Micro/Nanoplastics Alter Daphnia magna Life History by Disrupting Glucose Metabolism and Intestinal Structure

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Environmental pollution caused by plastic waste is a major concern in the modern world. The impact of plastic microparticles on living organisms and the functioning of ecosystems is still not fully understood. Therefore, the authors' research on the impact of plastic microparticles on the reproductive ability and changes in the gut microbiota of Daphnia magna seems to be relevant.

The text of the article needs some clarification and editing.

 

In the Graphical Abstract, Daphnia appears to be a chimera. Has microplastic pollution had this effect?

 

Paragraph 2.1: “scenedesmus” must be “Scenedesmus”

 

2.1–2.2: Why were these specific plastic particle sizes and concentrations selected for the study? What is the concentration of microplastics found in different water bodies? Is there any information available on particle size categories? This information could be included in the Introduction.

 

3.1: “Analysis via fluorescence microscopy imaging (at 0, 2, 7, 14, 21, 28, 35, and 49 days) of microsphere ingestion (Fig. 1) and fluorescence intensity (Fig. 2) in D. magna revealed significant differences in fluorescence intensity among the experimental groups (A, B, C, D) (P < 0.001).”

What criteria were used to determine the significance of the differences between the groups?

 

“This phenomenon was likely closely related to the feeding habits of Daphnia magna… “

The size of particles consumed by Cladocera is determined not by their feeding habits, but by the size of the mesh in the filtration apparatus. For Daphnia magna, the mesh size is 0.24–0.64 µm (Geller, Muller, 1981). Thus, 0.1 µm of particles at a low concentration do not become trapped by the setae of the filtering limbs of Daphnia.

 

Fig. 2: The axis labels are incomplete. What units are the fluorescence intensities measured in? Use the horizontal axis to label the letters and numbers (Group, Date).

It is not clear what the letters above the bars in the diagram represent.

 

Page 7: “The results of ANOVA analysis showed that there were significant differences in the body length of D. magna among different groups (CK, A, B, C, D) (P = 0.003).”

Apparently, in this case, the ANOVA analysis reveals significant differences between the control group and the groups containing plastic particles. How did the groups with different particle sizes and concentrations differ? Based on Figure 3, it appears that not all groups differ significantly from each other.

 

3.3: “Study has demonstrated that smaller-sized MPs accumulate more in organisms and remain for a longer time…”

Is there a contradiction between this statement and the fact that the level of fluorescence was minimal in Group A?

 

Fig. 4: Explanations to the details of the image are required. How is the mean, median, etc. indicated? The caption for Figure 4 seems to be inaccurate. It would be more accurate to use the term "Reproductive parameters" instead of "Changes in growth and development" in this context.

 

Conclusion: Since the experimental setup included plastic particles of different sizes and concentrations, it is necessary to draw a conclusion about the impact of these parameters on the reproductive ability and changes in the gut microbiota of Daphnia.

The list of references needs to be formatted.

The article can be accepted for publication after appropriate corrections have been made.

Comments for author File: Comments.pdf

Author Response

Reviewer 1: Environmental pollution caused by plastic waste is a major concern in the modern world. The impact of plastic microparticles on living organisms and the functioning of ecosystems is still not fully understood. Therefore, the authors' research on the impact of plastic microparticles on the reproductive ability and changes in the gut microbiota of Daphnia magna seems to be relevant. The text of the article needs some clarification and editing.

 

Comments 1: In the Graphical Abstract, Daphnia appears to be a chimera. Has microplastic pollution had this effect?

Response 1: Thank you for this question. In the Graphical Abstract, Daphnia appears to be a chimera, which is due to our insufficient ability to draw stick figures. We will replace it with a photograph of Daphnia instead.

Comments 2. Paragraph 2.1: “scenedesmus” must be “Scenedesmus”.

Response 2: Thank you very much for this suggestion. We will change "scenedesmus" to "Scenedesmus".

Comments 3. Why were these specific plastic particle sizes and concentrations selected for the study? What is the concentration of microplastics found in different water bodies? Is there any information available on particle size categories? This information could be included in the Introduction.

Response 3: The authors thanked for those questions. In this research, the fluorescent polystyrene green MPs was chosen to explore the distribution of MPs in Daphnia more intuitively. The 0.1 and 0.5 μm MPs were used due to the feeding habits of D. magna. And 0.1, and 1 mg/L MPs were chosen following the research [1, 2].

In addition, in nature water bodies, especially for lake water. The recent research [3] has demonstrated that the abundance of microplastics reached 0.01 × 10⁶-6.8 × 10⁶ items/km² in plankton net samples, 3.4-25.8 items/L in surface water, 11.0-234.6 items/kg dw in sediments. Considering the resuspension of sediment, the low and high concentration of MPs were chosen.

[1] Gao J.W., Zhao S.S., Li F.Y., et al. Effects of Microplastics on Feeding Behavior and Antioxidant System of Daphnia magna [J]. Research of Environmental Science, 34, 5, 1205-1212.

[2] Rist, S.; Baun, A.; Hartmann, N. B., Ingestion of micro- and nanoplastics in Daphnia magna – Quantification of body burdens and assessment of feeding rates and reproduction [J]. Environ. Pollut. 2017, 228, 398-407.

[3] Su L., Xue Y.G., Li LG., et al. Microplastics in Taihu Lake, China [J]. Environ. Pollut. 216 (2016) 711e719

Comments 4. “Analysis via fluorescence microscopy imaging (at 0, 2, 7, 14, 21, 28, 35, and 49 days) of microsphere ingestion (Fig. 1) and fluorescence intensity (Fig. 2) in D. magna revealed significant differences in fluorescence intensity among the experimental groups (A, B, C, D) (P < 0.001).” What criteria were used to determine the significance of the differences between the groups?

Response 4: Thank you for this question. The data of fluorescence intensity for different groups were collected and analyzed by software “Image J”. In the analysis, when the P-value is less than 0.001 according to One-way analysis of variance, (ANOVA), significant differences between the two groups are recognized.

Comments 5: “This phenomenon was likely closely related to the feeding habits of Daphnia magna…”. The size of particles consumed by Cladocera is determined not by their feeding habits, but by the size of the mesh in the filtration apparatus. For Daphnia magna, the mesh size is 0.24– 0.64 µm (Geller, Muller, 1981). Thus, 0.1 µm of particles at a low concentration do not become trapped by the setae of the filtering limbs of Daphnia.

Response 5: Thank you very much for this suggestion. Although the mesh size of Daphnia magna ranges is 0.24-0.64 μm, the size of the particles found in the daphnid gut or those which caused high feeding activities were larger than the smallest mesh sizes of the filters [1]. Sieving is unlikely process for routine particle collection, particle capture is not a simple mechanical process and not by sieving alone. The Daphnia’s feeding mechanism achieves particles abstraction not by sieving [2]. “Size-efficiency hypothesis” [3] have assumed that the upper size limit of particles that can be taken by large cladocerans probably accords roughly with the body size of the zooplankter. Therefore, MPs with larger particle sizes are more compatible with the food size preferences of D. magna in this study.

[1] Gophen, M.; Geller, W., Filter mesh size and food particle uptake by Daphnia. Oecologia (Berlin) 1984, 64, 408-412.

[2] Gophen, M., Internal water flows and particles abstraction in Daphnia Open Journal of Ecology 2022, 12, 742-755.

[3] Brooks, J. L.; Dodson, S. I., Predation, Body Size, and Composition of Plankton: The effect of a marine planktivore on lake plankton illustrates theory of size, competition, and predation. Science 1965, 150, (3692), 28-35.

Comments 6. The axis labels are incomplete. What units are the fluorescence intensities measured in? Use the horizontal axis to label the letters and numbers (Group, Date). It is not clear what the letters above the bars in the diagram represent.

Response 6: Thank you for this question. In the fluorescence intensity test experiment, the values of fluorescence intensity were derived from the relative changes analyzed by Image J software based on fluorescence images captured under a unified background, and thus no units were added. The letters a, b, c, and d on the bar chart respectively indicate significant differences (typically, different letters represent that there is a statistically significant difference between the corresponding groups, while the same letter indicates no significant difference).

Comments 7. “The results of ANOVA analysis showed that there were significant differences in the body length of D. magna among different groups (CK, A, B, C, D) (P = 0.003).” Apparently, in this case, the ANOVA analysis reveals significant differences between the control group and the groups containing plastic particles. How did the groups with different particle sizes and concentrations differ? Based on Figure 3, it appears that not all groups differ significantly from each other.

Response 7: Thank you for this question. The body length of D. magna followed the order: CK > C > A > B > D. For each group, the body length of over 150 D. magna individuals was measured every day, and there were significant individual differences. In Figure 3, only a line graph of "mean ± standard deviation" could be used to present the data. However, in reality, when all individual daily data were subjected to analysis of variance (ANOVA), a conclusion of significant differences could be drawn.

Comments 8. “Study has demonstrated that smaller-sized MPs accumulate more in organisms and remain for a longer time…” Is there a contradiction between this statement and the fact that the level of fluorescence was minimal in Group A?

Response 8: Thank you for this question. Exposure studies were carried out by Rist et al. (2017). The experiment was a 21 days exposure of D. magna to nano- (100 nm) and microplastics (2 mm), which concentrations were 0.1, 0.5 and 1 mg/L. During the 21 days exposure, individual daphnids were kept in 100 mL glass beakers with 50 mL M7 medium in a semi-static test setup, in which the medium was changed three times per week, and the exposure suspensions were freshly prepared before every medium exchange. Both particle sizes were readily ingested, but the ingested mass of particles was five times higher for the 2 μm particles than for the 100 nm particles, and higher amounts of the 2 μm particles were egested. Animal body burdens of particles were strongly reduced in the presence of food. Daphnid feeding rates decreased by 21% in the presence of 100 nm particles, and high body burdens of particles at the end of 21 days exposure. The lower egestion and decreased feeding rates, caused by the 100 nm particles, could indicate that particles in the nanometer size range are potentially more hazardous to D. magna compared to larger particle sizes[1].

1. magna could not completely egest MPs accumulated in its body within 24 h. In our experimental process, the exposure medium was replaced daily. Consequently, D. magna ingested new MPs before it could egest the previously accumulated ones. Furthermore, large-sized microplastics were more palatable as food for D. magna. This phenomenon manifested as a higher ingestion amount of large-sized microplastics, whose fluorescence intensity was far greater than that of small-sized microplastics. Therefore, the level of fluorescence was minimal in Group A. This phenomenon cannot obscure the fact that smaller-sized MPs accumulate more in organisms and remain for a longer time.

[1] Rist, S.; Baun, A.; Hartmann, N. B., Ingestion of micro- and nanoplastics in Daphnia magna – Quantification of body burdens and assessment of feeding rates and reproduction [J]. Environ. Pollut. 2017, 228, 398-407.

Comments 9. Explanations to the details of the image are required. How is the mean, median, etc. indicated? The caption for Figure 4 seems to be inaccurate. It would be more accurate to use the term "Reproductive parameters" instead of "Changes in growth and development" in this context.

Response 9: The authors thank for this suggestion. As suggested, the term "Reproductive parameters" instead of "Changes in growth and development" in this context.

Comments 10. Since the experimental setup included plastic particles of different sizes and concentrations, it is necessary to draw a conclusion about the impact of these parameters on the reproductive ability and changes in the gut microbiota of Daphnia.

Response 10: Thank you very much for this suggestion. We have added a conclusion about the impact of these parameters on the reproductive ability and changes in the gut microbiota of Daphnia as followed.

This study demonstrates that the deleterious effects of microplastics on D. magna's reproduction and gut microbiota are co-determined by particle size and concentration, with high concentrations of large (5 μm) particles exerting the most severe impact. As D. magna developed, it preferentially ingested these larger microplastics, which led to higher accumulation in the gut. This accumulation not only physically damaged the intestinal structure, accelerating aging, but also significantly disrupted the gut microbial community. Functional prediction analysis further revealed that these microbial changes impaired core glucose metabolism pathways, inducing a starvation-like state. Consequently, these intertwined microbial and physical injuries directly resulted in retarded growth and the most pronounced delays in reproduction. Moreover, the observed physiological and metabolic disruptions highlight a broader ecological concern: the potential decline in zooplankton-mediated ecosystem services. As key components of aquatic food webs and biogeochemical cycles, reduced zooplankton populations could impair water quality, alter species composition, and weaken the resilience of freshwater ecosystems. Thus, addressing microplastic pollution is critical not only for protecting sentinel species like D. magna, but also for sustaining the functional integrity and resilience of aquatic ecosystems in the face of environmental change.

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This study addresses a highly topical ecotoxicological issue: the effects of microplastics on Daphnia magna, a key organism in aquatic food webs. Particular emphasis is placed on comprehensive investigations into the impairment of the life cycle through disruption of glucose metabolism and structural changes in the microbiota and structure.

Introduction:

The introduction presents the relevance of microplastic pollution in aquatic ecosystems. It clearly describes the problem of microplastic uptake by organisms and highlights both direct and indirect risks. The choice of Daphnia magna as a model organism is well justified. The compilation of literature on the influence of microplastics on intestinal microbiota and metabolism provides a solid foundation.

Materials and Methods:

The description of materials, experimental setups, and analytical approaches is detailed and comprehensible. The use of fluorescent microplastic particles and advanced analytical methods (16S rRNA sequencing, scanning electron microscopy) is technically appropriate and innovative. The investigative methods are clearly presented.

Results and Discussion

The results are clearly structured and comprehensively presented. The statistical analysis of fluorescence intensities, growth and reproduction data and intestinal microbiota is statistically robust. Including functional predictions of metabolism and structural intestinal changes broadens our understanding of the mechanisms of action.

The legend of Figure 4 must be corrected.

Figure 4. Changes in growth and development parameters of D. magna during the experiment (Aa: Time to first brood, Bb: Number of offspring in first brood, Cc: Total number of broods, Dd: Total number of offspring).

The discussion meaningfully relates the observed effects to previous studies and emphasizes their ecological relevance.

Conclusion:

The conclusion summarizes the key results and underscores the importance of the study for ecological and toxicological research. References to potential impacts on aquatic ecosystems and implications for environmental management are well founded.

 

 

 

 

 

Author Response

Reviewer 2 This study addresses a highly topical ecotoxicological issue: the effects of microplastics on Daphnia magna, a key organism in aquatic food webs. Particular emphasis is placed on comprehensive investigations into the impairment of the life cycle through disruption of glucose metabolism and structural changes in the microbiota and structure.

Comments 1. Introduction: The introduction presents the relevance of microplastic pollution in aquatic ecosystems. It clearly describes the problem of microplastic uptake by organisms and highlights both direct and indirect risks. The choice of Daphnia magna as a model organism is well justified. The compilation of literature on the influence of microplastics on intestinal microbiota and metabolism provides a solid foundation.

Response 1: The authors thanked for this comment.

Comments 2. Materials and Methods:

The description of materials, experimental setups, and analytical approaches is detailed and comprehensible. The use of fluorescent microplastic particles and advanced analytical methods (16S rRNA sequencing, scanning electron microscopy) is technically appropriate and innovative. The investigative methods are clearly presented.

Response 2: The authors thanked for this comment.

Comments 3. Results and Discussion

The results are clearly structured and comprehensively presented. The statistical analysis of fluorescence intensities, growth and reproduction data and intestinal microbiota is statistically robust. Including functional predictions of metabolism and structural intestinal changes broadens our understanding of the mechanisms of action.

Response 3: The authors thanked for this comment.

Comments 4. The legend of Figure 4 must be corrected.

Figure 4. Changes in growth and development parameters of D. magna during the experiment (a: Time to first brood, b: Number of offspring in first brood, c: Total number of broods, d: Total number of offspring).

Response 4: The authors thanked for this point. As suggested, the relative parts were modified in Figure 4.

Comments 5. The discussion meaningfully relates the observed effects to previous studies and emphasizes their ecological relevance.

Conclusion:

The conclusion summarizes the key results and underscores the importance of the study for ecological and toxicological research. References to potential impacts on aquatic ecosystems and implications for environmental management are well founded.

Response 4: The authors thanked for this comment.

 

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

1. Recommendation
Minor Revision
Title: Microplastics Alter Daphnia magna Life History by Disrupting Glucose Metabolism and Intestinal Structure
Overview and General Recommendations:
Various environmental studies have been conducted using Daphnia. While published studies have examined the growth, reproduction, and accumulation of Daphnia in response to microplastics, few have clearly demonstrated the effects of gut microbiota disruption on glucose metabolism. This paper clearly demonstrates the effects of microplastics on the gut of Daphnia and will be of great help to researchers conducting related research. However, a few minor revisions are needed.

This paper is well-written, with a relatively detailed introduction, methods, results and discussion, and conclusion. Consequently, this paper is considered suitable for publication in Sustainability after minor revision.

The main comments on the paper are as follows:
Abstract
This paper summarizes the effects of microplastics on gut glucose metabolism and the structural damage and aging of gut tissue. (No corrections)

1. Introduction (NO revision)
Despite the limited number of papers on the effects of microplastics on the gut of Daphnia magna, this review was well-written.
The two research objectives, intestinal changes and structural damage caused by the microbiota, were clearly and concisely presented.

2. Materials and Methods (NO revision)

Overall, the test methods were clearly summarized.

3. Results

3.1. Changes in Fluorescence Intensity in D. magna

The differences in gut residence time and uptake efficiency according to the amount of microplastic ingested were well-described.

3.2. Effects of Microplastics on the Growth and Development of D. magna
ANOVA was used to determine significance and clearly demonstrate the effects of microplastics on growth and survival.

3.3 Effects of Microplastics on Offspring Development of D. magna

The results demonstrating the effects of high-concentration and large plastic particle exposure on Daphnia reproduction were well-described.

3.4 Effects of Microplastics on the Intestinal Microbiota of D. magna
The study examined the effects of changes in the intestinal microbiota on glucose metabolism.

3.5 Effects of Microplastics on the Intestinal Structure of D. magna
SEM examination of the intestines revealed villus loss, cell shedding, mucosal destruction, and accelerated aging, confirming that microplastics accelerate intestinal aging.

3.6 Implications for Aquatic Ecosystem Sustainability
While the contribution of microplastics to the decline in Daphnia populations was well demonstrated, at least two additional reviews of related papers are needed.

5. Conclusion

- The results of the paper were summarized relatively well. However, rather than stating "perferentially ingested larger MPs," it is recommended to include quantitative numbers.

6. References

- Written in MDPI format, some aspects require rechecking (e.g., corrections to DOI notation, including spacing).

Overall, this paper, which studies the ecotoxicity of microplastics to Daphnia, is highly readable and logically presented. Therefore, this paper is suitable for publication in Sustainability.

Author Response

Reviewer 3: Overview and General Recommendations:

Various environmental studies have been conducted using Daphnia. While published studies have examined the growth, reproduction, and accumulation of Daphnia in response to microplastics, few have clearly demonstrated the effects of gut microbiota disruption on glucose metabolism. This paper clearly demonstrates the effects of microplastics on the gut of Daphnia and will be of great help to researchers conducting related research. However, a few minor revisions are needed.

This paper is well-written, with a relatively detailed introduction, methods, results and discussion, and conclusion. Consequently, this paper is considered suitable for publication in Sustainability after minor revision.

The main comments on the paper are as follows:
Comments 1. Abstract

This paper summarizes the effects of microplastics on gut glucose metabolism and the structural damage and aging of gut tissue. (No corrections)

Response 1: The authors thanked for this comment.

Comments 2. 1. Introduction (NO revision)

Despite the limited number of papers on the effects of microplastics on the gut of Daphnia magna, this review was well-written.

The two research objectives, intestinal changes and structural damage caused by the microbiota, were clearly and concisely presented.

Response 2: The authors thanked for this comment.

Comments 3. Materials and Methods (NO revision)

Overall, the test methods were clearly summarized.

Response 3: The authors thanked for this comment.

Comments 4. 3. Results

3.1. Changes in Fluorescence Intensity in D. magna

The differences in gut residence time and uptake efficiency according to the amount of microplastic ingested were well-described.

3.2. Effects of Microplastics on the Growth and Development of D. magna

ANOVA was used to determine significance and clearly demonstrate the effects of microplastics on growth and survival.

3.3 Effects of Microplastics on Offspring Development of D. magna

The results demonstrating the effects of high-concentration and large plastic particle exposure on Daphnia reproduction were well-described.

3.4 Effects of Microplastics on the Intestinal Microbiota of D. magna

The study examined the effects of changes in the intestinal microbiota on glucose metabolism.

3.5 Effects of Microplastics on the Intestinal Structure of D. magna

SEM examination of the intestines revealed villus loss, cell shedding, mucosal destruction, and accelerated aging, confirming that microplastics accelerate intestinal aging.

3.6 Implications for Aquatic Ecosystem Sustainability

While the contribution of microplastics to the decline in Daphnia populations was well demonstrated, at least two additional reviews of related papers are needed.

Response 4: The authors thanked for this comment.

Comments 5. Conclusion

The results of the paper were summarized relatively well. However, rather than stating "perferentially ingested larger MPs," it is recommended to include quantitative numbers.

Response 5: Thank you for this suggestion. The ingestion of microplastics with different particle sizes by D. magna is expressed via fluorescence intensity as follows.

Time	Group A	Group B	Group C	Group D
2	1.57±0.20	24.12±3.41	15.76±0.95	57.17±6.68
7	3.13±0.39	8.68±1.23	9.02±1.85	52.56±2.82
14	3.63±1.53	5.50±1.17	10.21±2.10	58.43±4.378
21	1.42±0.14	8.25±0.05	20.12±3.12	61.38±2.18
28	1.97±0.13	6.31±0.88	22.74±2.11	68.01±0.92
35	3.16±0.53	5.36±0.47	9.10±1.02	72.08±5.51
49	1.64±0.48	6.81±1.27	22.82±6.27	72.34±3.12

Although the amount of MPs ingested by D. magna varies at different time points, it is still clearly observed that the ingestion amount of large-sized microplastics is several times that of small-sized ones.

Comments 6. References

Written in MDPI format, some aspects require rechecking (e.g., corrections to DOI notation, including spacing).

Response 6: As suggested, the References part was modified in manuscript.

 

Author Response File: Author Response.pdf