Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript addresses an important and timely question in aquatic ecotoxicology: the ingestion, fragmentation, and elimination of microplastics (MPs) in crustaceans, using Procambarus clarkii as a model species. The study is well-structured, with thorough microplastic characterisation, clear exposure and depuration designs, and multiple complementary analytical techniques (fluorescence, CLSM, SEM). The results provide novel insights into short-term MP accumulation dynamics, organ-specific distribution, and morphological alterations of ingested particles. Overall, the work is scientifically sound and has the potential to make a useful contribution to the field. However, aspects of the methodology, statistical analysis, and interpretation require clarification and tempering, particularly regarding mechanistic claims and ecological extrapolations. Addressing these issues will improve the rigour and ensure the conclusions remain well supported by the data.

Abstract

The Abstract states that “particle sizes decreased significantly, indicating active fragmentation” and that “dietary supplementation enhanced fragmentation efficiency.” While consistent with the reported data, the phrasing implies mechanistic certainty that was not directly demonstrated. It would be more accurate to say “consistent with fragmentation” or “suggesting enhanced fragmentation.”

The closing sentences emphasise ecological risks, pollutant fate, and trophic transfer. These are important broader implications, but extend beyond the scope of the presented short-term laboratory exposures at high concentrations. It would strengthen the Abstract to clearly frame these as potential implications rather than direct outcomes.

The high experimental concentration (100,000 particles/L) is described, but without contextualization. A brief acknowledgement that this is above environmentally relevant levels would improve transparency.

Minor - put Latin name in italic. 

M&M

The use of 100,000 particles/L is much higher than concentrations typically reported in natural environments. While this is acceptable for controlled laboratory studies, the limitation should be explicitly acknowledged in the manuscript to avoid overstating ecological realism.

During exposure, crayfish were not fed, but in the depuration phase, groups were compared as “fed” vs. “unfed.” It is important to clarify precisely how dietary supplementation was introduced, how it was standardised across individuals, and why starvation was applied during exposure but not depuration.

The text states that three replicates per group were established and three crayfish were sampled per time point, but the total number of individuals analysed per treatment and time point remains ambiguous. A clearer description, perhaps in a summary table or schematic, would ensure reproducibility and remove any concerns about pseudoreplication.

The manuscript states that ANOVA was applied to mean particle size, but it is unclear how many particles per sample were measured and whether size distributions were tested for normality. Given that particle size data are often non-normal, additional detail on data handling (e.g., transformations, distribution checks) is needed.

The authors conclude that KOH had no observable effect on MPs. However, since KOH can degrade some plastics under certain conditions, this conclusion should be cautiously worded and contextualised with reference to the limited conditions tested here (30% KOH, 25 °C, 48 h).

Both Tukey’s HSD and Fisher’s LSD were applied. Since Fisher’s LSD is relatively liberal and less commonly recommended, the rationale for using both post hoc tests should be explained. Ideally, the more conservative test is emphasised in the main results.

Overall, the methods are rigorous and well-designed, but clarifying these points will greatly improve transparency and ensure confidence in the reported results.

Results

The Results are presented in a structured and logical manner, closely aligned with the experimental design described in the Materials and Methods. The figures are generally clear and support the main conclusions: MP accumulation peaked at 48 h, declined by 96 h, and showed distinct patterns of fragmentation and tissue distribution. The inclusion of depuration and dietary treatments adds novelty, and the morphological imaging (fluorescence, CLSM, SEM) convincingly illustrates physical changes to MPs.

However, both Tukey’s HSD and Fisher’s LSD are presented for post hoc comparisons. Since Fisher’s LSD is liberal and prone to inflated Type I error, the rationale for using it should be explained, and the more conservative Tukey’s test should be emphasised in the main results.

While figures present means ± SD, the exact number of animals contributing to each bar is unclear. The manuscript should specify whether values are based on pooled or individual data, and explicitly state n for each treatment/timepoint to avoid concerns of pseudoreplication.

Figure 2 shows that the 48-h group contained larger average fragment sizes compared to 24 h and 96 h. This result is counterintuitive, as fragmentation typically produces smaller particles over time. Authors should discuss whether this pattern reflects aggregation, methodological detection limits, or biological variability, rather than a simple fragmentation trend.

Figure 3 demonstrates that dietary supplementation increased apparent fragmentation efficiency. However, since crayfish were starved during exposure and only fed during depuration, it is unclear whether this effect reflects true biological fragmentation or accelerated gut clearance due to feeding. This distinction should be carefully acknowledged in the interpretation.

SEM and CLSM images (Figure 4) show surface wrinkling, irregular edges, and particle shrinkage. While compelling, the manuscript currently attributes these changes directly to “digestion-induced” processes. Without abiotic digestion controls (e.g., MPs exposed to simulated gastric fluids), the more cautious conclusion would be that changes are “consistent with digestive processing.”

Figures are informative, but the pastel colour palette (pink, purple, orange) makes it difficult to visually separate intact vs. fragmented MPs. Stronger contrasts or patterned fills would improve clarity for readers.

Discussion & Conclusion

The Discussion provides a broad and well-structured interpretation of the results, situating the findings within existing literature on MP ingestion and transformation in aquatic organisms. The authors clearly link their observations of accumulation, tissue distribution, and fragmentation patterns to prior reports, and the Conclusions succinctly summarise the main outcomes of the study. Importantly, they also acknowledge limitations in temporal resolution, which is appropriate.

The manuscript frequently attributes observed size-dependent retention and fragmentation to specific physiological mechanisms such as peristaltic grinding in the gastric mill, enzymatic hydrolysis, and mucosal retention. While these are plausible explanations, they were not directly measured in this study. These should be reframed as hypotheses consistent with the data, not as demonstrated mechanisms.

The conclusion that dietary supplementation enhanced fragmentation “via gastric mill motility and enzymatic secretion” is speculative. The data show increased fragmentation in fed crayfish, but whether this reflects true biological fragmentation versus accelerated gut clearance cannot be distinguished here. A more cautious interpretation is recommended.

The discussion introduces oxidative stress, enzymatic responses, and potential metabolic disorders. Since this study did not measure biomarkers or physiological endpoints, these statements should be toned down or explicitly presented as context from prior literature, not as outcomes of the present work.

References to trophic transfer and risks to humans through seafood consumption are relevant, but currently overstated. The data are limited to short-term exposures in crayfish under laboratory conditions at high particle concentrations. The discussion should emphasise that human health implications are speculative and require further research.

The summary effectively captures the core findings (accumulation peak at 48 h, elimination by 96 h, organ-specific translocation, increased fragmentation in fed crayfish, morphological alterations). However, mechanistic explanations (e.g., gastric mill activity, enzymatic secretion) should be softened to reflect that these were not directly tested.

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

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Comments for author File: Comments.pdf

Author Response

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Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors fully and appropriately addressed my review