How suitable is freshwater sponge Ephydatia fluviatilis (Linnaeus, 1759) for time-integrated biomonitoring of microbial water quality?

  1. Allison Cartwright
  2. James S. G. Dooley
  3. Chris D. McGonigle
  4. Joerg Arnscheidt

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Abstract

Faecal pollution of water by bacteria has a negative effect on water quality and can pose a potential health hazard. Conventional surveillance of microbial water quality relies on the analysis of low-frequency spot samples and is thus likely to miss episodic or periodic pollution. This study aimed to investigate the potential of filter-feeding sponges for time-integrated biomonitoring of microbial water quality. Laboratory trials tested the effects of different ratios of bacterial abundance and the sequence of exposure on bacterial retention by the freshwater sponge Ephydatia fluviatilis (Linnaeus, 1759) to establish its potential to indicate bacterial exposure. Gemmule grown sponges were simultaneously exposed to Escherichia coli and Enterococcus faecalis but at different ratios (Trial 1) or individually exposed to each bacterial species but in different sequential order (Trial 2). The E. coli and E. faecalis retained in each sponge was quantified by culture on selective agars. Data analysis was conducted using the Kruskal–Wallis test and/or the Mann–Whitney U test to compare between the numbers of bacteria retained in each treatment. Additionally, the Wilcoxon matched-paired signed-rank test was used for comparison of the different bacterial abundances retained within each individual sponge. Sponges from all trials retained E. coli and E. faecalis in small numbers relative to the exposure (<0.05 % Trial 1 and <0.07 % Trial 2) but exhibited higher retention of E. coli . Higher abundance of either bacterial species resulted in significantly lower ( P <0.005) retention of the same species within sponges (Trial 1). An initial exposure to E. coli resulted in significantly higher ( P =0.040) retention of both bacterial species than when sponges were exposed to E. faecalis first (Trial 2).Bacterial retention by sponges was neither quantitatively representative of bacterial abundance in the ambient water nor the sequence of exposure. This implies either selective filtration or an attempt by sponges to prevent infection. However, freshwater sponges may still be useful in biomonitoring as qualitative time-integrated samplers of faecal indicator bacteria as they detect different bacteria present in the water even if their quantities cannot be estimated.

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  1. Version published to 10.1099/acmi.0.000691.v4 on Access Microbiology
  3. Version published to 10.1099/acmi.0.000691.v3 on Access Microbiology
  5. Access Microbiology

    Comments to Author

    The authors greatly improved the clarity of the manuscript and my concerns about the controls were also adressed. There remain just a few minor remarks: - Lines 40-43: I will try to convey why the conclusion in the abstract is not fully comprehensible. At line 40, the authors first conclude that there is NO relation, i.e. representativeness. It could be argued, since the conclusion at line 40 is based on significant differences (as mentioned in lines 33-38), that there is no relation between retention and abundance or sequence of exposure. After all, the authors then suggest at line 41 that there IS a relation: "This implies...". This comes across contradictory to the reader. (IMO there probably is a relation, however it is less suitable for quantitative determination). Then in line 42, the authors conclude that freshwater sponges may still be useful qualitatively whilst they just stated that there was no relation. Readers may read over the word "qualitatively". - Lines 206-208: To me, the altered sentence still does not make sense. Even worse, it now includes two times "between". - Lines 228-229: IMO, the legend does not make clear what the letters represent. E.g., does each letter represent a certain p value? - Lines 302-304: I indeed referred to the word preferably, which is now resolved. However now the phrase "retained more .. to .." does not appear to be grammatically correct.

    Please rate the manuscript for methodological rigour

    Good

    Please rate the quality of the presentation and structure of the manuscript

    Good

    To what extent are the conclusions supported by the data?

    Strongly support

    Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?

    No

    Is there a potential financial or other conflict of interest between yourself and the author(s)?

    No

    If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?

    Yes

  6. Access Microbiology

    Comments to Author

    I have reviewed the changes made to the manuscript. I appreciate the response received from the authors and the amendments made to the manuscript.

    Please rate the manuscript for methodological rigour

    Good

    Please rate the quality of the presentation and structure of the manuscript

    Satisfactory

    To what extent are the conclusions supported by the data?

    Strongly support

    Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?

    No

    Is there a potential financial or other conflict of interest between yourself and the author(s)?

    No

    If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?

    Yes

  7. Access Microbiology

    Comments to Author

    It is satisfactory that the authors incorporated all the suggestions and comments. After all amendments, the article can be accepted.

    Please rate the manuscript for methodological rigour

    Good

    Please rate the quality of the presentation and structure of the manuscript

    Good

    To what extent are the conclusions supported by the data?

    Strongly support

    Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?

    No

    Is there a potential financial or other conflict of interest between yourself and the author(s)?

    No

    If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?

    Yes

  8. Version published to 10.1099/acmi.0.000691.v2 on Access Microbiology
  10. Access Microbiology

    Comments to Author

    Authors have described a good possible approach for biomonitoring of microbial water quality. However, I have few queries : 1) What is the rationale of using Ephydatia fluviatilis for this study? Or to reform my question I would like the authors to justify the motive for selecting E. fluviatilis for their study. How and why out of the many sponges it became authors first choice for this experimentation? 2) E. coli is the most commonly tested/ best fecal bacteria indicators. In this study, experiments were conducted with E. coli and E. faecalis. This raises a couple of queries: a) Why E. faecalis was used in the study? Why not other coliform bacteria such as Enterobacter, Klebsiella, Citrobacter? Moreover, If E. coli is detected then there is strong evidence that sewage/fecal pollutant is present; therefore, a greater potential for pathogenic organisms exists. b) What is the importance of testing other microbial forms when E. coli is a well-known indicator? 3) Line 133-134, one sponge exposed to E. coli first died. Lines 293-294? These claims are quite ambiguous and contradict one other; could you please clarify? 4) Membrane filtration is the method of choice for the analysis of fecal coliforms in water. Please explain the novelty of using freshwater sponges for microbiological water quality biomonitoring or could you perhaps explain why employing freshwater sponge is an innovative approach to microbiological water quality biomonitoring? (considering Lines 286-289) sponges can succumb to bacterial infection, causing death of the organism).

    Please rate the manuscript for methodological rigour

    Satisfactory

    Please rate the quality of the presentation and structure of the manuscript

    Satisfactory

    To what extent are the conclusions supported by the data?

    Strongly support

    Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?

    No

    Is there a potential financial or other conflict of interest between yourself and the author(s)?

    No

    If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?

    Yes

  11. Access Microbiology

    Comments to Author

    Comments on manuscript Manuscript Number: ACMI-D-23-00161 Title: How suitable is the freshwater sponge Ephydatia fluviatilis (Linnaeus, 1759) for time-integrated biomonitoring of microbial water quality? Comments: The authors demonstrated the novel usage of freshwater sponges as an indication of water contamination, aiming to identify the water quality. The experimental data and statistical analysis strongly align with the interpretation of the results. However, this work lacks the time interval analysis of bacterial exposure and bacterial retention in sponges. Also, for the manuscript, some major improvements should be made before it is considered for publication. 1. Page 2 Line 90- Authors may consider explaining choosing E. fluviatilis by citing previous research works. 2. The methods section is significantly lacking references and needs to be thoroughly referenced to support their choice of bacterial ratio and methodology used in the current study. 3. Also may need to input rationalization for why this study lacks the time interval incubation (short time or longer time) rather than 24 hours. Batch culture incubation is widely used in this type of sponge study or a flow system would more represent the real freshwater environment? 4. Either bacteria or sponges growing during incubation experiments? 5. Similar to the above comment the figures showed in the manuscript discuss the retained bacteria in the sponge, is there a decrease or increase of bacteria while incubation? Did the authors take a look at the incubation supernatant solutions for further bacterial count before and after experiments? 6. Is high abundance exposure of bacteria toxic to the sponges? Any data showing the death of sponges in 10:90, and 90:10 incubation? It may be an explanation for the low retention of bacteria to higher cell density exposure. 7. Page 7, lines 269-271. The sentence needs to be rewritten for clear understanding. 8. In the Discussion section, the authors need add an explanation for experimental data differences comparing the retention levels with previous studies. Minor points: * The overall manuscript needs to be uniformly arranged especially in terms of reference style according to journal requirements.

    Please rate the manuscript for methodological rigour

    Good

    Please rate the quality of the presentation and structure of the manuscript

    Good

    To what extent are the conclusions supported by the data?

    Strongly support

    Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?

    No

    Is there a potential financial or other conflict of interest between yourself and the author(s)?

    No

    If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?

    Yes

  12. Access Microbiology

    Comments to Author

    I commend the authors with this interesting research. I have the following suggestions and remarks on the manuscript which I hope to be constructive. - line 34: The word 'preference' suggests the sponges like it. I would suggest another choice of words. - line 41: This seems contradictory with the observation of significantly higher retention as mentioned in line 37. - line 42: This should be motivated. - line 47: Be consequent, writing species names in full should also apply to Escherichia coli. - line 88-90: This sentence does not go so well. - line 123: How was this concentration chosen? Is it comparable to in situ wastewater pollution? - line 125: It is not clear what is ment with 'replicates'. - line 125: This sentence is not clear. Does this mean 3 trials x 3 ratios = 9? And where does the number 22 come into? - line 131: Ditto as remark at line 123. - line 140: The authors don't mention the inclusion of any controls in the trials. I would have liked to see a control of the whole procedure, including the bacteria but without addition of sponges. This would give an indication as to how much bacteria actually survived the procedure in mineral water or did not sink to the bottom of the tube, for an overnight broth would be more saltier than mineral water. Possibly this number would even have been better suited as a baseline (100%) to calculate percentages with. The percentages that are calculated by this study are that of the bacteria that both survived the mineral water, did not sink to the bottom of the tube AND were retained in the sponge. This needs to be adressed. - line 158: This seems incorrect and highly confusing. Bs is described as bacteria per sponge, whilst in the sentence above the authors describe they wanted to calculate the number of cfu/mm2. N usually is a total number, not per ml. It would also not fit with the parameter units on both sides of the equation. - line 180: Could it possibly also be due to a low survival rate of the bacteria in mineral water? This should be in the Discussion section. - line 183: This sentence does not make sense. A difference is between two items. Difference between the bacterial retention and ... what? - line 185: The choice of the word 'relative' seems wrong. Was the retention relative? - line 195: This is a conclusion and should not be in the results section. - line 195: Is it due to the sponge or due to E. coli? - line 200-205: Again, this should be in the Discussion section. - line 209-210: "Different letters indicate...": this is not clear. - line 212: Title should be clearer, indicating it was sequential exposure - line 215: Same remark as that in section 3.1. - line 221-223: This seems contradictory with the statement in line 216: "The sponges showed higher retention of E.coli ..." - line 225-226: Again, this should be in the Discussion section. - line 242-243: Sentence does not go so well. - line 244-245""retained ...": This is a conclusion that can not be drawn from the results because information is lacking. The sponges can only retain what is present in the water. Again, the amount of bacteria that survived transfer to the mineral water is unknown due to the lack of the control I described previously. Part of the bacteria will have lysed upon addition to the mineral water, which likely also differs per species. - line 251: Which concentration goes with which species? Also, these numbers should be moved to the results section. - line 252: To my understanding, the ON cultures were also diluted with an approximate factor 10 when they were added to the mineral water. The most important question here is: what was the concentration of living bacteria in the test tube surrounding the sponge. This would have been determined by the lacking control or baseline I have mentioned previously. - line 273: Suggest other word choice. - line 280: I suggest to move this to the beginning of the paragraph: it is what makes this study unique what the readers want to know first. Perhaps it should also be emphasised more elsewhere in the document. - line 281: This is a vague formulation. It is not immediately clear that the authors mean enterococcus with "this faecal indicator". I suggest a rephrase. - line 293: I do wonder at this point how the one sponge in the current study died. - line 294: "While ...": The picture may be more complicated. In humans, E. faecalis has been shown to possess probiotic properties. - line 309: I suggest choosing another word than 'prevent'. - line 323: 3 times "for" in a sentence is confusing. This sentence does not go so well. - line 325: I suggest another choice of words for 'preferential'. - line 328: What about labour intensity? - line 334-336: Vague formulation.

    Please rate the manuscript for methodological rigour

    Poor

    Please rate the quality of the presentation and structure of the manuscript

    Good

    To what extent are the conclusions supported by the data?

    Partially support

    Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?

    No

    Is there a potential financial or other conflict of interest between yourself and the author(s)?

    No

    If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?

    Yes

  13. Version published to 10.1099/acmi.0.000691.v1 on Access Microbiology