Quantitative Detection of Microplastics in Water through Fluorescence Signal Analysis

Round 1

Reviewer 1 Report

The article is interesting and well-written and i only have made a few minor suggestions below: 

The term “microplastics” was not coined by Prof. Thompson, it was indeed coined much earlier by Ryan, P. G. and C. L. Moloney (1990). ‘Plastic and other artefacts on South African beaches - temporal trends in abundance and composition’. South African Journal of Science 86(7-10): 450-452.

 

Line 244: two decades? Can you please explain what you mean here? This is unclear. Perhaps you meant two-fold?

 

Lines 306-310: how can you explain this? Any hypothesis? Perhaps this can be explained by the fact that tap water was not pre-filtered before the experiment and so also non-spiked particles were counted by the sensor? Can you elaborate a little bit more on this unexpected result? What are the most likely explanations? 

 

Also, it would be good to briefly discuss the reason for choosing to test only PS particles in this experiment. Why use PS only, given that PE and PP are the two most abundant polymers in the environment? Can you briefly elaborate on the reasoning behind this single polymer choice?

 

 

Author Response

Reviewer 1

The article is interesting and well-written and i only have made a few minor suggestions below:

First, we would like to thank the reviewer for her/his nice words of appreciation, for the time and effort invested into the review of our manuscript, for the constructive and valuable comments which were of great help in revising and improving the manuscript. Accordingly, the revised version has been systematically revised and/or integrated as follows: 

The term “microplastics” was not coined by Prof. Thompson, it was indeed coined much earlier by Ryan, P. G. and C. L. Moloney (1990). ‘Plastic and other artefacts on South African beaches - temporal trends in abundance and composition’. South African Journal of Science 86(7-10): 450-452.

We thank the reviewer for his/her suggestion. We revised the text accordingly and added the reference.

 

Line 244: two decades? Can you please explain what you mean here? This is unclear. Perhaps you meant two-fold?

The term was replaced with two orders of magnitude.

 

Lines 306-310: how can you explain this? Any hypothesis? Perhaps this can be explained by the fact that tap water was not pre-filtered before the experiment and so also non-spiked particles were counted by the sensor? Can you elaborate a little bit more on this unexpected result? What are the most likely explanations?

We thank the reviewer for his/her suggestion, and we decided to perform measurements after pre-filtration of the tap water by using a 0.7 µm glass-fibre filter in order to reduce possible MPs originally present in the real samples. Moreover, we measured what are called “the negative controls” that are counts in water before spiking with MPs (NR + water without MPs). In addition, inspired by the very recent paper by Hernandez et al. (2023), we also reduced the Nile Red concentration to 0.1 mg/l to reduce dye aggregation and false counts, but this didn’t improve the situation, instead prefiltration reduced the counts and the fluctuations. These new data are reported in the new graph (Figure 7) that replaced the old one. A comparison with unfiltered water samples, is reported in Figure S5 ed S6 in Supplementary Information.

 

Also, it would be good to briefly discuss the reason for choosing to test only PS particles in this experiment. Why use PS only, given that PE and PP are the two most abundant polymers in the environment? Can you briefly elaborate on the reasoning behind this single polymer choice?

We agree that PS is not the most abundant polymer in the environment but, at a first stage, we wanted to use the same plastic as the one used for calibration. Measurements are in progress on PC, PET, PE and PP but we think they are out of the purpose of this first study which essentially intended to demonstrate the proof of concept of the new method. A sentence has been added to Materials and Methods.

Reviewer 2 Report

In this manuscript, the authors developed an optical platform to detect the microplastic particles in water quantitatively. The performance of the platform has been calibrated and validated with fluorescence microsphere and samples in real water sample. Overall, it is an interesting study for environmental detection and monitoring. I have the following suggestions and comments that hope the authors can help address:

1. In cell study, people use flow cytometry to quantify the fluorescence objects in a fast and efficient way. How does the proposed method performance compared to the state of art flow cytometry?

2. In real applications, the test results will be significantly impacted by other particles also response to fluorescence or the background of fluorescence emission from the real water sample will dramatically reduce the contrast. How will that be resolved?

3. In real applications, will the microplastic particles have similar level of fluorescence response? Will that impact the performance?

 

Author Response

Reviewer 2

In this manuscript, the authors developed an optical platform to detect the microplastic particles in water quantitatively. The performance of the platform has been calibrated and validated with fluorescence microsphere and samples in real water sample. Overall, it is an interesting study for environmental detection and monitoring. I have the following suggestions and comments that hope the authors can help address:

First, we would like to thank the reviewer for her/his nice words of appreciation, for the time and effort invested into the review of our manuscript, for the constructive and valuable comments which were of great help in revising and improving the manuscript. Accordingly, the revised version has been systematically revised and/or integrated as follows: 

1. In cell study, people use flow cytometry to quantify the fluorescence objects in a fast and efficient way. How does the proposed method performance compared to the state of art flow cytometry?

Indeed, the present setup was inspired by the typical scheme of a flow cytometer based on fluorescence analysis to be implemented on a cheaper and more user-friendly scale and applied to MPs. In addition, we arrived at 100 ml/h but we aim to reach 2000 ml/h with a larger capillary in order to analyse large volume samples reasonably quickly, e.g. for drinking water with very low MP concentrations. That flow range is out of the typical range of flow cytometers. Moreover, our system is centred on time analysis that is not generally used by cytometers.

 

2. In real applications, the test results will be significantly impacted by other particles also response to fluorescence or the background of fluorescence emission from the real water sample will dramatically reduce the contrast. How will that be resolved?

With regard to drinking water, one of the real applications our system is mainly addressed to, our study demonstrated that the background can easily be handled by the software. Actually, it is designed for signals with much worse noise-to-signal ratio. Water samples from lakes, rivers or wastewater treatment plant can have a much greater amount of organic matter and thus a much higher fluorescence background. That is a common problem to visual observation and, for this reason, a large variety of pre-treatment processes based on digestion, etc., have long been developed as largely discussed and explained in the literature. Some considerations about this problem have been added to Conclusions.

 

3. In real applications, will the microplastic particles have similar level of fluorescence response? Will that impact the performance?

We are quite confident that real microplastic particles will have a similar brightness as that of PS MPs used in the present paper, obtained from PS object. Measurements performed with the same method on other types of MPs (PETand PE, not reported here) showed approximately the same fluorescence intensity. In a previous work we successfully performed measurements on real samples of wastewater from treatment plants by using a different fluorescence-based method (Nicolai, Eleonora, et al. "A New Optical Method for Quantitative Detection of Microplastics in Water Based on Real-Time Fluorescence Analysis." Water 14.20 (2022): 3235.). In addition, our system has a broad dynamic range not completely exploited in the present study. Finally, as stated in the abstract and in the conclusion, the present paper reports a proof of concept aiming to demonstrate the feasibility of the method. Further study will explore all the open questions.

Reviewer 3 Report

This manuscript is devoted to a very relevant environmental topic related to the determination of contamination of drinking water with microplastics. The authors have conducted serious research, which, although preliminary, can be published in the journal Photonics.

There are the following questions, comments and suggestions on the text of the Manuscript.

1. Citations [5-9], [28-31] should preferably be divided into 2-3 references of 1-3 sources of information.

2. Figures 1, 2 and others should be placed in the Manuscript immediately after the first mention, and not on the next page.

3. Why is the exciting radiation of 520nm used? What are the excitation spectra of MPs, NR?

4. If the laser radiation is continuous (line 116), then why is the fluorescence pulsed (lines 126-127, 133)? Explanations are needed in the text.

5. It is necessary to provide (at least in Additional materials) the spectral characteristics of the system components (radiation receiver, light filters).

6. Is it possible to get rid of the need to use a dichroic mirror when applying exciting radiation at an angle of 90 ° to the axis of the receiving optical system? This will reduce the cost of the system and simplify the adjustment.

7. Lines 319-321 should be deleted from the Conclusion section, since they are not conclusions.

Author Response

Reviewer 3

This manuscript is devoted to a very relevant environmental topic related to the determination of contamination of drinking water with microplastics. The authors have conducted serious research, which, although preliminary, can be published in the journal Photonics.

First, we would like to thank the reviewer for her/his nice words of appreciation, for the time and effort invested into the review of our manuscript, for the constructive and valuable comments which were of great help in revising and improving the manuscript. Accordingly, the revised version has been systematically revised and/or integrated as follows: 

 

There are the following questions, comments and suggestions on the text of the Manuscript.

 

1. Citations [5-9], [28-31] should preferably be divided into 2-3 references of 1-3 sources of information.

Done

 

2. Figures 1, 2 and others should be placed in the Manuscript immediately after the first mention, and not on the next page.

We replaced the figure immediately after the first mention in the text. Only for figure 2 this wasn’t possible due to the dimensions of the figure. The editorial office will take care of it.

 

3. Why is the exciting radiation of 520nm used? What are the excitation spectra of MPs, NR?

The wavelength of 520 nm was used since it lies in the excitation range of NR, see the spectrum added to Supplementary Material, without being too close to the cut-off wavelength of the dichroic mirror. MPs are not absorbing at 520 nm.

 

4. If the laser radiation is continuous (line 116), then why is the fluorescence pulsed (lines 126-127, 133)? Explanations are needed in the text.

We have added the following sentence “The MPs that cross the excitation volume emit fluorescence pulses since they travel across a limited excitation volume with a certain speed. For this reason, the effective excitation is limited in time and the fluorescence is pulsed even under a continuous light source.”

 

5. It is necessary to provide (at least in Additional materials) the spectral characteristics of the system components (radiation receiver, light filters).

The spectra have been added in supplementary information as figure S7.

 

6. Is it possible to get rid of the need to use a dichroic mirror when applying exciting radiation at an angle of 90 ° to the axis of the receiving optical system? This will reduce the cost of the system and simplify the adjustment.

We thank the Reviewer for this suggestion. That is possible and what we are planning to try. However, that modification, which would reduce the number of optical components and simplify the alignment, would also increase the bulk of the system in the perpendicular direction. We will try and check for the best trade-off.

 

7. Lines 319-321 should be deleted from the Conclusion section, since they are not conclusions.

Done