primary metabolite albendazole sulfoxide (ABZSO) are rapidly absorbed and eliminated, although tissue distribution patterns and elimination rates may vary depending on the species, temperature, and dosage regimen. Regarding toxicity, existing studies have reported that although ABZ has a wide safety margin in fish, high or prolonged exposure may affect liver function and induce oxidative stress [32]. However, research on the gut microbiota response to ABZ exposure in fish is still extremely limited.

And new citations are listed below:

[12] Min, J.K., Ji, Y.K., Dong, W.S., Mi, O.E. Application potential of albendazole as an aquatic animal drug based on its safety, efficacy, and residue profiles. Toxicological Research. 2024, 40, 519–531. https://doi.org/10.1007/s43188-024-00244-1.

[25] Raimundo, R.J.K., Carliane, M.G.A., Marcos, S.B.O., Clara, B.S., Abthyllane, A.C., Marcela, N.V., Eliane, T.O.Y., Marcos, T.D. Albendazol tem eficácia em controlar monogenéticos em Colossoma macropomum (Serrasalmidae): banhos terapêuticos e seus efeitos fisiológicos e histopatológicos. Revista Brasileira de Parasitologia Veterinária. 2024, 33, 3, e004924. https://doi.org/10.1590/S1984-29612024044.

[26] Carliane, M.G.A., Joziele, N.N., Isaac, B.B., Joilson, R.D.S., Gracienhe, G.S., Marcos, T.D. Albendazole, levamisole and ivermectin are effective against monogeneans of Colossoma macropomum (Pisces: Serrasalmidae). Journal of Fish Diseases. 2019, 42, 3, 405-412. https://doi.org/10.1111/jfd.12952.

[27] Luis, E.E.B. Ángel, G.L.C., Gustavo, A.R.M.O., Selene, M.A.R., Daniela, A.M.I., Edén, A.R.V., Zohar, I.Z., Mayra, I.G.M. Effective control and treatment of Rhabdosynochus viridisi (Monogenea: Diplectanidae) in Centropomus viridis (Teleostei: Centropomidae) in marine aquaculture. Latin American Journal of Aquatic Research. 2025, 53, 3, 375-387. https://doi.org/10.3856/vol53-issue3-fulltext-3338.

[28] Speare, D.J., Athanassopoulou, F., Daley, J., Sanchez, J.G. A Preliminary Investigation of Alternatives to Fumagillin for the Treatment of Loma salmonae Infection in Rainbow Trout. Journal of Comparative Pathology. 1999, 121, 3, 241-248. https://doi.org/10.1053/jcpa.1999.0325.

[29] Caio, F.S.F., Franmir, R.B., Fernanda, A.S., Damy, C.M.., Patrícia, C.M., Cláudia, M., Edsandra, C.C. Albendazole and praziquantel for the control of Neoechinorhynchus buttnerae in tambaqui (Colossoma macropomum). Aquaculture International. 2021, 29, 1495–1505. https://doi.org/10.1007/s10499-021-00687-5.

[30] Rafaelle, P. C.; Patrícia, A. C. B.; Maria, J. S. R.; Edsandra, C. C. and Felix, G. R. R. Depletion study and estimation of the withdrawal period for albendazole in tambaqui (Colossoma macropomum) parasitised by acanthocephalan (Neoechinorhynchus buttnerae) treated with albendazole-containing feed. Food Additives & Contaminants: Part A. 2021, 38, 11, 1883-1896. https://doi.org/10.1080/19440049.2021.1954700.

[32] Lacrămioara, G.N., Lorena, D., Mirela, C., Cristian, R., Angelica, D., Iulia, G., Floricel, M.D., Maria, D.S., Camelia, V. The Protective Effects of Korill Product on Carp Fingerlings Reared in High Densities and Challenged with Albendazole Treatment. Fishes. 2023, 8, 3, 153. https://doi.org/10.3390/fishes8030153.

 

  1. Comment: Line 119: Did the authors really use albendazole powder (6 g/100 g)? The drug purity is only 6% which is considered too low for this kind of study. Otherwise, other unidentified ingredients (94%) can interfere the results. In other word, the observed effects on tissue distribution and gut microbiota might be due to other components rather than albendazole per se. Please recheck this information.

Response: Thank you for your thoughtful comment and for raising this important point. We confirm that the albendazole used in this study was 6% albendazole powder, which is a commercially approved formulation widely used in aquaculture practices in China. We understand the concern regarding the potential influence of excipients on both pharmacokinetic behavior and gut microbiota composition.

To address this, we consulted the manufacturer of the product used in this study. According to the manufacturer's specifications, the remaining 94% of the formulation consists of standard pharmaceutical excipients, such as dispersing agents and fillers, that are commonly used in veterinary formulations and are not known to possess antimicrobial or pharmacological activity that would interfere with albendazole metabolism or intestinal microbial ecology.

Additionally, it is important to emphasize that the direct use of raw albendazole (≥98% purity) in aquaculture is strictly prohibited under Chinese regulatory guidelines. Only approved commercial formulations are permitted for use in fish, and our study adhered to this requirement to ensure compliance with national regulations and to reflect real-world usage conditions in aquaculture settings.

We acknowledge that using a formulation rather than pure albendazole introduces some complexity. However, our study design was aimed at evaluating the practical implications of ABZ administration in aquaculture, including both tissue disposition and potential microbiota impact. Therefore, we believe the use of the commercial formulation enhances the translational value of our findings for actual veterinary application in fish farming. Based on your suggestion, we have updated the relevant information (Lines 170 to 175).

 

  1. Comment: Line 155: Why did the authors select the dose of 12 mg/kg? Please provide some references to support this dose selection.

Response: Thank you very much for your valuable comment. The dosage of 12 mg/kg BW was determined based on the recommended dosage provided in the instructions for the commercial 6% albendazole soluble powder approved for use in aquaculture in China. According to the label, the recommended administration is 0.2 g/kg BW of the formulation, which corresponds to 12 mg/kg BW of active albendazole. This dosage is in line with standard therapeutic practices for antiparasitic treatment in aquaculture and reflects real-world usage conditions. We have clarified this point and added the relevant information to the revised manuscript (Lines 170 to 175).

 

  1. Comment: Line 156-157: The method of drug administration was unclear (either via oral gavage or medicated feed?). How could the authors mix the drug with “feed” to prepare a “suspension”?

Response: Thank you for your constructive comment. We apologize for the confusion in the description of the administration method. In our study, albendazole powder was accurately weighed and suspended in an appropriate volume of 1% sodium carboxymethyl cellulose (CMC-Na) solution to prepare a suspension containing 12 mg/mL of ABZ (Lines 176 to 178). The suspension was freshly prepared each day before administration and delivered to the fish via oral gavage using a sterile syringe fitted with a flexible feeding needle. Thank you.

 

7.Comment: Line 159-164: Please specify whether the authors used anticoagulant and anesthetics during the blood sampling.

Response: Thank you for your comment. We confirm that heparin sodium was used as an anticoagulant during blood collection to prevent coagulation. However, no anesthetics were used during the sampling process in order to avoid potential interference with the pharmacokinetics and disposition of albendazole and its metabolites. We have clarified this information in the revised manuscript to improve methodological transparency (Lines 186 to 189).

 

8.Comment: Line 202: Please clarify whether “99%” means “99th percentile tolerance”?

Response: Thank you for pointing this out. The “99%” refers to the 99th percentile of the tolerance limit (MRL), meaning that 99% of the population is expected to have residue concentrations below the MRL (Lines 230 to 231).

 

9.Comment: Table 5 and 6: Please use 2 significant figures for the elimination rate constant (λz). For instance, use 0.024 instead of 0.02, or use 0.010 instead of 0.01.

Response: Done (Tables 5 and 6). Thanks.

 

10.Comment: Figure 4 (Line 325): Please remove “….in ruminant muscle”.

Response: Done (Figure 4; Lines 360-362). Thanks.

 

11.Comment: Figure 5 and 6: Please specify what are “a” and “b” in these figures. For example, “a” in Figure 5 is Phylum, and “b” is genus.

Response: Done in Figure 5. And a new Figure 6 was provided. Thanks.

 

12.Comment: Line 479-480: Please remove the phrase “…,confirming that the liver and kidney are key organs for ABZ metabolism” because the present data was not enough to support this conclusion. The detection of high drug residue in the kidney does not necessarily imply that the kidney is the major organ for drug metabolism.

Response: Done (Line 517). Thank you.

 

13.Comment: The discussion on gut microbiota (Line 515-564) is terrible, with a lot of misinformation. Please consult experts in this field and revise the discussion accordingly.

Response: Thank you very much for your detailed comments and insightful feedback on the gut microbiota discussion section. We deeply appreciate your attention to detail, especially given the importance of this subject. We have carefully revised the relevant sections to address each of your specific concerns.

 

13.1. Comment: Line 518-532: Please note that the phylum Pseudomonadota (or Proteobacteria) is a very big phylum of Gram-negative bacteria. This phylum is not only include Pseudomonas spp., but also contain several important pathogenic bacteria of fish and human such as Vibrio, Aeromonas, Salmonella, E. coli, Edwardsiella, Franciscella. The authors should not emphasize Pseudomonas too much while ignore other more important bacteria of fish.

Response: We were really sorry for our careless mistakes and we have made changes to this section (Lines 553 to 566). Thank you for your reminder and expounding.

13.2. Comment: Line 538: Mycobacterium is a bacteria in the phylum Actinomycetota (or Actinobacteria), not belonging to the phylum Pseudomonadota or Bacteroidota.

Response: Thank you for pointing out this taxonomic inaccuracy. We have removed the incorrect statement regarding Mycobacterium and its classification.

 

13.3. Comment: Line 553: This sentence is totally wrong! Streptococcus spp. such as S. iniae and S. agalactiae are one of the most significant pathogens of fish.

Response: We were really sorry for our careless mistakes and we have made changes to this sentence (Lines 595 to 601). Thank you for your reminder.

New citations:

[46] Terutoyo, Y. Streptococcosis in aquaculture. Fish Pathology. 2016, 51, 2, 44–48. https://doi.org/10.3147/jsfp.51.44.

 

13.4. Comment: Line 562: This is misconception. Co-administration with suitable antibiotics or probiotics is strongly discouraged (not advisable!). Antibiotics or probiotics are mutually antagonistic. Co-administration of antibiotics and probiotics can leading to treatment failure and increasing drug resistance in bacteria.

Response: We fully agree with your professional opinion. In accordance with your suggestion, we have removed the ambiguous sentence to avoid any misconceptions regarding the co-administration of antibiotics and probiotics (Lines 608-609).

 

14.Comment: Conclusion is too long. Please make it more concise.

Response: We sincerely appreciate your expert review of this manuscript. Based on your invaluable suggestions, we have revised the conclusion section (Lines 611-623).

Reviewer 2 Report

Comments and Suggestions for Authors

This study examined the study titled "Depletion of Albendazole and Its Metabolites and Their Impact on the Gut Microbial Community Following Multiple Oral Dosing in Yellow River Carp (Cyprinus Carpio Haematopterus)". The pharmacokinetic profile of albendazole was determined in plasma, liver, muscle, and kidney tissues after seven days of once-daily administration. Furthermore, the gut microbiota profile was determined from samples taken on four different days following albendazole administration compared to a control group. Considering the data provided, it is believed that this study will contribute to scientific research. However, it is crucial to address the issues and answer the questions listed below.

Corrections and questions:

  1. Why were five fish selected at each sampling time for pharmacokinetic and tissue residue determination?
  2. The importance of the Yellow River carp (Cyprinus carpio haematopterus), a fish farmed in China, could be mentioned in the introduction.
  3. The statement "Forty healthy Yellow River carp (Cyprinus carpio haematopterus) with an average BW of 132 of 0.73 kg (range: 0.53–1.02 kg) were used for the residue depletion study." Body weight is as important as water temperature and cumulative dose administration in fish, as are the pharmacokinetics of drugs.        Why did you use such a wide weight scale in your study? Could you provide this value as the mean plus standard deviation?
  4. How were the sampling times and number chosen?
  5. The tables should be uniform. In some places, two numbers have been mistakenly placed after the decimal point (e.g. 1.334 ± 0.16, 0.733 ± 0.25); these should be increased to three.
  6. What is the reason for the AUC being above 10%?
  7. Although its half-life in tissues varies between 38 and 55 hours, and the conclusions section (568 lines) states that it is slowly excreted and widely distributed, how can it fall below the MRL in 16 days?
  8. It would be helpful to include information on the use of albendazole for seven days and the diseases it is used to treat.
  9. In the cited article "Dai, Y.; Yang, H.Y.; Yang, F.; Li, X.P.; Liu, Y.; Jin, Y.G.; Li, Z.E.; Duan, M.H.; Zhang, Y.N.; Yang, F. Pharmacokinetics and Tissue Distribution of Albendazole and Its Three Metabolites in Yellow River Carp (Cyprinus carpio haematopterus) after Single Oral Administration. Journal of Agricultural and Food Chemistry. 2025, 73, 3, 1824–1834. 668 https://doi.org/10.1021/acs.jafc.4c08959.", the pharmacokinetic profile is quite variable despite similar water temperatures. Why?
  10. Body weight should be added to the factors listed in line 449.

Author Response

Comment: This study examined the study titled "Depletion of Albendazole and Its Metabolites and Their Impact on the Gut Microbial Community Following Multiple Oral Dosing in Yellow River Carp (Cyprinus Carpio Haematopterus)". The pharmacokinetic profile of albendazole was determined in plasma, liver, muscle, and kidney tissues after seven days of once-daily administration. Furthermore, the gut microbiota profile was determined from samples taken on four different days following albendazole administration compared to a control group. Considering the data provided, it is believed that this study will contribute to scientific research. However, it is crucial to address the issues and answer the questions listed below.

Response: Thank you for your thoughtful summary and for recognizing the potential contribution of our study to scientific research. We appreciate your constructive feedback and have carefully addressed all issues and questions raised in your detailed comments. Below, we provide specific responses and clarifications, and have revised the manuscript accordingly to improve its scientific accuracy, methodological transparency, and overall clarity. We hope that the revisions meet your expectations and enhance the quality and impact of the manuscript.

 

1.Comment: Why were five fish selected at each sampling time for pharmacokinetic and tissue residue determination?

Response: Thank you for your comment. We selected five fish per sampling time point for pharmacokinetic and tissue residue analysis based on standard practices in aquatic pharmacokinetic and residue depletion studies. This sample size provides a balance between statistical robustness and ethical considerations, including the minimization of animal use. Furthermore, this approach is consistent with several previously published studies that used five fish per time point for similar analyses (e.g., DOI: 10.1016/j.etap.2011.12.001; DOI: 10.1111/jvp.13425; DOI: 10.1111/jvp.12689). These references demonstrate that five fish per time point is generally adequate to characterize pharmacokinetic profiles and support withdrawal time estimation under controlled experimental conditions.

 

2.Comment: The importance of the Yellow River carp (Cyprinus carpio haematopterus), a fish farmed in China, could be mentioned in the introduction.

Response: Your suggestion is very valuable. We have briefly described this aspect in the manuscript (Lines 60 to 62). Thank you.

New citations:

[9] Jiang, X.N.; Qu, F.H.; Ge, Y.L.; Li, C.T.; Shi, X.D.; Hu, X.S.; Cheng, L.; Zhao, X.Y.; Jia, Z.Y. Effects of Dietary Protein Levels on the Growth, Physiological, and Biochemical Indices of Juvenile Yellow River Carp (Cyprinus carpio haematopterus). Animals. 2025, 15, 12, 1800. https://doi.org/10.3390/ani15121800.

 

3.Comment:The statement "Forty healthy Yellow River carp (Cyprinus carpio haematopterus) with an average BW of 132 of 0.73 kg (range: 0.53–1.02 kg) were used for the residue depletion study." Body weight is as important as water temperature and cumulative dose administration in fish, as are the pharmacokinetics of drugs. Why did you use such a wide weight scale in your study? Could you provide this value as the mean plus standard deviation?

Response: Thank you for your valuable comment. We agree that body weight is an important factor influencing drug pharmacokinetics and residue depletion in fish. In this study, we selected fish to reflect the weight variation commonly encountered in practical aquaculture settings. As suggested, we have now provided the mean body weight with standard deviation (0.73 ± 0.085 kg) in the revised manuscript (Line 147).

To minimize variability, we selected 35 fish with relatively uniform body weights for the experimental groups. The remaining 5 fish, which had larger weight deviations, were assigned to the blank control group. We acknowledge this potential source of variability and will take greater care to control weight range more strictly in future studies. Thank you again for your helpful suggestion.

 

4.Comment: How were the sampling times and number chosen?

Response: Thank you for your insightful comment. The selection of sampling times and number was based on the pharmacokinetic data obtained from our previous laboratory study titled “Pharmacokinetics and Tissue Distribution of Albendazole and Its Three Metabolites in Yellow River Carp (Cyprinus carpio haematopterus) after Single Oral Administration” (DOI: 10.1021/acs.jafc.4c08959) (Lines 184-186). This earlier study provided crucial insights into the absorption, distribution, and elimination patterns of albendazole and its metabolites following a single oral dose, which informed the sampling strategy and design of the current multiple-dose residue depletion study. We appreciate your attention to this important aspect of the methodology.

 

5.Comment: The tables should be uniform. In some places, two numbers have been mistakenly placed after the decimal point (e.g. 1.334 ± 0.16, 0.733 ± 0.25); these should be increased to three.

Response: Thank you for your valuable comment. We have carefully reviewed all tables in the manuscript and identified inconsistencies in the number of decimal places, particularly in the representation of standard deviations. As suggested, we have revised the formatting so that both the mean values and their corresponding standard deviations are consistently reported with three decimal places throughout all tables. However, for ABZ-2-NH₂−SO₂ (Table 4), due to its particularly low concentration, we have retained four decimal places to maintain appropriate numerical resolution. These corrections have been implemented to improve clarity and maintain uniformity in data presentation.

 

6.Comment: What is the reason for the AUC being above 10%?

Response: Thank you for your comment. We apologize for the confusion caused by the lack of a detailed explanation regarding the AUC% parameter. In this study, AUC% refers to the percentage of the area under the curve extrapolated from the last quantifiable concentration to infinity (i.e., (AUC0-∞–AUC0-last)/AUC0-∞×100%). This extrapolated portion indicates how much of the total AUC (AUC0–∞) is estimated rather than directly calculated from observed data. In our results, the maximum AUC% was 12.50%, which is within the generally accepted threshold of ≤20%. Values below this limit are considered acceptable and indicate that the AUC0-∞ is reliable and accurately estimated.

 

7.Comment: Although its half-life in tissues varies between 38 and 55 hours, and the conclusions section (568 lines) states that it is slowly excreted and widely distributed, how can it fall below the MRL in 16 days?

Response: Thank you very much for your insightful comment. We agree that the t1/2λZ of albendazole and its metabolites in tissues (ranging from 38 to 55 hours) indicates relatively slow elimination. However, the calculated withdrawal period of 16 days is based specifically on residue levels in skin-on muscle, which is the target tissue for regulatory monitoring in most countries. This calculation was performed using the WT 1.4 software, following the EMA guidelines, and reflects the time required for residues in edible tissue to fall below the established MRL.

Although higher concentrations may persist longer in certain internal organs (e.g., liver and kidney), these tissues are not typically consumed and are not considered in regulatory residue monitoring. Therefore, the 16-day withdrawal period is scientifically justified and complies with international standards, which focus on residues in consumable muscle tissues.

Furthermore, the current calculated withdrawal period of 16 days corresponds to approximately 7 times the t1/2λZ of ABZSO in skin-on muscle (54.19 hours). At this point, the drug concentration would be reduced to only about 0.78% of its initial value, which is negligible and far below regulatory thresholds.

 

8.Comment: It would be helpful to include information on the use of albendazole for seven days and the diseases it is used to treat.

Response: Thank you for your valuable suggestion. Based on your feedback, we consulted the official instructions for the 6% albendazole soluble powder used in this study and integrated information relevant to practical aquaculture use.

According to the product label and common breeding practices in China, the 7-day oral dosing regimen (12 mg/kg BW) is employed to treat ectoparasitic infestations caused primarily by monogenean parasites, such as Dactylogyrus spp. and Trichodina spp., which frequently infect freshwater carp species in aquaculture. These parasites commonly attach to the gills and skin, causing significant morbidity and mortality by damaging gill tissue, impairing respiration, and promoting secondary infections. Albendazole is reported to effectively target and reduce these monogenean populations when administered orally at this dosage over a short-term period.

We have added this context into the revised Introduction and Materials and Methods sections to better explain the rationale and relevance of the treatment protocol:

“The administered 7-day regimen corresponds to the recommended therapeutic practice using 6% albendazole powder for freshwater fish in China, targeting monogenean parasites such as Dactylogyrus and Trichodina spp., which commonly infect carp species.” (Lines 172-175)

 

9.Comment: In the cited article "Dai, Y.; Yang, H.Y.; Yang, F.; Li, X.P.; Liu, Y.; Jin, Y.G.; Li, Z.E.; Duan, M.H.; Zhang, Y.N.; Yang, F. Pharmacokinetics and Tissue Distribution of Albendazole and Its Three Metabolites in Yellow River Carp (Cyprinus carpio haematopterus) after Single Oral Administration. Journal of Agricultural and Food Chemistry. 2025, 73, 3, 1824–1834. 668 https://doi.org/10.1021/acs.jafc.4c08959.", the pharmacokinetic profile is quite variable despite similar water temperatures. Why?

Response: Thank you for raising this insightful question. The variability observed in the pharmacokinetic profiles between the cited single-dose study and our current multiple-dose study stems from key differences in dosing regimens and resulting accumulation behavior.

In the previous single-dose work, a single 12 mg/kg ABZ administration was evaluated, revealing absorption and elimination characteristics over a short period. In contrast, our present study involved seven consecutive daily doses, which resulted in drug accumulation, particularly of the active metabolite ABZSO. Repeated administration can lead to higher steady‑state concentrations and altered pharmacokinetic parameters compared to a single dose.

Additionally, drug accumulation kinetics depend on factors such as the elimination half-life, dosing interval, and linearity of metabolism. Multi-dose pharmacokinetics often produce longer apparent half-lives or higher area-under-the-curve (AUC) values at steady state. These effects are consistent with established pharmacokinetic principles and have been described in both clinical and veterinary drug studies.

In summary, the observed differences in pharmacokinetic parameters between the single-dose study and our repeated‑dose design are expected and reflect the impact of drug accumulation, steady-state dynamics, and potentially non-linear elimination following multiple administrations.

 

10.Comment: Body weight should be added to the factors listed in line 449.

Response: Thank you for your thoughtful suggestion. We agree that body weight is an important factor influencing drug pharmacokinetics in fish, as it can affect absorption, distribution, metabolism, and excretion processes. In response to your comment, we have revised the manuscript to explicitly include body weight as one of the contributing factors to the pharmacokinetic differences observed between studies. (Line 494 to 496). Thank you.

Reviewer 3 Report

Comments and Suggestions for Authors

Your manuscript presents a valuable two‐part study comparing the pharmacokinetics and withdrawal periods of albendazole and its metabolites alongside gut microbiota changes, but because it lacks the necessary analytical validation data for the drug and its metabolites and does not include statistical analysis of the experimental results, it is declined at this stage and may be considered for review once these elements are fully addressed.

Author Response

Comment: Your manuscript presents a valuable two‐part study comparing the pharmacokinetics and withdrawal periods of albendazole and its metabolites alongside gut microbiota changes, but because it lacks the necessary analytical validation data for the drug and its metabolites and does not include statistical analysis of the experimental results, it is declined at this stage and may be considered for review once these elements are fully addressed.

Response: Thank you very much for your thoughtful review and constructive comments. We appreciate your recognition of the value of the two-part study comparing the pharmacokinetics and withdrawal periods of albendazole and its metabolites, alongside gut microbiota changes.

Regarding the analytical validation data, we acknowledge its importance. The method validation, including calibration, accuracy, precision, recovery, and detection limits, was extensively described in a recent study published by our laboratory: “Pharmacokinetics and Tissue Distribution of Albendazole and Its Three Metabolites in Yellow River Carp (Cyprinus carpio haematopterus) after Single Oral Administration” (DOI: 10.1021/acs.jafc.4c08959). Since the method used in this current study is the same, we have referenced this publication and decided not to duplicate the validation details in the current manuscript. However, if needed, we are happy to provide the full validation data or include a more detailed summary in the revised manuscript.

Regarding the statistical analysis, we would like to clarify that the current study involved a single dosing regimen and was conducted under constant water temperature conditions. As such, there were no experimental groups or variables requiring comparative statistical testing. The concentration data were summarized and analyzed using Microsoft Excel, and the withdrawal period was calculated using WT 1.4 software, which follows established EMA guidelines for residue depletion studies.

Therefore, we did not perform additional statistical tests such as ANOVA or multiple comparisons, as the experimental design did not include variable treatment groups. However, if the reviewers believe it would strengthen the manuscript, we are open to including descriptive statistics (e.g., coefficient of variation or confidence intervals) or expanding on the analytical approach in the revised version.

We hope this clarification addresses your concern and thank you again for your valuable comments.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors responded to my comments properly. I have no further questions.

Author Response

Comment: The authors responded to my comments properly. I have no further questions.

Response: Thanks very much.

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Author;

The study titled "Depletion of Albendazole and Its Metabolites and Their Impact on the Gut Microbial Community Following Multiple Oral Dosing in Yellow River Carp (Cyprinus Carpio Haematopterus" has been reviewed after corrections. Firstly, I would like to thank the researchers for their kind and scientific responses to the suggestions and corrections provided.

I wish you continued success.


Author Response

Comment: The study titled "Depletion of Albendazole and Its Metabolites and Their Impact on the Gut Microbial Community Following Multiple Oral Dosing in Yellow River Carp (Cyprinus Carpio Haematopterus" has been reviewed after corrections. Firstly, I would like to thank the researchers for their kind and scientific responses to the suggestions and corrections provided. I wish you continued success.

Response: Thanks very much.

Reviewer 3 Report

Comments and Suggestions for Authors

Authors have revised the manuscript. But one thing, the authors need to consider the following; It is necessary to supplement the statistical description of the WT1.4 program’s analysis. For the authors reference-“Comparison of statistical approaches recommended by FDA and EU for withdrawal time estimation of veterinary drugs.” Food and Chemical Toxicology. 2012;50(3–4):773–778.

Author Response

Comments: Authors have revised the manuscript. But one thing, the authors need to consider the following; It is necessary to supplement the statistical description of the WT1.4 program’s analysis. For the authors reference-“Comparison of statistical approaches recommended by FDA and EU for withdrawal time estimation of veterinary drugs.” Food and Chemical Toxicology. 2012;50(3–4):773–778.

 

Response: Thank you for your helpful suggestion. In response, we have supplemented the statistical description of the WT 1.4 program’s analysis in the revised manuscript to enhance clarity and transparency.

 

The analysis was performed using WT 1.4 software at a 95% confidence level, ensuring that the 99th percentile of the tolerance limit (MRL) was not exceeded. Prior to the withdrawal time calculation, the dataset was evaluated using WT 1.4 to confirm compliance with the four fundamental assumptions of linear regression: (1) linearity of logₑ(concentration) versus time, (2) independence of observations, (3) normality of residuals on a log scale, and (4) homoscedasticity [32].

 

Additionally, we have cited the recommended reference—“Comparison of statistical approaches recommended by FDA and EU for withdrawal time estimation of veterinary drugs” (Food and Chemical Toxicology, 2012; 50(3–4):773–778)—to support our methodology and provide context for readers.

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