Microplastics Affect Rates of Locomotion and Reproduction via Dietary Uptake in Globally Invasive Snail Physa acuta

Round 1

Reviewer 1 Report

The authors investigated the rates of microplastic ingestion (polystyrene), effects on locomotion, and reproduction in invasive snail Physa acuta. Although this research and the results presented are interesting, there are minor concerns that should be corrected/improved.

Some specific comments are listed below:

2. Materials and Methods-2.1 Preparation and Characterization of MPs

(L78-86) The authors stated that they used discarded macro plastic procured from the plastic processing industry and identified the plastic as polystyrene (PS). Considering the origin of macro plastic, were other types of microplastic also found, or was just PS identified?

Concentrations 500×104, 750×104 and 1000×104 particles/200mL respectively – do authors mean 500×10^4? If so, it should be corrected throughout the manuscript.

Authors stated that the common occurrence of MPs in freshwater is about 5-34 particles/L (L34-L35), and their lowest concentration of MPS is 20 mg/200 mL or 2.5 x104 particles/mL (again I suppose authors mean 2.5 x 10^4 (2.5 x 10⁴)) which is quite high compared to environmentally relevant concentrations. Authors should provide some justification for the concentrations used and put them in context with relevant concentrations. Also, in the discussion section, authors should provide some justification for why they find that their results are environmentally relevant (or in which cases they can be relevant).

Experiment II (Fecundity) – a. Egg capsule production

What number of organisms was used in this part of the experiment?

Results

Fig.3

L147 -148 If I understand, there are 3 replicates (samples) per concentration? This number of test organisms is quite small. Considering that, can authors provide confidence intervals for fit (Michaelis-Menten) in Fig. 3 (ingestion rate)?

Fig. 4.

Please mark significant differences on the graph. Also, for the y-axis unit, I suppose that speed is mm/min?

Fig 7.

The figure description says that results are presented as mean ± SD, however standard deviations are very small, so I would suspect those are possibly standard errors? In previous graphs, authors used standard error (mean ± SE) which is always smaller than SD. Please check. Please mark significant differences on the graph.

Supplementary materials

What is the difference between supplementary files and Non-published Material?

Author Response

Author’s point by point response to Reviewer’s comment

Open Review

The authors investigated the rates of microplastic ingestion (polystyrene), effects on locomotion, and reproduction in invasive snail Physa acuta. Although this research and the results presented are interesting, there are minor concerns that should be corrected/improved.

Author: Thanks for appreciation and encouraging remarks. These positive remarks have been encouraging us to further study along the line

Some specific comments are listed below:

2. Materials and Methods-2.1 Preparation and Characterization of MPs

(L78-86) The authors stated that they used discarded macro plastic procured from the plastic processing industry and identified the plastic as polystyrene (PS). Considering the origin of macro plastic, were other types of microplastic also found, or was just PS identified?

Author: Just PS were identified. We have clearly given the FT IR spectrum of the polymers.

L83, Page 2, 2. Materials and Methods, 2.1 Preparation and Characterization of MPs:

Specified raw material discards as the initial plastic, ground and sieved, to produce MPs.

Revised as suggested; Page: 2 line 85

Concentrations 500×104, 750×104 and 1000×104 particles/200mL respectively – do authors mean 500×10^4? If so, it should be corrected throughout the manuscript.

Corrections made throughout the manuscript.

Authors stated that the common occurrence of MPs in freshwater is about 5-34 particles/L (L34-L35), and their lowest concentration of MPS is 20 mg/200 mL or 2.5 x104 particles/mL (again I suppose authors mean 2.5 x 10^4 (2.5 x 10⁴)) which is quite high compared to environmentally relevant concentrations. Authors should provide some justification for the concentrations used and put them in context with relevant concentrations. Also, in the discussion section, authors should provide some justification for why they find that their results are environmentally relevant (or in which cases they can be relevant).

L70-L76, Page 5, 1. Introduction: Stated the justification for the concentration used, in relation to the concentrations discharged at wastewater effluent sites.

Experiment II (Fecundity) – a. Egg capsule production

What number of organisms was used in this part of the experiment?

A total of 40 freshly matured oragsnisms were used . explained in the Ms tex Page: 5: line 209-210

L206, Page 5 , 2. Materials and methods, 2.3 Experimental setup, 2.3.2 Long duration experimental set up, Experiment II (Fecundity)- a. Egg capsule production

A total of 40 freshly matured oragsnisms were used . explained in the Ms tex Page: 5: line 209-210

Results

Fig.3

L147 -148 If I understand, there are 3 replicates (samples) per concentration? This number of test organisms is quite small. Considering that, can authors provide confidence intervals for fit (Michaelis-Menten) in Fig. 3 (ingestion rate)?

Figure 3, Page 7: We are currently working for an intendent objective under the effect of MPs on feeing on natural food by P acuta, for this objective we have estimated functional response for 45 individuals at each MP concentration. The number of data is sufficient for z test to test the confidence interval.  For the present work where feeding trial is just a confirmatory test MP ingestion and differential effect of concentration we have shown Functional response curve. We fully agree that 3 data points are not enough to estimate confidence interval, in the revised version we have use data of 10 individual snails for each concentration and clearly shown the Standard error of 10 data points, Confidence interval is estimated and t test can be applied.

Fig. 4.

Please mark significant differences on the graph. Also, for the y-axis unit,

Figure 4, Page 7: Thanks for the suggestion. Significant differences are marked in the revise figures.

I suppose that speed is mm/min?

Thanks! The speed is mm/min indicated as Y axis legend of figure 4 and legend of Figure 5.

Fig 7.

The figure description says that results are presented as mean ± SD, however standard deviations are very small, so I would suspect those are possibly standard errors? In previous graphs, authors used standard error (mean ± SE) which is always smaller than SD. Please check. Please mark significant differences on the graph.

Thanks! We have corrected Figure 7, L349, L367-L371, Page 11: changes made accordingly

Supplementary materials

What is the difference between supplementary files l?

Non published materials have been remove the file is supplementary materials.

We are thankful for thorough reviewing the Ms accordingly we have followed all suggestions made.

Reviewer 2 Report

The article submitted for review presents research on investigating the ingestion probability, locomotory and reproductive performance of P. acuta, exposed to a range of MPs concentrations (0 to 40 mg/200mL) in laboratory controlled condition. The article was written with great scientific care, however, it requires a few corrections:
1. Please specify the fact of documenting the occurrence of MP in aquatic ecosystems. The abstract mentions that this is poorly documented and the introduction contradicts this.
2. How was the aeration described in section 2.2 controlled (what was the oxygen concentration?).
3. I suggest renumbering the experiments so that there is no doubt which to refer to later.
4. Please omit the dot in the description of the figures (unless an abbreviation is used). Please check and correct this entry in the text as well. This note applies to all figures. Alternatively, I would suggest putting a dot after the number.
5. Shouldn't chapter 3.3.3 be numbered 3.2.3?
6. I propose to improve the quality of the presented figures.

Moreover, the publication is a valuable source of information and forms the basis for further research and implementation of new solutions.

Thank you for considering my opinion. I encourage the authors to continue working on improving the manuscript.

Author Response

The article submitted for review presents research on investigating the ingestion probability, locomotory and reproductive performance of P. acuta, exposed to a range of MPs concentrations (0 to 40 mg/200mL) in laboratory controlled condition. The article was written with great scientific care, however, it requires a few corrections:

Author: Thanks for the appreciation and encouraging remarks. These positive remarks have been encouraging us to further study along the line. we have followed all suggestions made.

1. 
   Please specify the fact of documenting the occurrence of MP in aquatic ecosystems. The abstract mentions that this is poorly documented and the introduction contradicts this.

Page1, L14-L17, Abstract: Changes made as suggested. We intend to state that no study has looked into long term reproduction and age specific locomotion,

2. 
   How was the aeration described in section 2.2 controlled (what was the oxygen concentration?).

Page 3, L116, 2. Material and Methods, 2.2. Test Organism:  Aeration was done to maintain Dissolved Oxygen concentration >6mg/. added Page 3: line 118.

3. 
   I suggest renumbering the experiments so that there is no doubt which to refer to later.

Thanks experiments have been renumbered and same referred throughout the Ms.

4. 
   Please omit the dot in the description of the figures (unless an abbreviation is used). Please check and correct this entry in the text as well. This note applies to all figures. Alternatively, I would suggest putting a dot after the number.

Yes ! Edited accordingly.

5. Shouldn't chapter 3.3.3 be numbered 3.2.3?

Thanks! Yes corrected Page 11, L354:

6. I propose to improve the quality of the presented figures.

We have redrawn all the figures improving the quality. We are also giving separate figures in tif version with higher resolution (>400pi)   Moreover, the publication is a valuable source of information and forms the basis for further research and implementation of new solutions.

Thank you for considering my opinion. I encourage the authors to continue working on improving the manuscript.

We gratefully thank for encouraging remarks we will continue the work along this line.