Liver Damage in Ctenopharyngodon idellus Induced by Nanoplastics and Cadmium Exposure

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

General Comments

This manuscript investigates the combined effects of PS-NPs and Cd on liver injury in grass carp using biochemical, molecular, histological and transcriptomic approaches. The topic is relevant to current concerns regarding combined pollution in aquatic environments. However, several conclusions are not fully supported by the presented data, and some interpretations appear speculative. 

specific comments:

1. On Page 4, Lines 120–128, the authors selected 1 mg/L PS-NPs and 1 mg/L Cd based mainly on previous toxicity studies. However, the manuscript aims to provide references for ecological risk assessment (Page 2, Lines 22–23; Page 3, Lines 98–99). The selected concentrations appear substantially higher than those typically reported in natural freshwater systems.
2. In the Abstract (Page 2, Lines 16–20) and Conclusions (Page 13, Lines 392–394), the authors state that co-exposure caused synergistic effects. However, several biochemical endpoints do not support this conclusion. For example, GSH-ST activity in the co-exposure group was not significantly different from the control (Page 5, Lines 187–189), and LDH activity showed no significant difference among treatments (Lines 190–190). Even in the Discussion (Page 11, Lines 307–309), the authors acknowledge antagonistic interactions at the biochemical level. Therefore, describing the overall response simply as "synergistic" may be an oversimplification. The interaction appears endpoint-dependent and should be discussed more carefully.
3. On Page 6, Lines 218–221, the authors observed that HO-1 expression increased under single exposure but returned to near-control levels under co-exposure. Subsequently, they suggest an uncoupling of antioxidant and anti-inflammatory pathways (Page 12, Lines 337–339). However, no direct evidence for pathway uncoupling was provided. Alternative explanations, such as transcriptional inhibition or feedback regulation under severe stress, cannot be excluded. The authors should moderate the interpretation. There are also similar situations like this, e.g., sections 4.2 and 4.3.
4. The manuscript identifies lipid metabolism and steroid biosynthesis as major pathways affected by co-exposure (Page 9, Lines 266–277). Based on these findings, the Discussion extensively focuses on lipid remodeling and endocrine disruption (Page 12–13, Lines 356–372). However, no physiological measurements, such as lipid accumulation, triglyceride content, cholesterol levels, or hormone concentrations, were conducted. Therefore, the biological significance of these enriched pathways remains uncertain. 
5. The authors propose that disruption of lipid and steroid metabolism explains the severe liver injury observed in the co-exposure group (Page 13, Lines 381–384). However, the study only demonstrates an association between these observations. Causality remains unclear. The manuscript should avoid implying direct mechanistic links that were not experimentally verified
6.  

Author Response

Responses to Reviewer #1 (Comments from the reviewer are italicized and the referred texts are in blue color):

 

This manuscript investigates the combined effects of PS-NPs and Cd on liver injury in grass carp using biochemical, molecular, histological and transcriptomic approaches. The topic is relevant to current concerns regarding combined pollution in aquatic environments. However, several conclusions are not fully supported by the presented data, and some interpretations appear speculative. 

Response: Thanks for the critical comments and help suggestions. We have improved the MS concerning presentation, statistical analysis, and logical coherence, following the comments and suggestions. Please see our point-to-point responses below.

 

Specific comments:

1. On Page 4, Lines120–128, the authors selected 1 mg/L PS-NPs and 1 mg/L Cd based mainly on previous toxicity studies. However, the manuscript aims to provide references for ecological risk assessment (Page 2, Lines22–23; Page 3, Lines 98–99). The selected concentrations appear substantially higher than those typically reported in natural freshwater systems.

 

Response: We thank the reviewer for raising this important issue. We fully agree that environmentally relevant concentrations are critical for ecological risk assessment. In this study, the concentrations of polystyrene nanoplastics (1 mg/L) was selected based on a comprehensive review of existing literature and environmental monitoring data. The quantity of larger-sized microplastics in the environment ranges from ng/L to μg/L, while the content of nanoplastics cannot be precisely measured due to the limitation of actual detection methods. It had been estimated that the abundance of nanoplastics could be several orders of magnitude greater than that of microplastics (Alimi et al., 2018; Birch et al., 2020; Boakye et al., 2025; Stenzel, 2026; Ko et al., 2025) and much high putative risk has to be allocated to these smaller plastics (Triebskorn et al., 2019; Nawaz et al., 2025). As mentioned in the review (Bao et al., 2024), 1 mg/L concentration represents a moderate level of exposure. This concentration is commonly adopted as a reference point in the experimental design of NPs’ toxicology assessments (Wu et al., 2022; Wu et al., 2025; Zhang et al., 2024a; Zhang et al., 2024b).

The primary aim of this study was to elucidate the mechanisms of combined hepatotoxicity under sublethal but clearly toxic conditions, rather than to directly mimic ambient environmental levels. Acute exposure at moderate concentrations allows for the identification of key molecular pathways and histopathological endpoints, which can subsequently guide chronic, environmentally relevant studies. 1 mg/L Cd is a commonly used sublethal concentration in acute toxicity tests for freshwater fish, as it consistently induces detectable hepatotoxicity without high mortality. We have revised the description of research significance and experimental setup. Please see the revised MS or below blue texts.

“This study clarified the toxicological interaction of NPs and Cd on the liver of grass carp, and provided a theoretical basis for understanding the combined toxicity of NPs and heavy metal pollution in extreme contamination scenarios or accidental pollution events.” (Lines 21–23 in the revised manuscript)

“The results would elucidate the joint toxicity of NPs and Cd on aquatic organisms under high-exposure conditions, thereby offering a methodological reference for future research focused on environmentally relevant concentrations and long-term exposure in freshwater aquaculture environments.” (Lines 86–89 in the revised manuscript)

“The concentrations of NPs (1 mg/L) was selected based on a comprehensive review of existing literature and environmental monitoring data ^[31-33]. The Cd concentration of 1 mg/L was selected based on preliminary toxicity tests and published data on Cd toxicity in grass carp ^{[34, 35]}.” (Lines 110–113 in the revised manuscript)

 

References:

Alimi, O.S.; Budarz, J.F.; Hernandez, L.M.; Tufenkji, N. Microplastics and Nanoplastics in Aquatic Environments: Aggregation, Deposition, and Enhanced Contaminant Transport. Environ. Sci. Technol. 2018, 52, 1704-1724, doi: 10.1021/acs.est.7b05559.

Bao, S.; Yi, J.; Xian, B.; Rao, C.; Xiang, D.; Tang, W.; Fang, T. Global Analysis of the Adverse Effects of Micro- and Nanoplastics on Intestinal Health and Microbiota of Fish. J. Hazard. Mater. 2024, 470, doi: 10.1016/j.jhazmat.2024.134157.

Potter, P.M.; Pinto, P.X.; Dionysiou, D.D.; Al-Abed, S.R. Sources, Transport, Measurement and Impact of Nano and Microplastics in Urban Watersheds. Reviews in Environmental Science and Bio/Technology 2020, 19, 275-336, doi: 10.1007/s11157-020-09529-x.

Boakye, G.A.; Trotta, E.; Ambagahawatta, N.; Venkataraman, A.; Cheeptham, N.; Papadopoulos, C. Nanoscale Plastic Pollution: Sources, Identification and Potential Mitigation. Nanotechnology 2025, 36, doi: 10.1088/1361-6528/ae0c1b.

Nawaz, F.; Islam, Z.U.; Ghori, S.A.; Bahadur, A.; Ullah, H.; Ahmad, M.; Khan, G.U. Microplastic and Nanoplastic Pollution: Assessing Translocation, Impact, and Mitigation Strategies in Marine Ecosystems. Water Environment Research  a Research Publication of the Water Environment Federation 2025, 97, e70032, doi: 10.1002/wer.70032.

Ko, J.O.; Kadac-Czapska, K.; Ewska, K.J.D.; Nowak, N.; Kowalczyk, P.; Grembecka, M.G. Nanoplastics: from Separations to Analysis—Challenges and Limitations. Separations 2025, 12, doi: 10.3390/separations12070185.

Stenzel, M.H. The Dark Matter in Water, Air and Land: from Microplastic to Invisible Nanoplastics. Mater. Horiz. 2026, 13, 1628-1648, doi: 10.1039/d5mh01544h.

Triebskorn, R.; Braunbeck, T.; Grummt, T.; Hanslik, L.; Huppertsberg, S.; Jekel, M.; Knepper, T.P.; Krais, S.; Mueller, Y.K.; Pittroff, M.; et al. Relevance of Nano- and Microplastics for Freshwater Ecosystems: A Critical Review. Trac-Trends Anal. Chem. 2019, 110, doi: 10.1016/j.trac.2018.11.023.

Wu, C.; Zhang, H.; Ma, H.; Ji, R.; Pan, K.; Yue, T.; Miao, A. Mechanisms Underlying the Size-Dependent Neurotoxicity of Polystyrene Nanoplastics in Zebrafish. Environ. Sci. Technol. 2025, 59, 7419, doi: 10.1021/acs.est.4c12148.

Wu, H.; Guo, J.; Yao, Y.; Xu, S. Polystyrene Nanoplastics Induced Cardiomyocyte Apoptosis and Myocardial Inflammation in Carp by Promoting ROS Production. Fish Shellfish Immunol. 2022, 125, 1-8, doi: 10.1016/j.fsi.2022.04.048.

Zhang, C.; Bao, F.; Wang, F.; Xue, Z.; Lin, D. Toxic Effects of Nanoplastics and Microcystin-Lr Coexposure on the Liver-Gut Axis of Hypophthalmichthys Molitrix. Sci. Total Environ. 2024, 916, 170011, doi: 10.1016/j.scitotenv.2024.170011.

Zhang, C.; Wang, F.; Bao, F.; Zhu, J.; Xu, J.; Lin, D. The Effects of Nanoplastics and Microcystin-Lr Coexposure on Aristichthys Nobilis at the Early Developmental Stages. Aquat. Toxicol. 2024, 273, doi: 10.1016/j.aquatox.2024.107006.

31. Ou, M.; Zhang, C.; Wang, X.; Zheng, C.; Zhang, H.; Xue, Y. Automated Video Analysis of the Specific Effects of the Size and Concentration of Polystyrene Microplastics on the Swimming Behavior of the Crucian Carp (Carassius carassius). IEEE Access2024, 12, doi: 10.1109/access.2024.3452774.
32. Bao, S.; Yi, J.; Xian, B.; Rao, C.; Xiang, D.; Tang, W.; Fang, T. Global Analysis of the Adverse Effects of Micro- and Nanoplastics on Intestinal Health and Microbiota of Fish. J. Hazard. Mater.2024, 470, doi: 10.1016/j.jhazmat.2024.134157.
33. Zhang, C.; Bao, F.; Wang, F.; Xue, Z.; Lin, D. Toxic Effects of Nanoplastics and Microcystin-Lr Coexposure on the Liver-Gut Axis of Hypophthalmichthys Molitrix. Sci. Total Environ.2024, 916, 170011, doi: 10.1016/j.scitotenv.2024.170011.
34. Radwan, M.; Malki, J.S.A.; Waheed, R.M.; Moussa, M.A. Mitigation Exposed to Acute Cadmium Stress in Nile Tilapia by Dietary Quercetin: Exploring the Growth Performance, Intestinal Health, Stress Biomarkers, and Immune Responses. Biol. Trace Elem. Res.2025, 1-17, doi: 10.1007/s12011-025-04686-y.
35. Zhang, Y.; Duan, X.; Feng, L.; Jiang, W.; Wu, P.; Liu, Y.; Kuang, S.; Tang, L.; Zhou, X. Soybean Glycinin Disrupted Intestinal Structural Integrity Related to Aggravation of Apoptosis and Downregulated Transcription of Tight Junction Proteins in the Intestine of Juvenile Grass Carp (Ctenopharyngodon idella). Aquaculture2021, 531, doi: 10.1016/j.aquaculture.2020.735909.

 

2. In the Abstract (Page 2, Lines16–20) and Conclusions (Page 13, Lines392–394), the authors state that co-exposure caused synergistic effects. However, several biochemical endpoints do not support this conclusion. For example, GSH-ST activity in the co-exposure group was not significantly different from the control (Page 5, Lines 187–189), and LDH activity showed no significant difference among treatments (Lines 190–190). Even in the Discussion (Page 11, Lines 307–309), the authors acknowledge antagonistic interactions at the biochemical level. Therefore, describing the overall response simply as "synergistic" may be an oversimplification. The interaction appears endpoint-dependent and should be discussed more carefully.

Response: We agree with the reviewer that the overall interaction between NPs and Cd is not uniformly synergistic. We have revised the Abstract and Conclusions to replace “synergistic” with more precise descriptions such as “endpoint‑dependent interactions” and “aggravated hepatotoxicity at the histopathological and transcriptomic levels despite antagonistic or additive biochemical responses.” In the Discussion, we have added a paragraph explicitly acknowledging that some biochemical parameters (GSH‑ST, LDH) showed antagonistic or non‑significant changes, and that the toxic interaction is endpoint‑specific. Please see the revised MS or below blue texts.

 

Abstract

“The results revealed that co-exposure synergistically interactively suppressed interleukin-10 (IL-10) expression and heme oxygenase-1 (HO-1) antioxidant response…” (Lines 17)

Sections 4.1

“Importantly, the combined effects of PS‑NPs and Cd were not uniformly synergistic across all endpoints; while some biochemical responses (GSH‑ST and LDH) exhibited antagonistic or additive patterns, histopathological damage and transcriptomic disruptions (particularly in lipid and steroid metabolic pathways) were clearly aggravated. This endpoint‑dependent nature underscores that the overall toxicity of combined exposure should be viewed as a complex integration of multiple, sometimes opposing, interaction mechanisms, and evaluating such toxicity therefore requires integrating multiple endpoints across different biological scales rather than relying on a limited set of enzyme activities.” (Lines 244–251)

Conclusions

“Overall, PS‑NPs and Cd exhibited endpoint‑dependent interactions in grass carp liver, with aggravated hepatotoxicity at histopathological and transcriptomic levels despite some biochemical parameters showing antagonistic or additive effects. This indicated that the combined toxicity was not simply synergistic but rather multi‑faceted and endpoint‑specific.” (Lines 323–327)

 

3. On Page 6, Lines218–221, the authors observed that HO-1 expression increased under single exposure but returned to near-control levels under co-exposure. Subsequently, they suggest an uncoupling of antioxidant and anti-inflammatory pathways (Page 12, Lines337–339). However, no direct evidence for pathway uncoupling was provided. Alternative explanations, such as transcriptional inhibition or feedback regulation under severe stress, cannot be excluded. The authors should moderate the interpretation. There are also similar situations like this, e.g., sections 4.2 and 4.3.

 

Response: We appreciate this careful criticism. We agree that our original interpretation “uncoupling” was speculative without direct molecular evidence. We have now moderated the language throughout the Discussion, replacing definitive statements with more cautious phrasing. We also explicitly mention other plausible explanations (transcriptional inhibition, feedback regulation, shift to ferroptosis/apoptosis pathways). Please see the revised MS or below blue texts.

 

Sections 4.2

“…further supporting the view that co-exposure leads to an uncoupling of antioxidant/anti-inflammatory signaling pathways or a shift toward other stress pathways such as ferroptosis and apoptosis. This pattern might suggest a disruption of the coordinated antioxidant/anti‑inflammatory response, although alternative explanations such as transcriptional inhibition of HO‑1 under severe stress or a shift toward other stress pathways (e.g., ferroptosis, apoptosis) cannot be ruled out.” (Lines 271–274)

Sections 4.3

“…points to a possible interference raised the possibility of interference…” (Lines 296–297)

“Nevertheless, direct measurements of lipid and hormone levels are required to validate these functional alterations.” (Lines 299–300)

“It should be noted that transcriptomic changes indicated pathway enrichment, but did not prove functional alteration. All mechanistic interpretations are presented as hypotheses requiring experimental validation.” (Lines 313–315)

 

4. The manuscript identifies lipid metabolism and steroid biosynthesis as major pathways affected by co-exposure (Page 9, Lines266–277). Based on these findings, the Discussion extensively focuses on lipid remodeling and endocrine disruption (Page 12–13, Lines356–372). However, no physiological measurements, such as lipid accumulation, triglyceride content, cholesterol levels, or hormone concentrations, were conducted. Therefore, the biological significance of these enriched pathways remains uncertain.

 

Response: Thanks for your comment. We have toned down the biological claims and now clearly state that transcriptomic data provide hypothesis‑generating evidence rather than functional validation. We have added a limitation paragraph in the Discussion explicitly acknowledging the lack of direct biochemical/hormonal measurements and suggesting future directions. Please see the revised MS or below blue texts.

 

Sections 4.3

“It is important to emphasize that transcriptomic enrichment analysis indicates potential molecular perturbations, but does not directly measure functional outcomes, such as lipid accumulation, triglyceride or cholesterol levels, or hormone concentrations.” (Lines 307–310)

Conclusions

“However, direct measurements of lipid and hormone levels are needed to confirm these functional changes.” (Lines 322–323)

 

5. The authors propose that disruption of lipid and steroid metabolism explains the severe liver injury observed in the co-exposure group (Page 13, Lines381–384). However, the study only demonstrates an association between these observations. Causality remains unclear. The manuscript should avoid implying direct mechanistic links that were not experimentally verified.

 

Response: Thanks for your comment. We have revised all statements that implied causality, changing them to associative or correlational language. Please see the revised MS or below blue texts.

 

Sections 4.3

“…which provides an upstream mechanistic explanation for the severe liver injury observed in histopathology which may contribute to the severe liver injury observed in histopathology.” (Lines 312–313)

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

This manuscript presents data on the effects of a polystyrene-based nanoplastic and cadmium ions on fish liver. The authors conducted a comprehensive study, including biochemical analysis, gene expression level analysis, histopathological analysis, and transcriptome sequencing. In my opinion, the experimental data demonstrating the complexity and ambiguity of the effects of the combination of nanoparticles and cadmium ions is new and interesting. For example, the authors demonstrated that the combined exposure group exhibited severe lesions, such as hepatocyte necrosis, melanization, and diffuse fibrinoid necrosis, whereas relatively milder damage was observed in the groups exposed to only polystyrene nanoparticles or only cadmium ions. This suggests that although some enzymatic indicators exhibit antagonism (suppression of expression), tissue-level damage tends to be aggravated, which may be associated with metabolic disturbances. Furthermore, interesting data also indicate that the simultaneous exposure to polystyrene nanoparticles and cadmium ions non-additively and systemically disrupted the liver lipid metabolism network in carp.

In my opinion, the manuscript deserves the attention of researchers, and I can recommend it for publication after the authors have made some revisions to the manuscript.
There are a number of questions and comments regarding the manuscript:
1. In the introduction, the authors write that polystyrene nanoparticles are characterized by "...and strong hydrophobicity...." This statement should be clarified. It is clear that polystyrene itself is characterized by hydrophobic properties. However, when plastic products are broken down to nanometer size, humic acids are localized in the surface layer of such particles, and functional groups are formed under the influence of UV radiation. As a result, the structure of the surface layer of polystyrene nanoparticles cannot be described as hydrophobic.
2. The authors write "Cd has an extremely long half-life in aquatic organisms...." What does this mean? An explanation is needed. Where does the cadmium go?

3. "...the hydrophobic and π–π interactions on NP surfaces enable them to act as effective carriers for metal ions like Cd2+." The authors should clarify this statement. How can ions bind via π–π interactions?
4. Reference 14 should be explained. On which cells was this effect demonstrated?
5. In the experimental section, the description of the Fluorescent Polystyrene Nanoparticles (PS-NPs, PS-7-3-0010) should be supplemented. Are there functional surface groups, and if so, which ones? If not, how are the particles stabilized?
6. What does the statement "...that antagonism at the biochemical level does not equate to an overall attenuation of toxicity" mean? What is overall attenuation of toxicity?
7. Figure 4 should be formatted. It is difficult to read as is, and the text is too small.

8. The authors write that future studies should consider a longer-term experiment. This is certainly the right direction. However, they would appreciate comment on whether the system they studied is close to the concentrations of nanoplastics and cadmium ions present in environmental aquatic systems.

Author Response

Responses to Reviewer #2 (Comments from the reviewer are italicized and the referred texts are in blue color):

This manuscript presents data on the effects of a polystyrene-based nanoplastic and cadmium ions on fish liver. The authors conducted a comprehensive study, including biochemical analysis, gene expression level analysis, histopathological analysis, and transcriptome sequencing. In my opinion, the experimental data demonstrating the complexity and ambiguity of the effects of the combination of nanoparticles and cadmium ions is new and interesting. For example, the authors demonstrated that the combined exposure group exhibited severe lesions, such as hepatocyte necrosis, melanization, and diffuse fibrinoid necrosis, whereas relatively milder damage was observed in the groups exposed to only polystyrene nanoparticles or only cadmium ions. This suggests that although some enzymatic indicators exhibit antagonism (suppression of expression), tissue-level damage tends to be aggravated, which may be associated with metabolic disturbances. Furthermore, interesting data also indicate that the simultaneous exposure to polystyrene nanoparticles and cadmium ions non-additively and systemically disrupted the liver lipid metabolism network in carp. In my opinion, the manuscript deserves the attention of researchers, and I can recommend it for publication after the authors have made some revisions to the manuscript.

 

Response: Thanks for the critical comments and help suggestions. We have improved the MS concerning presentation, statistical analysis, and logical coherence, following the comments and suggestions. Please see our point-to-point responses below.

 

There are a number of questions and comments regarding the manuscript:

1. In the introduction, the authors write that polystyrene nanoparticles are characterized by "...and strong hydrophobicity...." This statement should be clarified. It is clear that polystyrene itself is characterized by hydrophobic properties. However, when plastic products are broken down to nanometer size, humic acids are localized in the surface layer of such particles, and functional groups are formed under the influence of UV radiation. As a result, the structure of the surface layer of polystyrene nanoparticles cannot be described as hydrophobic.

 

Response: We thank the reviewer for this important clarification. We have revised the sentence to acknowledge that although pristine polystyrene is hydrophobic, environmental aging and corona formation can alter surface properties. Please see the revised MS or below blue texts.

 

“Owing to their extremely small size, large specific surface area, and intrinsic hydrophobicity of polystyrene, PS-NPs possess a far greater capacity for pollutant adsorption and biological barrier penetration than MPs. However, it should be noted that in natural aquatic environments, the surface properties of NPs can be altered by the adsorption of humic substances and the formation of oxygen-containing functional groups due to UV irradiation, which may modify their hydrophobicity and pollutant carrier behavior^{[4, 5]}.” (Lines 35–40)

 

References:

4. Zhu, M.; Zhang, Z.; Zhang, T.; Hofmann, T.; Chen, W. Eco-Corona Dictates Mobility of Nanoplastics in Saturated Porous Media: the Critical Role of Preferential Binding of Macromolecules. Environ. Sci. Technol.2023, 57, doi: 10.1021/acs.est.2c07376.
5. Li, X.; Ji, S.; He, E.; Peijnenburg, W.J.G.M.; Cao, X.; Xu, X.; Zhang, P.; Qiu, H. UV/ozone Induced Physicochemical Transformations of Polystyrene Nanoparticles and Their Aggregation Tendency and Kinetics with Natural Organic Matter in Aqueous Systems. J. Hazard. Mater.2022, 433, doi: 10.1016/j.jhazmat.2022.128790.

 

2. The authors write "Cd has an extremely long half-life in aquatic organisms...." What does this mean? An explanation is needed. Where does the cadmium go?

 

Response: Thanks for your comment. We agree that this statement needed clarification. Please see the revised MS or below blue texts. 

 

“Cd is poorly metabolized and excreted in aquatic organisms, resulting in a long biological half-life (years to decades in some fish species)^[10]. It accumulates mainly in the liver, kidney, and gills, and causes sustained toxicity by inducing oxidative stress, interfering with essential metal ion homeostasis, and inhibiting antioxidant enzymes and DNA repair systems.” (Lines 48–51)

 

References:

10. Lee, J.; Jo, A.; Kang, Y.; Lee, D.; Choi, C.; Kang, J.; Kim, J. Review of Cadmium Bioaccumulation in Fish Exposed to Cadmium. Toxics2024, 13, doi: 10.3390/toxics13010007.

 

3. "...the hydrophobic and π–π interactions on NP surfaces enable them to act as effective carriers for metal ions like Cd²⁺." The authors should clarify this statement. How can ions bind via π–π interactions?
   

Response: Thanks for your comment. The direct evidence for π-π interaction in the adsorption of Cd²⁺ by nano-plastics is extremely limited. Therefore, we have revised the wording in the text. Please see the revised MS or below blue texts.

 

“…the hydrophobic and π–π interactions on NP surfaces enable them to act as effective carriers for metal ions like Cd²⁺ the surface properties of NPs, including their surface charge and oxygen-containing functional groups, enable them to act as effective carriers for metal ions like Cd²⁺…” (Lines 53–54)

 

4. Reference 14 should be explained. On which cells was this effect demonstrated?
   

Response: Thanks for your comment. The hepatocyte used in Reference 14 (Now it is Reference 17) were of a specific type. We have already made the necessary additions in the article. Please see the revised MS or below blue texts.

 

“Li demonstrated that NPs significantly increased the uptake of Cd by hepatocyte, and the intracellular desorption of loaded Cd was the key prerequisite for amplifying cytotoxicity^[17].” (Lines 55–57)

 

References:

17. Li, X.; Hu, S.; Yu, Z.; He, F.; Zhao, X.; Liu, R. New Evidence for the Mechanisms of Nanoplastics Amplifying Cadmium Cytotoxicity: Trojan Horse Effect, Inflammatory Response, and Calcium Imbalance. Environ. Sci. Technol.2025, 59, 9471-9485, doi: 10.1021/acs.est.5c01254.

 

5. In the experimental section, the description of the Fluorescent Polystyrene Nanoparticles (PS-NPs, PS-7-3-0010) should be supplemented. Are there functional surface groups, and if so, which ones? If not, how are the particles stabilized?
   

Response: We thank the reviewer for the valuable suggestion to improve the characterization of the fluorescent polystyrene nanoplastics (PS-NPs) used in this study. According to the manufacturer’s specifications, the nanoparticles are plain (non-functionalized) polystyrene without any charged surface functional groups. The particles are stabilized by anionic surfactants adsorbed onto the particle surface during the synthesis process, which generates a negative zeta potential and prevents aggregation via electrostatic repulsion. In response, we have supplemented the Section 2.1. Please see the revised MS or below blue texts.

 

“According to the manufacturer, the particles are plain polystyrene with no charged surface groups. The nanoparticles are stabilized by anionic surfactants, resulting in a negatively charged surface (zeta potential: -16.6 ± 1.8 mV, as reported by the manufacturer). The fluorescence excitation/emission peak are 518/458 nm.” (Lines 93–97)

 

6. What does the statement "... that antagonism at the biochemical level does not equate to an overall attenuation of toxicity" mean? What is overall attenuation of toxicity?
   

Response: We thank the reviewer for this insightful comment. In the revised manuscript, we have substantially clarified this point. By “overall toxicity”, we refer to the integrated adverse outcome across multiple biological levels — including histopathological damage, immune dysregulation, transcriptomic disruption, and multi‑biomarker integrated indices (e.g., IBR) — rather than any single biochemical endpoint.

We cited Wen et al. to illustrate that similar antagonistic effects at the biochemical level have been observed in other fish species, yet the authors themselves emphasised that overall toxicity cannot be judged from enzyme activities alone. In that study on discus fish (Symphysodon aequifasciatus), co‑exposure to microplastics and Cd led to antagonistic or additive effects on several antioxidant enzymes (e.g., SOD and CAT), but the combined treatment caused significantly higher Cd accumulation and more pronounced alterations in innate immunity parameters (e.g., lysozyme and complement activities) than did single exposures. This clearly demonstrated that biochemical antagonism in one set of endpoints did not equate to an attenuation of the overall toxic burden. We have now incorporated this detailed explanation into the revised discussion. Please see the revised MS or below blue texts.

 

“Similar antagonistic effects were also reported by Wen in a combined exposure study on discus fish (Symphysodon aequifasciatus)^[42], indicating that antagonism at the biochemical level does not equate to an overall attenuation of toxicity. Similar antagonistic effects were also reported by Wen et al. in discus fish (Symphysodon aequifasciatus), who found that co‑exposure to MPs and Cd resulted in antagonistic interactions for several biochemical parameters (catalase, acid phosphatase, alkaline phosphatase, and complement 3), yet also produced synergistic increases in protein carbonyl content and lysozyme activity, ultimately inducing severe oxidative stress and stimulating innate immunity^[42]. ” (Lines 239–244)

 

References:

42. Wen, B.; Jin, S.; Chen, Z.; Gao, J.; Liu, Y.; Liu, J.; Feng, X. Single and combined effects of microplastics and cadmium on the cadmium accumulation, antioxidant defence and innate immunity of the discus fish (Symphysodon aequifasciatus). Environ. Pollut.2018, 243, 462-471, doi: 10.1016/j.envpol.2018.09.029.

 

7. Figure 4 should be formatted. It is difficult to read as is, and the text is too small.

 

Response: Thanks for your comment. We have made modifications to Figure 4. Please see the revised MS.

 

8. The authors write that future studies should consider a longer-term experiment. This is certainly the right direction. However, they would appreciate comment on whether the system they studied is close to the concentrations of nanoplastics and cadmium ions present in environmental aquatic systems.

 

Response: We thank the reviewer for this valuable comment. We fully acknowledge that environmentally relevant concentrations are essential for long-term ecological risk assessment. In the present study, the exposure concentrations (1 mg/L for both PS-NPs and Cd) were not intended to directly replicate typical background levels in natural freshwater systems, but rather to establish a sublethal, clearly toxic condition suitable for elucidating the mechanisms of combined hepatotoxicity within a short-term acute exposure framework.

Current analytical techniques (e.g., Raman spectroscopy, electron microscopy) still face significant challenges in accurately quantifying NPs in complex environmental matrices. Consequently, reported environmental concentrations of NPs are extremely limited, and many existing studies rely on extrapolations from microplastic data. It has been estimated that the abundance of NPs could be several orders of magnitude higher than that of microplastics due to continuous fragmentation (Alimi et al., 2018; Birch et al., 2020; Boakye et al., 2025; Stenzel, 2026; Ko et al., 2025). Moreover, in extreme contamination scenarios (e.g., accidental spillage, industrial discharge, or during high-flow events in plastic-polluted rivers), local concentrations of NPs may transiently reach mg/L levels. Thus, the 1 mg/L concentration used in this study represents a worst-case or environmentally plausible high-exposure scenario, which is commonly adopted in mechanistic nanotoxicology studies to enable the detection of key molecular and histopathological endpoints.

The Cd concentration of 1 mg/L is a well-established sublethal dose in acute toxicity tests for freshwater fish, including grass carp, as it consistently induces hepatic oxidative stress and histopathological changes without causing excessive mortality (Radwan et al., 2025; Zhang et al., 2021). While this level is higher than the average Cd concentrations reported in most natural waters (typically in the µg/L range) (Mao et al., 2025), it is relevant for simulating localized pollution events, such as those near mining sites, electroplating facilities, or areas with historical heavy metal contamination.

The primary goal of this study was to identify the interactive toxicity mechanisms of NPs and Cd co-exposure on the liver of grass carp under conditions where clear toxic effects are observable. Acute exposure at moderate concentrations allows for the efficient detection of pathway-level disruptions and histopathological lesions, which can then serve as mechanistic benchmarks for designing future long-term, environmentally relevant chronic studies. To avoid overstating the direct environmental implications, we have carefully revised the relevant statements in the manuscript. Please see the revised MS or below blue texts.

“This study clarified the toxicological interaction of NPs and Cd on the liver of grass carp, and provided a theoretical basis for understanding the combined toxicity of NPs and heavy metal pollution in extreme contamination scenarios or accidental pollution events.” (Lines 21–23)

“The results would elucidate the joint toxicity of NPs and Cd on aquatic organisms under high-exposure conditions, thereby offering a methodological reference for future research focused on environmentally relevant concentrations and long-term exposure in freshwater aquaculture environments.” (Lines 86–89)

“The concentrations of NPs (1 mg/L) was selected based on a comprehensive review of existing literature and environmental monitoring data ^[31-33]. The Cd concentration of 1 mg/L was selected based on preliminary toxicity tests and published data on Cd toxicity in grass carp ^{[34, 35]}.” (Lines 110–113)

 

References:

Alimi, O.S.; Budarz, J.F.; Hernandez, L.M.; Tufenkji, N. Microplastics and Nanoplastics in Aquatic Environments: Aggregation, Deposition, and Enhanced Contaminant Transport. Environ. Sci. Technol. 2018, 52, 1704-1724, doi: 10.1021/acs.est.7b05559.

Bao, S.; Yi, J.; Xian, B.; Rao, C.; Xiang, D.; Tang, W.; Fang, T. Global Analysis of the Adverse Effects of Micro- and Nanoplastics on Intestinal Health and Microbiota of Fish. J. Hazard. Mater. 2024, 470, doi: 10.1016/j.jhazmat.2024.134157.

Boakye, G.A.; Trotta, E.; Ambagahawatta, N.; Venkataraman, A.; Cheeptham, N.; Papadopoulos, C. Nanoscale Plastic Pollution: Sources, Identification and Potential Mitigation. Nanotechnology 2025, 36, doi: 10.1088/1361-6528/ae0c1b.

Stenzel, M.H. The Dark Matter in Water, Air and Land: from Microplastic to Invisible Nanoplastics. Mater. Horiz. 2026, 13, 1628-1648, doi: 10.1039/d5mh01544h.

Ko, J.O.; Kadac-Czapska, K.; Ewska, K.J.D.; Nowak, N.; Kowalczyk, P.; Grembecka, M.G. Nanoplastics: from Separations to Analysis—Challenges and Limitations. Separations 2025, 12, doi: 10.3390/separations12070185.

Radwan, M.; Malki, J.S.A.; Waheed, R.M.; Moussa, M.A. Mitigation Exposed to Acute Cadmium Stress in Nile Tilapia by Dietary Quercetin: Exploring the Growth Performance, Intestinal Health, Stress Biomarkers, and Immune Responses. Biol. Trace Elem. Res. 2025, 1-17, doi: 10.1007/s12011-025-04686-y.

Zhang, Y.; Duan, X.; Feng, L.; Jiang, W.; Wu, P.; Liu, Y.; Kuang, S.; Tang, L.; Zhou, X. Soybean Glycinin Disrupted Intestinal Structural Integrity Related to Aggravation of Apoptosis and Downregulated Transcription of Tight Junction Proteins in the Intestine of Juvenile Grass Carp (Ctenopharyngodon idella). Aquaculture 2021, 531, doi: 10.1016/j.aquaculture.2020.735909.

Mao, B.; Shi, L.; Che, R.; Chen, G. Assessment of Heavy Metal Pollution in an Urbanized Waterway of the Pearl River Delta, China. Water & Ecology 2025, doi: 10.1016/j.wateco.2025.100016.

 

31. Ou, M.; Zhang, C.; Wang, X.; Zheng, C.; Zhang, H.; Xue, Y. Automated Video Analysis of the Specific Effects of the Size and Concentration of Polystyrene Microplastics on the Swimming Behavior of the Crucian Carp (Carassius carassius). IEEE Access2024, 12, doi: 10.1109/access.2024.3452774.
32. Bao, S.; Yi, J.; Xian, B.; Rao, C.; Xiang, D.; Tang, W.; Fang, T. Global Analysis of the Adverse Effects of Micro- and Nanoplastics on Intestinal Health and Microbiota of Fish. J. Hazard. Mater.2024, 470, doi: 10.1016/j.jhazmat.2024.134157.
33. Zhang, C.; Bao, F.; Wang, F.; Xue, Z.; Lin, D. Toxic Effects of Nanoplastics and Microcystin-Lr Coexposure on the Liver-Gut Axis of Hypophthalmichthys Molitrix. Sci. Total Environ.2024, 916, 170011, doi: 10.1016/j.scitotenv.2024.170011.
34. Radwan, M.; Malki, J.S.A.; Waheed, R.M.; Moussa, M.A. Mitigation Exposed to Acute Cadmium Stress in Nile Tilapia by Dietary Quercetin: Exploring the Growth Performance, Intestinal Health, Stress Biomarkers, and Immune Responses. Biol. Trace Elem. Res.2025, 1-17, doi: 10.1007/s12011-025-04686-y.
35. Zhang, Y.; Duan, X.; Feng, L.; Jiang, W.; Wu, P.; Liu, Y.; Kuang, S.; Tang, L.; Zhou, X. Soybean Glycinin Disrupted Intestinal Structural Integrity Related to Aggravation of Apoptosis and Downregulated Transcription of Tight Junction Proteins in the Intestine of Juvenile Grass Carp (Ctenopharyngodon idella). Aquaculture2021, 531, doi: 10.1016/j.aquaculture.2020.735909.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

All the suggestions and comments have been meticulously addressed and revised, and the paper is now suitable for acceptance.