the Creative Commons Attribution 4.0 License.
the Creative Commons Attribution 4.0 License.
| 06 Nov 2024
Combining benzalkonium chloride addition with filtration to inhibit dissolved inorganic carbon alteration during the preservation of seawater in radiocarbon analysis
Abstract. Benzalkonium chloride (BAC) addition has shown great promise as a disinfectant for measuring δ13C and 14C of dissolved inorganic carbon (DIC) in freshwater samples. However, it was reported that the effectiveness of BAC to prevent DIC change was reduced for the use of seawater samples. The present study aimed to evaluate the effectiveness of adding BAC as a disinfectant in carbon isotopic analyses of DIC in seawater samples. We compared the efficacy of BAC addition, filtration (0.22 μm PTFE or 0.2–0.45 μm PES filters), and a combination of BAC addition and filtration in preventing DIC alterations caused by biological activity. The combined procedure was effective in preserving seawater, although this assessment was based on results from a single seawater sample. The 14C concentration of samples treated with both BAC addition and filtration exhibited minimal changes, ranging from 0.2–0.4 pMC over 41 weeks, despite the addition of sugar included to increase biological activity several-fold. Although the complete elimination of biological effects may be challenging with the combined method, the observed changes remained within practical limits. Concerns about CO2 contamination during sample filtration were also addressed and found to be negligible. These results suggest that combining filtration and BAC addition is an effective method for suppressing biological DIC alterations in 14C analysis, even in seawater samples.
- Preprint
(694 KB) - Metadata XML
-
Supplement
(108 KB) - BibTeX
- EndNote
Status: open (until 01 Jan 2025)
-
RC1: 'Comment on egusphere-2024-3349', Anonymous Referee #1, 17 Nov 2024
reply
General comments:
Overall this is an interesting study that may provide more data to inform our understanding of water sample collection and storage prior to analysis, and the ways in which different preservation methods may affect accuracy, pending the authors responses to the comments below.
Specific comments:
- The experimental design needs to be made explicitly clear. The manuscript must state how many independent replicates were used for each treatment in order for the reader to better understand what the results were. At present, it appears n = 1 or 2 for some or all experiments which is too low to warrant publication.
- Where appropriate, add 'groundwater' to the title and Abstract. The study tested both groundwater and seawater.
- Line 16: Edit for clarity. Microbial activity was not directly measured.
- Line 83. Please add information on the salinity of the SW and GW to Table S1 and in the Abstract. The reader should be able to view this information to better understand what this text is talking about and/or more easily make an assessment whether it is appropriate for the SW sample to be called seawater, estuarine water, brackish water or something else.
- Figure 1, Figure 2, Table 1, and all supplementary Table captions should state what the data is (means?) and the number of replicates.
- Figure 1. There appear to be duplicate data points shown for some treatments (e.g. unfiltered, GF) but there is no explanation of what each represents. Are these replicates or different treatments?
- Line 165. It is incorrect to conclude that filtration does not cause a change in 13C. There is not enough evidence for or against. Indeed, as there appears to be very low or no replication (n = 1 or 2) any differences in 13C among treatments may simply be an artefact.
- Line 186: “Beet sugar is more easily degraded”. Compared to what?
- Line 204: “ …reduced changes in DIC….”. Compared to the control treatment?
- Line 213: This is not true. Filtration + BAC essentially stopped changes in DIC for both GW and SW.
Technical corrections:
Line 36. Remove “have been” and “. They “
Line 63. Replace “is” with ‘are’
Line 210-211: Edit for clarity.
Line 273: Remove full stop after “However”Citation: https://doi.org/10.5194/egusphere-2024-3349-RC1 -
AC1: 'Reply on RC1', Hiroshi Takahashi, 13 Dec 2024
reply
We would like to thank the anonymous referee for their time and the thoughtful feedback on our manuscript. We will revise the manuscript according to the reviewer’s comments. The main points of revision are summarized as follows:
(1) The Materials and procedures section will be revised to clearly explain the experimental design, especially the number of samples.
(2) While the number of analyses conducted in this study was limited, we believe that the conclusions would remain unchanged even with an increase in the number of analyses. We will include an explanation of this in the Results and discussion section.
All responses to the reviewer’s comments are listed below. We believe that we have addressed all of the comments from the reviewer.
<Specific comment from RC1>
The experimental design needs to be made explicitly clear. The manuscript must state how many independent replicates were used for each treatment in order for the reader to better understand what the results were. At present, it appears n = 1 or 2 for some or all experiments which is too low to warrant publication.
<Answer>
The description of the experimental design will be revised to include the number of samples that were processed or measured. The measured values excluding GW-Control2, GW-PES2 (0.2 µm), and GW-PES2 (0.45 µm) were derived from a single measurement of an individual treatment or a preservation bottle. The three excluded samples are presented as the mean and standard deviation of the analytical values from the three preservation bottles of each. The description will be revised to include the number of samples, and errors.
In response to the comment that the number of analyses is insufficient, we offer the following counterargument:
Background assessment: While not previously delineated in the initial manuscript, the filtration background assessment was conducted using a single NaHCO3 solution, with the order of unfiltered, PES, GF, GF, and unfiltered samples. The 14C concentrations and δ13C values of the NaHCO3 solution before and after the filtration experiment were identical, indicating that the DIC of the NaHCO3 solution itself did not change during the evaluation experiment. A comparison of the filtered and unfiltered samples showed a high degree of consistency in the analytical values, indicating that filtration did not change the 14C concentration. While the individual data points were obtained from a single experiment, the results of the assessment should be evaluated as being derived from the results of a series of experiments. Therefore, we believe it is reasonable to conclude that the results are not merely due to chance but reflect the absence of an influence of filtration. The revised manuscript will include the processing order for both unfiltered and filtered samples and a discussion of these results.
Comparison of treatments: As same above, the results were obtained as a time-series, although the individual data points were derived from a single experiment. While the detailed values pertaining to isotopic changes may not be entirely accurate, it is possible to discern the presence or absence of alterations and trends. If the objective is to ascertain an exact amount of change, repeated measurements are necessary. However, if the objective is to determine whether change has occurred or to identify its direction of change, the results of this study are sufficient for this purpose.
<Specific comment from RC1>
Where appropriate, add 'groundwater' to the title and Abstract. The study tested both groundwater and seawater.
<Answer>
The title will be revised from "seawater" to "water sample" to encompass both seawater and groundwater. The result of groundwater will be incorporated into the abstract as follows: “The freshwater sample that had undergone a BAC addition treatment showed the no alteration of DIC”.
<Specific comment from RC1>
Line 16: Edit for clarity. Microbial activity was not directly measured.
<Answer>
As the reviewer commented, the degree of biological activity was not directly measured in this study. Instead, changes in dissolved inorganic carbon (DIC) were attributed to biological activity, and thus the expression "biological activity" was used in the text. However, this description is not entirely accurate and will be revised to "DIC changes" for clarity.
<Specific comment from RC1>
Line 83. Please add information on the salinity of the SW and GW to Table S1 and in the Abstract. The reader should be able to view this information to better understand what this text is talking about and/or more easily make an assessment whether it is appropriate for the SW sample to be called seawater, estuarine water, brackish water or something else.
<Answer>
The salinities of the SW and GW will be added to the abstract, the Materials and procedures section, and Table S1. The salinities of SW and GW were 20.4% and less than 0.05%, respectively. Therefore, SW is categorized as brackish water, and GW is categorized as freshwater.
<Specific comment from RC1>
Figure 1, Figure 2, Table 1, and all supplementary Table captions should state what the data is (means?) and the number of replicates.
<Answer>
All values indicated in Figures 1 and 2 and Tables S2 and S3 are derived from a single measurement taken for each respective treatment. In contrast, the values shown in Table 1 represent the mean values of the respective measurements taken of the three preservation bottles for each period.
The figure and table captions will be revised to clearly indicate whether the analytical values represent mean values or individual treatment sample values.
<Specific comment from RC1>
Figure 1. There appear to be duplicate data points shown for some treatments (e.g. unfiltered, GF) but there is no explanation of what each represents. Are these replicates or different treatments?
<Answer>
The respective data points were obtained from a single treatment, therefore, the duplicate points of unfiltered and GF indicate two measurements from two individual respective treatments.
Figure 1 will be rearranged to show the order of filtration treatments using the NaHCO3 solution. Please refer to the supplement file for additional details.
<Specific comment from RC1>
Line 165. It is incorrect to conclude that filtration does not cause a change in 13C. There is not enough evidence for or against. Indeed, as there appears to be very low or no replication (n = 1 or 2) any differences in 13C among treatments may simply be an artefact.
<Answer>
As you correctly noted, the description that filtration does not cause a δ13C change was not accurate and therefore will be deleted. Moreover, we concur with your comment that the observed differences in δ13C among treatments may be attributed to an artefact. However, we maintain that the changes in δ13C were not caused by atmospheric CO2 contamination or degassing, and we propose that the observed δ13C change may be attributed to an unidentified artifact factor other than filtration. This conclusion and a clarification stating that the effect of filtration on 14C analysis is negligible will be added.
<Specific comment from RC1>
Line 186: “Beet sugar is more easily degraded”. Compared to what?
<Answer>
The materials under comparison were BAC or other organic materials suspended in water. This description will be added.
<Specific comment from RC1t>
Line 204: “ …reduced changes in DIC….”. Compared to the control treatment?
<Answer>
Yes. It is compared to the Control sample. The description will be revised.
<Specific comment from RC1t>
Line 213: This is not true. Filtration + BAC essentially stopped changes in DIC for both GW and SW.
<Answer>
The description was inaccurate and will be deleted.
< Technical corrections from RC1t>
<Answer>
The manuscript will be revised in accordance with all technical recommendations.
Viewed
| HTML | XML | Total | Supplement | BibTeX | EndNote | |
|---|---|---|---|---|---|---|
| 91 | 22 | 6 | 119 | 22 | 5 | 2 |
- HTML: 91
- PDF: 22
- XML: 6
- Total: 119
- Supplement: 22
- BibTeX: 5
- EndNote: 2
Viewed (geographical distribution)
| Country | # | Views | % |
|---|
| Total: | 0 |
| HTML: | 0 |
| PDF: | 0 |
| XML: | 0 |
- 1