Preconcentration with Chlorella vulgaris combined with energy dispersive X-ray fluorescence spectrometry for rapid determination of Cd in water

Freshwater green algae Chlorella vulgaris was selected as an adsorbent, and a simple, rapid, economical and environmentally friendly method for the detection of heavy metal Cd in water samples based on preconcentration with C. vulgaris combined with energy dispersive X-ray fluorescence (EDXRF) spectrometry was proposed. Chlorella vulgaris could directly and rapidly adsorb Cd2+ without any pretreatment, and the maximum adsorption efficiency could be obtained when the contact time was 1 min with an optimal pH of 10. The obtained Cd-enriched thin samples after preconcentration with C. vulgaris by suction filtration of reaction solution had very good uniformity, which could be directly measured by EDXRF spectrometry, and the net integral fluorescence intensity of Cd Kα characteristic peak had a very good linear relationship with the initial concentration of Cd in the range of 0.703–74.957 µg ml−1 with a correlation coefficient of 0.9979. When the Cd thin samples with a Cd-enriched region of 15.1 mm in diameter were formed by the developed preconcentration method with suction filtration of 10 ml reaction solution, the detection limit of this method was 0.0654 µg ml−1, which was lower than the maximum allowable discharge concentration of Cd in various industrial wastewaters. The proposed method was simple to operate, and could effectively remove the influence of matrix effect of water samples and effectively improve the sensitivity and stability of EDXRF spectrometry directly detecting heavy metals in water samples, which was successfully applied to detect Cd in real water samples with satisfactory results, and the recoveries ranged from 94.80% to 116.94%. Moreover, this method can be applied to the rapid detection and early warning of excessive Cd in discharged industrial wastewaters. This work will provide a methodological basis for the development of rapid and online monitoring technology and instrument of heavy metal pollutants in water.

# Experimental -The C. vulgaris culture solution need to be characterized. The mass of adsorbent in the suspension need to be determined. -Please add more information about the mixed cellulose membranes. -Preparation of the calibration standards and blank need to be described.
-The sample volume needs to be informed in the procedure.
# Results and discussion -Discuss why the use of the C. vulgaris in culture solution was more efficient.
-The effect of pH is not clear. How was the pH control performed in the system? For example, the authors affirm that the method can applied in discharged industrial wastewaters. However, the final pH of the system can be affected by the sample acidity.
-Please present the study of the effect of matrix. -The C. vulgaris could adsorb other inorganic species from water. In this way, a study of potential interferents need to be presented. -Inform how the detection limit was calculated.
-The method was unable to determine cadmium in the water samples without adding the analyte. However, the detection limit of the method can be improved and decreased by varying, for example, the sample volume. Additional experiments need to be present with this intention. -The method is not sufficiently validated. For example: the results obtained need to be compared with a comparative method or a water certified reference material needs to be analyzed.

06-Mar-2020
Dear Dr Gan: Title: Preconcentration with Chlorella vulgaris combined with energy dispersive X-ray fluorescence spectrometry for rapid determination of Cd in water Manuscript ID: RSOS-200182 Thank you for your submission to Royal Society Open Science. The chemistry content of Royal Society Open Science is published in collaboration with the Royal Society of Chemistry.
The editor assigned to your manuscript has now received comments from reviewers. We would like you to revise your paper in accordance with the referee and Subject Editor suggestions which can be found below (not including confidential reports to the Editor). Please note this decision does not guarantee eventual acceptance.
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When submitting your revised manuscript, you must respond to the comments made by the referees and upload a file "Response to Referees" in "Section 6 -File Upload". Please use this to document how you have responded to the comments, and the adjustments you have made. In order to expedite the processing of the revised manuscript, please be as specific as possible in your response. Comments to the Author(s) The manuscript presents a preconcentration procedure with Chlorella vulgaris for posterior determination of Cd in water samples using energy dispersive X-ray fluorescence spectrometry. The subject is interesting and important and has merit for publication. There are comments/ addings, and perhaps additional experiments, which could be considered prior to publication: # Experimental -The C. vulgaris culture solution need to be characterized. The mass of adsorbent in the suspension need to be determined. -Please add more information about the mixed cellulose membranes. -Preparation of the calibration standards and blank need to be described.
-The sample volume needs to be informed in the procedure.
# Results and discussion -Discuss why the use of the C. vulgaris in culture solution was more efficient.
-The effect of pH is not clear. How was the pH control performed in the system? For example, the authors affirm that the method can applied in discharged industrial wastewaters. However, the final pH of the system can be affected by the sample acidity.
-Please present the study of the effect of matrix. -The C. vulgaris could adsorb other inorganic species from water. In this way, a study of potential interferents need to be presented. -Inform how the detection limit was calculated.
-The method was unable to determine cadmium in the water samples without adding the analyte. However, the detection limit of the method can be improved and decreased by varying, for example, the sample volume. Additional experiments need to be present with this intention. -The method is not sufficiently validated. For example: the results obtained need to be compared with a comparative method or a water certified reference material needs to be analyzed.

30-Mar-2020
Dear Dr Gan: Title: Preconcentration with Chlorella vulgaris combined with energy dispersive X-ray fluorescence spectrometry for rapid determination of Cd in water Manuscript ID: RSOS-200182.R1 It is a pleasure to accept your manuscript in its current form for publication in Royal Society Open Science. The chemistry content of Royal Society Open Science is published in collaboration with the Royal Society of Chemistry.
The comments of the reviewer(s) who reviewed your manuscript are included at the end of this email.
Thank you for your fine contribution. On behalf of the Editors of Royal Society Open Science and the Royal Society of Chemistry, I look forward to your continued contributions to the Journal. We appreciate for Editor and Reviewers' warm work earnestly. We would like to resubmit this revised manuscript to Royal Society Open Science, and hope it is acceptable for publication in the journal. In what follows, we respond to these comments and we list the changes to the original manuscript.
Once again, thank you very much for your comments and suggestions. Looking forward to hearing from you soon.

Tingting Gan
Anhui Institute of Optics and Fine Mechanics, Chinese Academy of Sciences Appendix A

Responses to the comments made by the Reviewers
Reviewer 1: Comment: This paper is an interesting study. The data support the conclusion, and I am very interested to read the paper.
Response: Thank you very much for your interest and carefully reading our manuscript, and giving us a positive comment on this paper. And we have further revised and improved our manuscript during this revision stage in order to make it better and more acceptable for this journal. Once again, special thanks to you for your comment.

Reviewer 2:
Comments: The manuscript presents a preconcentration procedure with Chlorella vulgaris for posterior determination of Cd in water samples using energy dispersive X-ray fluorescence spectrometry. The subject is interesting and important and has merit for publication. There are comments/ addings, and perhaps additional experiments, which could be considered prior to publication:

Comment:
Please add more information about the mixed cellulose membranes.
Response: Thank you very much for your suggestion. In our original manuscript, the information about the mixed cellulose membranes was indeed too simple and not adequate. So according to your suggestion, in our revised manuscript, we have added more detailed information about the mixed cellulose membranes in the section "2.

Comment:
Preparation of the calibration standards and blank need to be described.

Comment:
The effect of pH is not clear. How was the pH control performed in the system? For example, the authors affirm that the method can applied in discharged industrial wastewaters. However, the final pH of the system can be affected by the sample acidity.
Response: Thank you very much for your comment. We are very sorry that in our original manuscript the effect of pH and the pH control of reaction solution system were not described clearly. So according to your comment, in our revised manuscript, the sentences in line 7-23 have been carefully modified so that we can more clearly describe how pH affected the adsorption of Cd by C. vulgaris. And in order to clearly describe how to control the pH of the reaction solution, we have supplemented the Effect of reaction solution pH". In addition, because when the pH value of the reaction solution system of C. vulgaris and Cd was 10, the adsorption capacity of C.
vulgaris was the greatest, and the adsorption efficiency of Cd was the highest. So we consider when this method is applied to the detection of heavy metals Cd in discharged industrial wastewaters, 0.05 mol L -1 Na2B4O7-NaOH buffer solution with a pH of 10 will be used to adjust the pH value of reaction solution to 10. This way the final pH of the reaction system can't be affected by the sample acidity.

Comment:
Please present the study of the effect of matrix.
Response: Thank you very much for your comment. In this paper, we used C.
vulgaris in the culture solution after 8 days of cultivation to enrich heavy metal Cd.
Therefore, according to your suggestion, we have carefully considered that the matrix you said should refer to the matrix in the algae culture solution at this time. We think that the matrix in the culture solution at this time mainly includes some remaining nutrients in the culture medium, some substances secreted by the algae cells during the culture process, and some dead algae cells. According to the research results in the section "3.2.1 Effects of solution environment of algae cells and contact time" in our revised manuscript, after removing the original culture solution by centrifugation and washing, the adsorption capability of C. vulgaris re-dispersed in ultrapure water was significantly lower than that of C. vulgaris in culture solution. Since C. vulgaris cells were centrifuged and washed in order to remove the original culture solution, the matrix in the culture solution was also removed, so this experimental result shows that the matrix in the original culture solution would not reduce the adsorption capability of C. vulgaris for Cd, on the contrary, it may increase the adsorption capability of C.
vulgaris for Cd. Of course, it also may be because the centrifugation and washing processes affected the activity of C. vulgaris, and then the adsorption capability of C.
vulgaris for Cd was affected. On the other hand, for the preconcentration experiment, the preconcentration process of heavy metal Cd was as follows: Firstly, a reaction solution was formed by mixing 25 mL of algae culture solution, 0.5 mL buffer solution and a certain amount of Cd test samples, and using ultrapure water to dilute to 40 mL, and then the subsequent adsorption reaction and preconcentration process were carried out. For this adsorption reaction process, the matrix in the C. vulgaris culture solution was present in the reaction solution. And the experimental results of "3.2.3 Effect of initial Cd concentration" in our revised manuscript also showed that under the optimal adsorption conditions, when the Cd concentration was in the range of 0.703-74.957 g mL -1 , the adsorption efficiencies were more than 90%, and the highest adsorption efficiency could reach 99.31%. Such a high adsorption efficiency also indicates that the matrix in the culture solution would not affect the adsorption of Cd by C. vulgaris. So in our manuscript, we didn't further describe the effect of matrix.

Comment:
The C. vulgaris could adsorb other inorganic species from water. In this way, a study of potential interferents need to be presented.

Response:
Thank you very much for your comment. We are very sorry that in our original manuscript we didn't provide the experimental results of potential interferences of other inorganic species from water for the adsorption of Cd by C.
vulgaris. Therefore, according to your comment, in our revised manuscript, we have carried out this experimental research, and the description of the experimental process has been added to the second paragraph in section "2.2 Preconcentration procedure", the experimental results also have been supplemented as a section "3.3 Influence of coexisting ions" in our revised manuscript. These results indicated that even at high concentrations, those coexisting inorganic ions (Na + , K + , NH4 + , Ca 2+ , Mg 2+ , Fe 3+ , Mn 2+ , Zn 2+ , NO3 -, SO4 2and Cl -) had no significant influence on the adsorption of Cd by C. vulgaris.

Comment: Inform how the detection limit was calculated.
Response: Thank you very much for your suggestion. According to IUPAC criteria, the calculation formula of detection limit is: LOD=3/k, where LOD is the detection limit,  is the standard deviation of multiple measurements for blank sample, and k is the slope of the calibration curve. In this study, when the initial concentration of Cd was in the range of 0.703-74.957 g mL -1 , for the obtained Cd-enriched thin samples, the net integral fluorescence intensity of Cd Kα characteristic peak had a very good linear relationship with the initial concentration of Cd, and the linear calibration plot was F=447.53061C+1193.35273 with the linear correlation coefficient R 2 of 0.9979, where F was the net integral fluorescence intensity of Cd Kα characteristic peak, and C was the initial concentration of Cd (the unit was g mL -1 ). So the slope of the calibration curve was 447.503061 mL g -1 . For the blank filter membrane sample loaded C. valgaris cells, the EDXRF spectrum measurement was repeated 11 times under the same experimental conditions, and the standard deviation () of the integrated fluorescence intensity obtained from 11 repeated measurements was 9.7617. So according to the calculation formula of detection limit, the detection limit of this proposed method was LOD=(3×9.7617)/(447.503061 mL g -1 )=0.0654 g mL -1 . In our revised manuscript, we have supplemented the sentences "For the blank filter

mL g -1 . So according to the 3/k IUPAC criteria ( is the standard deviation of multiple measurements for blank sample, k is the slope of linear
calibration plot), ......" in line 23-32 in the section "3.5 Detection of Cd by EDXRF" in order to explain in detail how the detection limit was calculated according to the above calculation process.

Comment:
The method was unable to determine cadmium in the water samples without adding the analyte. However, the detection limit of the method can be improved and decreased by varying, for example, the sample volume. Additional experiments need to be present with this intention.
Response: Thank you very much for your comment. In this paper, for the preconcentration method, the Cd thin samples were obtained by filtering 15 mL of the reaction solution, and the diameter of the Cd-enriched region on these formed Cd thin samples was 15.1 mm. Combined with this preconcenration method, the detection limit of this proposed EDXRF detection method for Cd was 0.0654 g mL -1 .
Although this detection limit was lower than the maximum allowable discharge concentration of Cd in various industries wastewaters, it was higher than the standard limit for heavy metals Cd in other waters such as surface water, drinking water, and urban water supply, and so on. So as you mentioned, at present, the proposed method was unable to determine Cd in these water samples without adding the analyte Cd.
However, for the preconcentration process, if we increase the volume of the reaction solution during filtration, and try to make the diameter of the Cd-enriched region on the Cd thin samples smaller, in this way, for the same water sample to be tested, we can obtain a Cd thin sample with a higher Cd mass per unit area by this improved preconcentration method. And this new Cd thin sample can obtain a stronger spectral signal by EDXRF spectrum measurement compared with the Cd thin sample obtained by the preconcentration method already used in this paper. Moreover, the slope (k) value in the linear calibration plot will be increased compared with the slope value in our manuscript. Therefore, the calculated detection limit will be lower. This is a very interesting and worthwhile research work. At present, we have already preliminarily tried this research. But more in-depth and detailed research work will be carried our in the future, hoping to obtain a lower detection limit in order to meet the detection requirements of heavy metal Cd in some water samples such as surface water, drinking water, urban water supply, and so on.

Comment:
The method is not sufficiently validated. For example: the results obtained need to be compared with a comparative method or a water certified reference material needs to be analyzed.

Response:
Thank you very much for your suggestions. It is really true as Reviewer suggested that in our original manuscript, the proposed method was not sufficiently validated. So according to your suggestion, in our revised manuscript, we have used two certified Cd reference water samples, GBW(E)082822a-1 and GBW(E)082822b-2 to validate the accuracy of our proposed method, and the related descriptions and results have been supplemented in line 36-46 in section "3.5

Detection of Cd by EDXRF".
We have try our best to improve the manuscript and carefully revised our manuscript according to your comments and suggestions. So some changes have been made in the manuscript but they will not influence the content and framework of the paper. Once again, special thanks to you for your good comments and suggestions.

influences of different coexisting ions on the adsorption efficiency of Cd by C.
vulgaris were investigated." was added in line 9 in our revised manuscript. (see

Introduction)
8. In the last paragraph of section "1. Introduction", the description "...... on the mixed cellulose membranes" in our original manuscript has been corrected as "......  5, 7, 18, 56, 108,   230, 480, 595, 812 L and 1.5, 3.4, 6, 11 mL)  25. In section "3.5 Detection of Cd by EDXRF", the sentences "In addition, the accuracy of the proposed method for detection of Cd was evaluated by analyzing two certified reference water samples, GBW(E)082822a-1 (certified Cd concentration: 100 g mL -1 ) and GBW(E)082822b-2 (certified Cd concentration: 500 g mL -1 ). Prior to preconcentration, the reaction solution composed of 25 mL of C. vulgaris culture solution, 0.5 mL of Na2B4O7-NaOH buffer solution (pH 10) and 1 mL Cd certified reference water sample was diluted to 40 mL with ultrapure water. Then preconcentration and detection of Cd were performed according to the proposed method. And compared with the certified Cd concentrations of the two reference water samples, the relative errors of the detection results were 4.7% and 3.1%, respectively." have been added in line 36-46 in our revised manuscript in order to validate the accuracy of our proposed method.
(see section 3.5) 26. The numbers of "Table1" and "Table2" in our original manuscript have been corrected as "Table2" and "Table3" in our revised manuscript. (see Results and discussion) 27. In section "4. Conclusions", the sentence "the maximum adsorption efficiency could be obtained when the contact time was 1min" in line 2-3 in our original manuscript has been corrected as "the maximum adsorption efficiency could be obtained when the contact time was 1min and the pH of reaction solution was 10" in our revised manuscript. (see Conclusions) 28. The format of the reference numbers in the main text of our revised manuscript has been modified. For example: "cardiovascular system and reproductive system of human body will be seriously affected, 1 " and "In addition, Cd has been known as a human carcinogen by the International Agency for Research on Cancer. 2,3 " in our original manuscript have been corrected as "cardiovascular system and reproductive system of human body will be seriously affected [1]," and "In addition, Cd has been known as a human carcinogen by the International Agency for Research on Cancer [2,3]." in our revised manuscript.
29. The format of the references in our revised manuscript has been modified according to the reference format in the Royal Soiety Open Science template.
30. In our revised manuscript, the data deposited in Dryad Digital Repository have been added as reference [32] in the reference list and cited in the data accessibility section. 31. Some grammar or spelling mistakes have been carefully corrected in our revised manuscript.