the Creative Commons Attribution 4.0 License.
the Creative Commons Attribution 4.0 License.
| 23 Mar 2026
Long-term storage of air-dried samples compromises water-extractable organic carbon as a soil health indicator
Abstract. The assessment of soil health relies on sensitive indicators to detect management-induced changes, yet the analytical reliability of these indicators following long-term storage is rarely assessed. We investigated how multi-year storage of air-dried samples influenced the concentrations of several common soil health indicators, including water-extractable organic carbon and nitrogen (WEOC, WEN), mineralizable carbon (Cmin), and permanganate-oxidizable carbon (POX-C), using archived samples from a cover crop experiment. Concentrations of WEOC nearly doubled after three years of storage, while WEN decreased by 19%. A small but significant 6% increase in Cmin concentration was also observed. In contrast, POX-C concentrations remained stable, indicating robustness to storage effects. These storage effects were consistent among three treatments with different cover crop species. In addition, WEOC concentrations consistently declined over time in this experiment and four long-term agricultural sites in the USA, but bulk soil organic carbon (SOC) or soil organic matter (SOM) did not. These results suggest that multi-year storage of air-dried samples inflates the WEOC pool. Therefore, we caution the use of WEOC as a soil health indicator in archived samples, as the observed variations might reflect storage artifacts rather than genuine management impacts.
-
Notice on discussion status
The requested preprint has a corresponding peer-reviewed final revised paper. You are encouraged to refer to the final revised version.
-
Preprint
(826 KB)
-
Supplement
(500 KB)
-
The requested preprint has a corresponding peer-reviewed final revised paper. You are encouraged to refer to the final revised version.
- Preprint
(826 KB) - Metadata XML
-
Supplement
(500 KB) - BibTeX
- EndNote
- Final revised paper
Journal article(s) based on this preprint
Interactive discussion
Status: closed
-
RC1: 'Comment on egusphere-2026-980', Anonymous Referee #1, 26 Mar 2026
-
AC1: 'Reply on RC1', Swarnali Mahmood, 25 Jun 2026
We appreciate the reviewer’s thoughtful comments and recognize that the primary concern centers on the clarity of our experimental design, sampling scheme, and interpretation of storage versus management effects. In response, we have developed a revision plan to improve the manuscript by systematically clarifying the study design, explicitly distinguishing the Florida experiment from the other long-term experiments, and revising the terminology throughout the manuscript to clearly identify the drivers of the observed trends. In addition, we plan to include new data on the residual moisture content of archived air-dried soils, which supports our interpretation of storage effects and allows us to directly address the reviewer’s concerns regarding continued microbial activity during storage. We will also expand our discussion of the methodological limitations of POX-C. Overall, we believe these revisions can improve the clarity, rigor, and interpretation of the manuscript while directly addressing the reviewer's comments.
Comment:
The manuscript “Long-term storage of air-dried samples compromises water-extractable organic carbon as a soil health indicator” authored by S. Mahmood and co-workers focused on the evolution with time of soil indicators with time of storage. The data refer to a common practice of “air drying” and storing at “room temperature”. Indeed, room temperatures are varying a lot across different locations and seasons, as for room moisture. In my opinion, air drying is rarely reaching moisture content below 5-10% in soils (at least in my region). This means that microbial communities will still have some residual activities at these moisture levels and room temperature, which is the main reason behind the modification of labile soil health indicators such as water-soluble C and N. For “labile” soil health indicators, other methods of drying (at controlled temperature and moisture) and storage (at colder temperature) are preferably, and this could be mentioned in the manuscript. Therefore, results from this study are relevant, but their novelty is relatively low.
Response:
Thank you for your feedback. We agree with the reviewer that residual moisture in air-dried soils can sustain microbial activity and contribute to changes in labile C pools during storage.
In the revised Introduction, we will emphasize that “air-drying prior to analysis and long-term storage is a standard and widely adopted procedure in soil science for both routine laboratory analyses and soil archiving (Kühnel et al., 2019; Stott, 2019). Despite being classified as air-dried, soils typically retain a small amount of residual water that remains adsorbed to mineral surfaces and within fine pores, particularly in clay-rich and organic matter-rich soils. Residual moisture content has been shown to be strongly related to soil texture and organic matter content, reflecting water retained at high matric potentials and on reactive mineral surfaces (Poeplau et al., 2015; Wäldchen et al., 2012). Microbial activity may persist even at the low water contents present in air-dried soils, potentially resulting in continued transformation of labile carbon and nitrogen pools during storage.” We believe these revisions will further corroborate the knowledge gap currently listed in lines 49-51: the combined effects of management and multi-year storage of air-dried soils on the repeatability and temporal trajectories of soil health indicators remain poorly understood.
To address the reviewer’s concern, we will expand the Materials and Methods section to provide additional details regarding sample preparation and storage as follows: Soil samples were air-dried in a climate-controlled laboratory on open trays until a constant mass was achieved before sieving and archiving. Samples are then stored in airtight plastic bags at controlled room temperature in the lab.
In the results, we will report the residual moisture content measured in a subset of air-dried samples from our cover crop study, which averaged 0.26% (n = 9; range: 0.20–0.32; CV = 16%). We will also discuss implications of these measurements in the discussion: the measured residual moisture contents are consistent with expectations for coarse-textured sandy soils, which retain substantially less residual water than fine-textured soils. Previous studies reported residual water contents ranging from approximately 0.3–6% in air-dried mineral soils, with the lowest values generally observed in sandy soils (Poeplau et al., 2015; Wäldchen et al., 2012). For example, Wäldchen et al. (2012) observed water contents as low as 2.8 g kg⁻¹ (0.28%) in air-dried sandy soils and demonstrated that residual water is primarily controlled by clay content and adsorption within fine pores. We will also discuss the potential contribution of residual moisture to variation among archived samples. We believe the revisions will complement existing discussion on how microbial dynamics, such as cell lysis and continued enzymatic depolymerization, could contribute to the observed accumulation of WEOC (lines 136-137 and 181-183).
Comment:
Another aspect is that permanganate oxidizable carbon (POX-C) is proposed as a more stable indicator, as suggested by USDA, but there are many concerns about the methods used for measuring POX-C, and this is not reported in the document. I suggest to include some of the studies questioning POX-C used methods to give a better overview of this indicator, as those by Margenot’s group Margenot et al., 2024 (https://doi.org/10.1002/ael2.20124).
Response:
We appreciate this important suggestion. We acknowledged the methodological limitations and interpretational uncertainties associated with POX-C in our Introduction. In contrast, POX-C is generally less sensitive to drying but may still be affected by methodological factors such as soil mass, sieve size, laboratory handling, and SOC content (Gasch et al., 2020; Wade et al., 2020) (lines 47-49). In our revised manuscript, we plan to present POX-C more cautiously as a relatively stable but method-dependent indicator highlighting recent studies. We will add in our Discussion that recent studies have highlighted methodological inconsistencies and concerns regarding the chemical specificity and interpretability of POX-C warranting caution against its interpretation as a proxy for biologically meaningful labile C pools (Gasch et al., 2020; Margenot et al., 2024; Woodings & Margenot, 2023) (after line 179).
Comment:
Lastly, the presentation of the experimental design, which comprises sample sets from 5 experiment, should be improved to clarify when samples were collected, when they were analysed the first time, when they where reanalysed and the variation occurred during the years of storage.]
Response:
We would like to revise the materials and methods section to clarify that the comparisons are within-sample (i.e., archived vs. original measurements), rather than across time or management treatments, in the cover crop study. The changes in these indicators were quantified as differences between original and reanalyzed values from the same archived samples, thereby isolating storage effects from field-driven temporal variability. This design is different from those of the other long-term experiments where we could not separate the storage effects from those due to management practices.
Comment:
Abstract: line 19 “four long term agricultural sites” is not clear if it is referred to comparison of long-term storage or real field experiments in which WEOC is decreasing. Please revise.
Response:
We plan to revise lines 19-20 for clarification as: In addition, WEOC concentrations consistently declined over time in this experiment along with four long-term agricultural sites in the USA, but their bulk soil organic carbon (SOC) or soil organic matter (SOM) did not.
Comment:
Lines 43-49: see my comment above about POX-C methodological problems
Response:
We would like to highlight these limitations in the revised discussion as per your suggestions as follows: Recent studies have highlighted methodological inconsistencies and concerns regarding the chemical specificity and interpretability of POX-C warranting caution against its interpretation as a proxy for biologically meaningful labile C pools (Gasch et al., 2020; Margenot et al., 2024; Woodings & Margenot, 2023) (after line 179).
Comment:
Lines 51-54: Why Florida and Midwest experiments are listed separately instead of simply saying “five long term experiments”? This will make things easier to understand, in my opinion.
Response:
Lines 51-54: The Florida experiment follows a different design from the other four long-term experiments. As discussed earlier, we were able to isolate storage effects in the Florida experiment by analyzing the same samples before and after storage. In the other experiments, however, storage effects were confounded with management effects.
Based on your comment and those of the other reviewer, we plan to clarify our rationale in the Methods section: Our intention in including the four long-term experiments was not to isolate storage effects independently from management history, but rather to evaluate the practical implications of using archived air-dried soils in long-term soil health assessments.
Comment:
Sections 2.2 and 2.3 and elsewhere: It is not clear the set-up of the re-analysis of stored samples. Which were the reference values for the samples stored from the other 4 long-term experiments? The comparison was done between the re-analysis values of stored samples against the freshly analysed soils?]
Response:
We plan to clarify in the revision that WEOC concentrations from the four long-term experiments were determined using the same extraction protocol in the same laboratory in 2023.
Comment:
Line 101: sampling time or storage duration?
Response:
As shown in Figure 2, we compared the temporal trends of SOM and WEOC using Pearson correlation analysis between sampling time and soil properties values reported in Nyabami et al. (2024). We repeated this analysis for SOC and WEOC in archived soils from the four long-term agricultural experiments (lines 101-103).
Comment:
Line 116: archived sample sets would better explain the setup of the experiment
In my opinion, section 3.2 and 2.2 should clearly state when archived samples where reanalysed, mentioning the storage time elapsed after sampling, in a scheme. I found that the experimental design is not fully explaining if the samples from long term experiments were collected in year X and reanalysed for this study. In this case, can the authors compare the WEOC data from the first time they were analysed and the reanalysis here? The increasing or decreasing trends can be driven by the land use over years, not only storage.
Response:
We appreciate the reviewer’s feedback regarding the lack of clarity in the sampling design. As discussed in our previous responses, we plan to systematically revise the Materials and Methods section to clearly indicate when the archived samples were reanalyzed and to explain how the sampling design differed between the Florida study and the other long-term experiments.
Comment:
line 139: why post-rewetting studies are cited to justify WEN decrease during storage?
Response:
We plan to clarify in the revision that measurements of WEN involve rewetting archived soils with water, which shared similarities with the post-wetting studies.
Comment:
Lines 147-150: as I said above, a quick mention to doubts about POX-C methods and “lability” should be mentioned here.
Response:
Besides our Introduction, we would like to highlight the methodological limitations and interpretational uncertainties associated with POX-C after lines 147-150 in our revision as follows: However, recent investigations have demonstrated that POX-C may not consistently represent biologically meaningful labile carbon fractions, owing to methodological inconsistencies and uncertainties in the specific carbon compounds targeted by the assay (Gasch et al., 2020; Margenot et al., 2024; Woodings & Margenot, 2023) (after line 150).
Comment:
Line 163: Again, the use of “temporal trends” could refer to trends driven by the agronomic practices inducing shifts in soil indicators or by the storage time at room temperature that is causing re-arrangements of C pools. I suggest to use different wording to avoid this confusion.
Response:
We fully understand the reviewer’s suggestion to clarify the drivers of these trends. Although we found it challenging to identify terminology that clearly distinguished the different effects, we plan to revise the Results and Discussion sections to explicitly identify the drivers of each trend and avoid potential confusion. These revisions will also complement our planned changes to the Materials and Methods section by providing a clearer explanation of the sampling design and analytical approach.
Comment:
Line 181: “controlled conditions” does not equal to optimized condition to minimize soil microbial activity. WEOC can be analysed in stored samples if the moisture is kept very low and the storage temperature is low (but not on frozen samples). But I agree that measuring WEOC as soon as possible is the best option.
Response:
Thank you for your positive feedback. We will include the point about frozen samples in our revision.
Comment:
Line 190: see my comment about POX-C. I think it has potential to became a soil health indicator, but since the last 15 years the method has marginally improved and actually is a worst indicator than SOC, for example.
Response:
We would like to highlight the caution against the use of POX-C for its methodological limitations and interpretational uncertainties in the revised discussion after line 190 as per your suggestion.
References
Gasch, C., Mathews, S., Deschene, A., Butcher, K., & DeSutter, T. (2020). Permanganate oxidizable carbon for soil health: Does drying temperature matter? Agricultural & Environmental Letters, 5(1), e20019. https://doi.org/10.1002/ael2.20019
Kühnel, A., Wiesmeier, M., Spörlein, P., Schilling, B., & Kögel-Knabner, I. (2019). Influence of drying vs. Freezing of archived soil samples on soil organic matter fractions. Journal of Plant Nutrition and Soil Science, 182(5), 772–781. https://doi.org/10.1002/jpln.201800529
Margenot, A. J., Wade, J., & Woodings, F. S. (2024). The misuse of permanganate as a quantitative measure of soil organic carbon. Agricultural & Environmental Letters, 9(1), e20124. https://doi.org/10.1002/ael2.20124
Nyabami, P., Weinrich, E., Maltais-Landry, G., & Lin, Y. (2024). Three years of cover crops management increased soil organic matter and labile carbon pools in a subtropical vegetable agroecosystem. Agrosystems, Geosciences & Environment, 7(1), e20454. https://doi.org/10.1002/agg2.20454
Poeplau, C., Eriksson, J., & Kätterer, T. (2015). Estimating residual water content in air-dried soil from organic carbon and clay content. Soil and Tillage Research, 145, 181–183. https://doi.org/10.1016/j.still.2014.09.021
Stott, D. E. (2019). Recommended Soil Health Indicators and Associated Laboratory Procedures (No. 450-03; Soil Health Technical Note). U.S. Department of Agriculture, Natural Resources Conservation Service. https://www.soils.org/files/napt/publications/method-papers/2019-nrcs-technote-450-03.pdf
Wade, J., Maltais-Landry, G., Lucas, D. E., Bongiorno, G., Bowles, T. M., Calderón, F. J., Culman, S. W., Daughtridge, R., Ernakovich, J. G., Fonte, S. J., Giang, D., Herman, B. L., Guan, L., Jastrow, J. D., Loh, B. H. H., Kelly, C., Mann, M. E., Matamala, R., Miernicki, E. A., … Margenot, A. J. (2020). Assessing the sensitivity and repeatability of permanganate oxidizable carbon as a soil health metric: An interlab comparison across soils. Geoderma, 366, 114235. https://doi.org/10.1016/j.geoderma.2020.114235
Wäldchen, J., Schöning, I., Mund, M., Schrumpf, M., Bock, S., Herold, N., Totsche, K. U., & Schulze, E. D. (2012). Estimation of clay content from easily measurable water content of air-dried soil. Journal of Plant Nutrition and Soil Science, 175(3), 367–376. https://doi.org/10.1002/jpln.201100066
Woodings, F. S., & Margenot, A. J. (2023). Revisiting the permanganate oxidizable carbon (POXC) assay assumptions: POXC is lignin sensitive. Agricultural & Environmental Letters, 8(1), e20108. https://doi.org/10.1002/ael2.20108
Citation: https://doi.org/10.5194/egusphere-2026-980-AC1
-
AC1: 'Reply on RC1', Swarnali Mahmood, 25 Jun 2026
-
RC2: 'Comment on egusphere-2026-980', Anonymous Referee #2, 02 Jun 2026
The authors investigated the effect of long-term storage on various soil-health-related indicators of air-dried soil samples. The topic should be interesting and will attract the readers. However, the quality of their manuscript needs substantial improvement for further processing. The worst point to revise is the fundamental lack of information regarding preparation in their air-dried samples. There is no information about the environmental conditions during air drying and the final soil moisture after the complete air-drying, which should substantially affect the characteristics of air-dried soils.
Furthermore, the scientific strategy and meaning of the additional soil analyses from four long-term agricultural experiments are very unclear, even after I have access to their data sets available for review. Fig 2 shows the results for those additional analyses. The data, however, likely reflect the long-term trends in soil characteristics during the agricultural experiments, rather than the effect of long-term storage on air-dried soils. If the authors still believe the analysis should work for evaluating the effect of long-term storage on air-dried soils, more detailed and dedicated descriptions are essential in the manuscript. Without fully resolving those issues, it is impossible to evaluate the relevance of the discussion and its conclusions properly. Thus, there is no meaning in achieving publication.Citation: https://doi.org/10.5194/egusphere-2026-980-RC2 -
AC2: 'Reply on RC2', Swarnali Mahmood, 25 Jun 2026
We appreciate the reviewer’s thoughtful comments and recognize that the primary concerns relate to the lack of methodological clarity and data interpretation. In response, we have developed a revision plan to clarify the experimental design, sample preparation, storage conditions, and the distinct objectives of the Florida vs. other long-term experiments. We also plan to incorporate new measurements of residual moisture in archived air-dried soils and revise the Results and Discussion to more clearly distinguish storage-related patterns from management-driven changes. Collectively, we believe these revisions will substantially improve the clarity, rigor, and interpretation of the manuscript while directly addressing the reviewer's concerns.
Comment:
The authors investigated the effect of long-term storage on various soil-health-related indicators of air-dried soil samples. The topic should be interesting and will attract the readers. However, the quality of their manuscript needs substantial improvement for further processing. The worst point to revise is the fundamental lack of information regarding preparation in their air-dried samples. There is no information about the environmental conditions during air drying and the final soil moisture after the complete air-drying, which should substantially affect the characteristics of air-dried soils.
Response:
Thank you for highlighting several important issues regarding the methodological description and the lack of residual moisture measurements.
In our revised Materials and Methods section, we will clarify that the soils from the cover crop experiment were air-dried to constant mass and stored in sealed airtight plastic bags at controlled room temperature (i.e., 24°C) in the lab (line 67). For the LTAR/LTER soils, we will include in our revision that after sampling, these soils were air-dried under laboratory room-temperature conditions to constant mass prior to archival storage. We will report the residual moisture content measured in a subset of air-dried samples from our cover crop study, which averaged 0.26% (n = 9; range: 0.20–0.32; CV = 16%). This value is consistent with expectations for coarse-textured sandy soils, which retain substantially less residual water than fine-textured soils. Previous studies reported residual water contents ranging from approximately 0.3–6% in air-dried mineral soils, with the lowest values generally observed in sandy soils and the highest values in clay-rich soils (Poeplau et al., 2015; Wäldchen et al., 2012). Wäldchen et al. (2012) observed water contents as low as 2.8 g kg⁻¹ (0.28%) in air-dried sandy soils and demonstrated that residual water is primarily controlled by clay content and adsorption within fine pores. We will also discuss the potential contribution of residual moisture to variation among archived samples.
Comment:
Furthermore, the scientific strategy and meaning of the additional soil analyses from four long-term agricultural experiments are very unclear, even after I have access to their data sets available for review. Fig 2 shows the results for those additional analyses. The data, however, likely reflect the long-term trends in soil characteristics during the agricultural experiments, rather than the effect of long-term storage on air-dried soils. If the authors still believe the analysis should work for evaluating the effect of long-term storage on air-dried soils, more detailed and dedicated descriptions are essential in the manuscript. Without fully resolving those issues, it is impossible to evaluate the relevance of the discussion and its conclusions properly. Thus, there is no meaning in achieving publication.
Response:
We appreciate the reviewer’s thoughtful comments and fully understand the concern regarding the interpretation of the four long-term agricultural experiments. We agree that the current presentation does not sufficiently distinguish storage effects from field-driven changes, which makes the scientific rationale and interpretation of these analyses unclear.
In the revised manuscript, we will systematically clarify the study design and the distinct objectives of the Florida experiment and the four long-term agricultural experiments. Specifically, we will emphasize that the Florida experiment was uniquely designed to isolate storage effects by comparing original and reanalyzed measurements from the same archived samples. In contrast, the four long-term experiments were not intended to isolate storage effects independently from management history. Rather, they were included to evaluate the practical implications of using archived air-dried soils in long-term soil health assessments, where storage effects are inevitably intertwined with management-induced changes.
We will revise the Results and Discussion section to clarify the interpretation of the four long-term agricultural experiments. Specifically, we will emphasize that, although long-term management practices influence soil properties, a common pattern emerged across all four archived datasets: WEOC declined with increasing storage duration, whereas bulk SOC and SOM did not. This finding indicates that labile carbon indicators may be more susceptible to storage-induced changes than bulk carbon measurements and underscores the importance of accounting for sample storage history when interpreting long-term soil health trends from archived soils.
References
Poeplau, C., Eriksson, J., & Kätterer, T. (2015). Estimating residual water content in air-dried soil from organic carbon and clay content. Soil and Tillage Research, 145, 181–183. https://doi.org/10.1016/j.still.2014.09.021
Wäldchen, J., Schöning, I., Mund, M., Schrumpf, M., Bock, S., Herold, N., Totsche, K. U., & Schulze, E. D. (2012). Estimation of clay content from easily measurable water content of air-dried soil. Journal of Plant Nutrition and Soil Science, 175(3), 367–376. https://doi.org/10.1002/jpln.201100066
Citation: https://doi.org/10.5194/egusphere-2026-980-AC2
-
AC2: 'Reply on RC2', Swarnali Mahmood, 25 Jun 2026
Peer review completion
Interactive discussion
Status: closed
-
RC1: 'Comment on egusphere-2026-980', Anonymous Referee #1, 26 Mar 2026
The manuscript “Long-term storage of air-dried samples compromises water-extractable organic carbon as a soil health indicator” authored by S. Mahmood and co-workers focused on the evolution with time of soil indicators with time of storage. The data refer to a common practice of “air drying” and storing at “room temperature”. Indeed, room temperatures are varying a lot across different locations and seasons, as for room moisture. In my opinion, air drying is rarely reaching moisture content below 5-10% in soils (at least in my region). This means that microbial communities will still have some residual activities at these moisture levels and room temperature, which is the main reason behind the modification of labile soil health indicators such as water-soluble C and N. For “labile” soil health indicators, other methods of drying (at controlled temperature and moisture) and storage (at colder temperature) are preferably, and this could be mentioned in the manuscript. Therefore, results from this study are relevant, but their novelty is relatively low.
Another aspect is that permanganate oxidizable carbon (POX-C) is proposed as a more stable indicator, as suggested by USDA, but there are many concerns about the methods used for measuring POX-C, and this is not reported in the document. I suggest to include some of the studies questioning POX-C used methods to give a better overview of this indicator, as those by Margenot’s group Margenot et al., 2024 (https://doi.org/10.1002/ael2.20124).
Lastly, the presentation of the experimental design, which comprises sample sets from 5 experiment, should be improved to clarify when samples were collected, when they were analysed the first time, when they where reanalysed and the variation occurred during the years of storage.
Detailed comments:
Abstract: line 19 “four long term agricultural sites” is not clear if it is referred to comparison of long-term storage or real field experiments in which WEOC is decreasing. Please revise.
Introduction
Lines 43-49: see my comment above about POX-C methodological problems
Lines 51-54: Why Florida and Midwest experiments are listed separately instead of simply saying “five long term experiments”? This will make things easier to understand, in my opinion.
Materials and Methods
Sections 2.2 and 2.3 and elsewhere: It is not clear the set-up of the re-analysis of stored samples. Which were the reference values for the samples stored from the other 4 long-term experiments? The comparison was done between the re-analysis values of stored samples against the freshly analysed soils?Line 101: sampling time or storage duration?
Results
Line 116: archived sample sets would better explain the setup of the experiment
In my opinion, section 3.2 and 2.2 should clearly state when archived samples where reanalysed, mentioning the storage time elapsed after sampling, in a scheme. I found that the experimental design is not fully explaining if the samples from long term experiments were collected in year X and reanalysed for this study. In this case, can the authors compare the WEOC data from the first time they were analysed and the reanalysis here? The increasing or decreasing trends can be driven by the land use over years, not only storage.
line 139: why post-rewetting studies are cited to justify WEN decrease during storage?Discussion
Lines 147-150: as I said above, a quick mention to doubts about POX-C methods and “lability” should be mentioned here.
Line 163: Again, the use of “temporal trends” could refer to trends driven by the agronomic practices inducing shifts in soil indicators or by the storage time at room temperature that is causing re-arrangements of C pools. I suggest to use different wording to avoid this confusion.
Line 181: “controlled conditions” does not equal to optimized condition to minimize soil microbial activity. WEOC can be analysed in stored samples if the moisture is kept very low and the storage temperature is low (but not on frozen samples). But I agree that measuring WEOC as soon as possible is the best option.
Conclusion
Line 190: see my comment about POX-C. I think it has potential to became a soil health indicator, but since the last 15 years the method has marginally improved and actually is a worst indicator than SOC, for example.Citation: https://doi.org/10.5194/egusphere-2026-980-RC1 -
AC1: 'Reply on RC1', Swarnali Mahmood, 25 Jun 2026
We appreciate the reviewer’s thoughtful comments and recognize that the primary concern centers on the clarity of our experimental design, sampling scheme, and interpretation of storage versus management effects. In response, we have developed a revision plan to improve the manuscript by systematically clarifying the study design, explicitly distinguishing the Florida experiment from the other long-term experiments, and revising the terminology throughout the manuscript to clearly identify the drivers of the observed trends. In addition, we plan to include new data on the residual moisture content of archived air-dried soils, which supports our interpretation of storage effects and allows us to directly address the reviewer’s concerns regarding continued microbial activity during storage. We will also expand our discussion of the methodological limitations of POX-C. Overall, we believe these revisions can improve the clarity, rigor, and interpretation of the manuscript while directly addressing the reviewer's comments.
Comment:
The manuscript “Long-term storage of air-dried samples compromises water-extractable organic carbon as a soil health indicator” authored by S. Mahmood and co-workers focused on the evolution with time of soil indicators with time of storage. The data refer to a common practice of “air drying” and storing at “room temperature”. Indeed, room temperatures are varying a lot across different locations and seasons, as for room moisture. In my opinion, air drying is rarely reaching moisture content below 5-10% in soils (at least in my region). This means that microbial communities will still have some residual activities at these moisture levels and room temperature, which is the main reason behind the modification of labile soil health indicators such as water-soluble C and N. For “labile” soil health indicators, other methods of drying (at controlled temperature and moisture) and storage (at colder temperature) are preferably, and this could be mentioned in the manuscript. Therefore, results from this study are relevant, but their novelty is relatively low.
Response:
Thank you for your feedback. We agree with the reviewer that residual moisture in air-dried soils can sustain microbial activity and contribute to changes in labile C pools during storage.
In the revised Introduction, we will emphasize that “air-drying prior to analysis and long-term storage is a standard and widely adopted procedure in soil science for both routine laboratory analyses and soil archiving (Kühnel et al., 2019; Stott, 2019). Despite being classified as air-dried, soils typically retain a small amount of residual water that remains adsorbed to mineral surfaces and within fine pores, particularly in clay-rich and organic matter-rich soils. Residual moisture content has been shown to be strongly related to soil texture and organic matter content, reflecting water retained at high matric potentials and on reactive mineral surfaces (Poeplau et al., 2015; Wäldchen et al., 2012). Microbial activity may persist even at the low water contents present in air-dried soils, potentially resulting in continued transformation of labile carbon and nitrogen pools during storage.” We believe these revisions will further corroborate the knowledge gap currently listed in lines 49-51: the combined effects of management and multi-year storage of air-dried soils on the repeatability and temporal trajectories of soil health indicators remain poorly understood.
To address the reviewer’s concern, we will expand the Materials and Methods section to provide additional details regarding sample preparation and storage as follows: Soil samples were air-dried in a climate-controlled laboratory on open trays until a constant mass was achieved before sieving and archiving. Samples are then stored in airtight plastic bags at controlled room temperature in the lab.
In the results, we will report the residual moisture content measured in a subset of air-dried samples from our cover crop study, which averaged 0.26% (n = 9; range: 0.20–0.32; CV = 16%). We will also discuss implications of these measurements in the discussion: the measured residual moisture contents are consistent with expectations for coarse-textured sandy soils, which retain substantially less residual water than fine-textured soils. Previous studies reported residual water contents ranging from approximately 0.3–6% in air-dried mineral soils, with the lowest values generally observed in sandy soils (Poeplau et al., 2015; Wäldchen et al., 2012). For example, Wäldchen et al. (2012) observed water contents as low as 2.8 g kg⁻¹ (0.28%) in air-dried sandy soils and demonstrated that residual water is primarily controlled by clay content and adsorption within fine pores. We will also discuss the potential contribution of residual moisture to variation among archived samples. We believe the revisions will complement existing discussion on how microbial dynamics, such as cell lysis and continued enzymatic depolymerization, could contribute to the observed accumulation of WEOC (lines 136-137 and 181-183).
Comment:
Another aspect is that permanganate oxidizable carbon (POX-C) is proposed as a more stable indicator, as suggested by USDA, but there are many concerns about the methods used for measuring POX-C, and this is not reported in the document. I suggest to include some of the studies questioning POX-C used methods to give a better overview of this indicator, as those by Margenot’s group Margenot et al., 2024 (https://doi.org/10.1002/ael2.20124).
Response:
We appreciate this important suggestion. We acknowledged the methodological limitations and interpretational uncertainties associated with POX-C in our Introduction. In contrast, POX-C is generally less sensitive to drying but may still be affected by methodological factors such as soil mass, sieve size, laboratory handling, and SOC content (Gasch et al., 2020; Wade et al., 2020) (lines 47-49). In our revised manuscript, we plan to present POX-C more cautiously as a relatively stable but method-dependent indicator highlighting recent studies. We will add in our Discussion that recent studies have highlighted methodological inconsistencies and concerns regarding the chemical specificity and interpretability of POX-C warranting caution against its interpretation as a proxy for biologically meaningful labile C pools (Gasch et al., 2020; Margenot et al., 2024; Woodings & Margenot, 2023) (after line 179).
Comment:
Lastly, the presentation of the experimental design, which comprises sample sets from 5 experiment, should be improved to clarify when samples were collected, when they were analysed the first time, when they where reanalysed and the variation occurred during the years of storage.]
Response:
We would like to revise the materials and methods section to clarify that the comparisons are within-sample (i.e., archived vs. original measurements), rather than across time or management treatments, in the cover crop study. The changes in these indicators were quantified as differences between original and reanalyzed values from the same archived samples, thereby isolating storage effects from field-driven temporal variability. This design is different from those of the other long-term experiments where we could not separate the storage effects from those due to management practices.
Comment:
Abstract: line 19 “four long term agricultural sites” is not clear if it is referred to comparison of long-term storage or real field experiments in which WEOC is decreasing. Please revise.
Response:
We plan to revise lines 19-20 for clarification as: In addition, WEOC concentrations consistently declined over time in this experiment along with four long-term agricultural sites in the USA, but their bulk soil organic carbon (SOC) or soil organic matter (SOM) did not.
Comment:
Lines 43-49: see my comment above about POX-C methodological problems
Response:
We would like to highlight these limitations in the revised discussion as per your suggestions as follows: Recent studies have highlighted methodological inconsistencies and concerns regarding the chemical specificity and interpretability of POX-C warranting caution against its interpretation as a proxy for biologically meaningful labile C pools (Gasch et al., 2020; Margenot et al., 2024; Woodings & Margenot, 2023) (after line 179).
Comment:
Lines 51-54: Why Florida and Midwest experiments are listed separately instead of simply saying “five long term experiments”? This will make things easier to understand, in my opinion.
Response:
Lines 51-54: The Florida experiment follows a different design from the other four long-term experiments. As discussed earlier, we were able to isolate storage effects in the Florida experiment by analyzing the same samples before and after storage. In the other experiments, however, storage effects were confounded with management effects.
Based on your comment and those of the other reviewer, we plan to clarify our rationale in the Methods section: Our intention in including the four long-term experiments was not to isolate storage effects independently from management history, but rather to evaluate the practical implications of using archived air-dried soils in long-term soil health assessments.
Comment:
Sections 2.2 and 2.3 and elsewhere: It is not clear the set-up of the re-analysis of stored samples. Which were the reference values for the samples stored from the other 4 long-term experiments? The comparison was done between the re-analysis values of stored samples against the freshly analysed soils?]
Response:
We plan to clarify in the revision that WEOC concentrations from the four long-term experiments were determined using the same extraction protocol in the same laboratory in 2023.
Comment:
Line 101: sampling time or storage duration?
Response:
As shown in Figure 2, we compared the temporal trends of SOM and WEOC using Pearson correlation analysis between sampling time and soil properties values reported in Nyabami et al. (2024). We repeated this analysis for SOC and WEOC in archived soils from the four long-term agricultural experiments (lines 101-103).
Comment:
Line 116: archived sample sets would better explain the setup of the experiment
In my opinion, section 3.2 and 2.2 should clearly state when archived samples where reanalysed, mentioning the storage time elapsed after sampling, in a scheme. I found that the experimental design is not fully explaining if the samples from long term experiments were collected in year X and reanalysed for this study. In this case, can the authors compare the WEOC data from the first time they were analysed and the reanalysis here? The increasing or decreasing trends can be driven by the land use over years, not only storage.
Response:
We appreciate the reviewer’s feedback regarding the lack of clarity in the sampling design. As discussed in our previous responses, we plan to systematically revise the Materials and Methods section to clearly indicate when the archived samples were reanalyzed and to explain how the sampling design differed between the Florida study and the other long-term experiments.
Comment:
line 139: why post-rewetting studies are cited to justify WEN decrease during storage?
Response:
We plan to clarify in the revision that measurements of WEN involve rewetting archived soils with water, which shared similarities with the post-wetting studies.
Comment:
Lines 147-150: as I said above, a quick mention to doubts about POX-C methods and “lability” should be mentioned here.
Response:
Besides our Introduction, we would like to highlight the methodological limitations and interpretational uncertainties associated with POX-C after lines 147-150 in our revision as follows: However, recent investigations have demonstrated that POX-C may not consistently represent biologically meaningful labile carbon fractions, owing to methodological inconsistencies and uncertainties in the specific carbon compounds targeted by the assay (Gasch et al., 2020; Margenot et al., 2024; Woodings & Margenot, 2023) (after line 150).
Comment:
Line 163: Again, the use of “temporal trends” could refer to trends driven by the agronomic practices inducing shifts in soil indicators or by the storage time at room temperature that is causing re-arrangements of C pools. I suggest to use different wording to avoid this confusion.
Response:
We fully understand the reviewer’s suggestion to clarify the drivers of these trends. Although we found it challenging to identify terminology that clearly distinguished the different effects, we plan to revise the Results and Discussion sections to explicitly identify the drivers of each trend and avoid potential confusion. These revisions will also complement our planned changes to the Materials and Methods section by providing a clearer explanation of the sampling design and analytical approach.
Comment:
Line 181: “controlled conditions” does not equal to optimized condition to minimize soil microbial activity. WEOC can be analysed in stored samples if the moisture is kept very low and the storage temperature is low (but not on frozen samples). But I agree that measuring WEOC as soon as possible is the best option.
Response:
Thank you for your positive feedback. We will include the point about frozen samples in our revision.
Comment:
Line 190: see my comment about POX-C. I think it has potential to became a soil health indicator, but since the last 15 years the method has marginally improved and actually is a worst indicator than SOC, for example.
Response:
We would like to highlight the caution against the use of POX-C for its methodological limitations and interpretational uncertainties in the revised discussion after line 190 as per your suggestion.
References
Gasch, C., Mathews, S., Deschene, A., Butcher, K., & DeSutter, T. (2020). Permanganate oxidizable carbon for soil health: Does drying temperature matter? Agricultural & Environmental Letters, 5(1), e20019. https://doi.org/10.1002/ael2.20019
Kühnel, A., Wiesmeier, M., Spörlein, P., Schilling, B., & Kögel-Knabner, I. (2019). Influence of drying vs. Freezing of archived soil samples on soil organic matter fractions. Journal of Plant Nutrition and Soil Science, 182(5), 772–781. https://doi.org/10.1002/jpln.201800529
Margenot, A. J., Wade, J., & Woodings, F. S. (2024). The misuse of permanganate as a quantitative measure of soil organic carbon. Agricultural & Environmental Letters, 9(1), e20124. https://doi.org/10.1002/ael2.20124
Nyabami, P., Weinrich, E., Maltais-Landry, G., & Lin, Y. (2024). Three years of cover crops management increased soil organic matter and labile carbon pools in a subtropical vegetable agroecosystem. Agrosystems, Geosciences & Environment, 7(1), e20454. https://doi.org/10.1002/agg2.20454
Poeplau, C., Eriksson, J., & Kätterer, T. (2015). Estimating residual water content in air-dried soil from organic carbon and clay content. Soil and Tillage Research, 145, 181–183. https://doi.org/10.1016/j.still.2014.09.021
Stott, D. E. (2019). Recommended Soil Health Indicators and Associated Laboratory Procedures (No. 450-03; Soil Health Technical Note). U.S. Department of Agriculture, Natural Resources Conservation Service. https://www.soils.org/files/napt/publications/method-papers/2019-nrcs-technote-450-03.pdf
Wade, J., Maltais-Landry, G., Lucas, D. E., Bongiorno, G., Bowles, T. M., Calderón, F. J., Culman, S. W., Daughtridge, R., Ernakovich, J. G., Fonte, S. J., Giang, D., Herman, B. L., Guan, L., Jastrow, J. D., Loh, B. H. H., Kelly, C., Mann, M. E., Matamala, R., Miernicki, E. A., … Margenot, A. J. (2020). Assessing the sensitivity and repeatability of permanganate oxidizable carbon as a soil health metric: An interlab comparison across soils. Geoderma, 366, 114235. https://doi.org/10.1016/j.geoderma.2020.114235
Wäldchen, J., Schöning, I., Mund, M., Schrumpf, M., Bock, S., Herold, N., Totsche, K. U., & Schulze, E. D. (2012). Estimation of clay content from easily measurable water content of air-dried soil. Journal of Plant Nutrition and Soil Science, 175(3), 367–376. https://doi.org/10.1002/jpln.201100066
Woodings, F. S., & Margenot, A. J. (2023). Revisiting the permanganate oxidizable carbon (POXC) assay assumptions: POXC is lignin sensitive. Agricultural & Environmental Letters, 8(1), e20108. https://doi.org/10.1002/ael2.20108
Citation: https://doi.org/10.5194/egusphere-2026-980-AC1
-
AC1: 'Reply on RC1', Swarnali Mahmood, 25 Jun 2026
-
RC2: 'Comment on egusphere-2026-980', Anonymous Referee #2, 02 Jun 2026
The authors investigated the effect of long-term storage on various soil-health-related indicators of air-dried soil samples. The topic should be interesting and will attract the readers. However, the quality of their manuscript needs substantial improvement for further processing. The worst point to revise is the fundamental lack of information regarding preparation in their air-dried samples. There is no information about the environmental conditions during air drying and the final soil moisture after the complete air-drying, which should substantially affect the characteristics of air-dried soils.
Furthermore, the scientific strategy and meaning of the additional soil analyses from four long-term agricultural experiments are very unclear, even after I have access to their data sets available for review. Fig 2 shows the results for those additional analyses. The data, however, likely reflect the long-term trends in soil characteristics during the agricultural experiments, rather than the effect of long-term storage on air-dried soils. If the authors still believe the analysis should work for evaluating the effect of long-term storage on air-dried soils, more detailed and dedicated descriptions are essential in the manuscript. Without fully resolving those issues, it is impossible to evaluate the relevance of the discussion and its conclusions properly. Thus, there is no meaning in achieving publication.Citation: https://doi.org/10.5194/egusphere-2026-980-RC2 -
AC2: 'Reply on RC2', Swarnali Mahmood, 25 Jun 2026
We appreciate the reviewer’s thoughtful comments and recognize that the primary concerns relate to the lack of methodological clarity and data interpretation. In response, we have developed a revision plan to clarify the experimental design, sample preparation, storage conditions, and the distinct objectives of the Florida vs. other long-term experiments. We also plan to incorporate new measurements of residual moisture in archived air-dried soils and revise the Results and Discussion to more clearly distinguish storage-related patterns from management-driven changes. Collectively, we believe these revisions will substantially improve the clarity, rigor, and interpretation of the manuscript while directly addressing the reviewer's concerns.
Comment:
The authors investigated the effect of long-term storage on various soil-health-related indicators of air-dried soil samples. The topic should be interesting and will attract the readers. However, the quality of their manuscript needs substantial improvement for further processing. The worst point to revise is the fundamental lack of information regarding preparation in their air-dried samples. There is no information about the environmental conditions during air drying and the final soil moisture after the complete air-drying, which should substantially affect the characteristics of air-dried soils.
Response:
Thank you for highlighting several important issues regarding the methodological description and the lack of residual moisture measurements.
In our revised Materials and Methods section, we will clarify that the soils from the cover crop experiment were air-dried to constant mass and stored in sealed airtight plastic bags at controlled room temperature (i.e., 24°C) in the lab (line 67). For the LTAR/LTER soils, we will include in our revision that after sampling, these soils were air-dried under laboratory room-temperature conditions to constant mass prior to archival storage. We will report the residual moisture content measured in a subset of air-dried samples from our cover crop study, which averaged 0.26% (n = 9; range: 0.20–0.32; CV = 16%). This value is consistent with expectations for coarse-textured sandy soils, which retain substantially less residual water than fine-textured soils. Previous studies reported residual water contents ranging from approximately 0.3–6% in air-dried mineral soils, with the lowest values generally observed in sandy soils and the highest values in clay-rich soils (Poeplau et al., 2015; Wäldchen et al., 2012). Wäldchen et al. (2012) observed water contents as low as 2.8 g kg⁻¹ (0.28%) in air-dried sandy soils and demonstrated that residual water is primarily controlled by clay content and adsorption within fine pores. We will also discuss the potential contribution of residual moisture to variation among archived samples.
Comment:
Furthermore, the scientific strategy and meaning of the additional soil analyses from four long-term agricultural experiments are very unclear, even after I have access to their data sets available for review. Fig 2 shows the results for those additional analyses. The data, however, likely reflect the long-term trends in soil characteristics during the agricultural experiments, rather than the effect of long-term storage on air-dried soils. If the authors still believe the analysis should work for evaluating the effect of long-term storage on air-dried soils, more detailed and dedicated descriptions are essential in the manuscript. Without fully resolving those issues, it is impossible to evaluate the relevance of the discussion and its conclusions properly. Thus, there is no meaning in achieving publication.
Response:
We appreciate the reviewer’s thoughtful comments and fully understand the concern regarding the interpretation of the four long-term agricultural experiments. We agree that the current presentation does not sufficiently distinguish storage effects from field-driven changes, which makes the scientific rationale and interpretation of these analyses unclear.
In the revised manuscript, we will systematically clarify the study design and the distinct objectives of the Florida experiment and the four long-term agricultural experiments. Specifically, we will emphasize that the Florida experiment was uniquely designed to isolate storage effects by comparing original and reanalyzed measurements from the same archived samples. In contrast, the four long-term experiments were not intended to isolate storage effects independently from management history. Rather, they were included to evaluate the practical implications of using archived air-dried soils in long-term soil health assessments, where storage effects are inevitably intertwined with management-induced changes.
We will revise the Results and Discussion section to clarify the interpretation of the four long-term agricultural experiments. Specifically, we will emphasize that, although long-term management practices influence soil properties, a common pattern emerged across all four archived datasets: WEOC declined with increasing storage duration, whereas bulk SOC and SOM did not. This finding indicates that labile carbon indicators may be more susceptible to storage-induced changes than bulk carbon measurements and underscores the importance of accounting for sample storage history when interpreting long-term soil health trends from archived soils.
References
Poeplau, C., Eriksson, J., & Kätterer, T. (2015). Estimating residual water content in air-dried soil from organic carbon and clay content. Soil and Tillage Research, 145, 181–183. https://doi.org/10.1016/j.still.2014.09.021
Wäldchen, J., Schöning, I., Mund, M., Schrumpf, M., Bock, S., Herold, N., Totsche, K. U., & Schulze, E. D. (2012). Estimation of clay content from easily measurable water content of air-dried soil. Journal of Plant Nutrition and Soil Science, 175(3), 367–376. https://doi.org/10.1002/jpln.201100066
Citation: https://doi.org/10.5194/egusphere-2026-980-AC2
-
AC2: 'Reply on RC2', Swarnali Mahmood, 25 Jun 2026
Peer review completion
Journal article(s) based on this preprint
Viewed
| HTML | XML | Total | Supplement | BibTeX | EndNote | |
|---|---|---|---|---|---|---|
| 738 | 271 | 64 | 1,073 | 114 | 61 | 57 |
- HTML: 738
- PDF: 271
- XML: 64
- Total: 1,073
- Supplement: 114
- BibTeX: 61
- EndNote: 57
Viewed (geographical distribution)
| Country | # | Views | % |
|---|
| Total: | 0 |
| HTML: | 0 |
| PDF: | 0 |
| XML: | 0 |
- 1
Swarnali Mahmood
Franky Celestin
Diego Arruda Huggins de Sá Leitão
Ryan E. Champiny
Xinlin Wang
JoAnn B. Donald
Audrey Plauche
S. Carolina Córdova
Christine D. Sprunger
Gabriel Maltais-Landry
Yang Lin
The requested preprint has a corresponding peer-reviewed final revised paper. You are encouraged to refer to the final revised version.
- Preprint
(826 KB) - Metadata XML
-
Supplement
(500 KB) - BibTeX
- EndNote
- Final revised paper
The manuscript “Long-term storage of air-dried samples compromises water-extractable organic carbon as a soil health indicator” authored by S. Mahmood and co-workers focused on the evolution with time of soil indicators with time of storage. The data refer to a common practice of “air drying” and storing at “room temperature”. Indeed, room temperatures are varying a lot across different locations and seasons, as for room moisture. In my opinion, air drying is rarely reaching moisture content below 5-10% in soils (at least in my region). This means that microbial communities will still have some residual activities at these moisture levels and room temperature, which is the main reason behind the modification of labile soil health indicators such as water-soluble C and N. For “labile” soil health indicators, other methods of drying (at controlled temperature and moisture) and storage (at colder temperature) are preferably, and this could be mentioned in the manuscript. Therefore, results from this study are relevant, but their novelty is relatively low.
Another aspect is that permanganate oxidizable carbon (POX-C) is proposed as a more stable indicator, as suggested by USDA, but there are many concerns about the methods used for measuring POX-C, and this is not reported in the document. I suggest to include some of the studies questioning POX-C used methods to give a better overview of this indicator, as those by Margenot’s group Margenot et al., 2024 (https://doi.org/10.1002/ael2.20124).
Lastly, the presentation of the experimental design, which comprises sample sets from 5 experiment, should be improved to clarify when samples were collected, when they were analysed the first time, when they where reanalysed and the variation occurred during the years of storage.
Detailed comments:
Abstract: line 19 “four long term agricultural sites” is not clear if it is referred to comparison of long-term storage or real field experiments in which WEOC is decreasing. Please revise.
Introduction
Lines 43-49: see my comment above about POX-C methodological problems
Lines 51-54: Why Florida and Midwest experiments are listed separately instead of simply saying “five long term experiments”? This will make things easier to understand, in my opinion.
Materials and Methods
Sections 2.2 and 2.3 and elsewhere: It is not clear the set-up of the re-analysis of stored samples. Which were the reference values for the samples stored from the other 4 long-term experiments? The comparison was done between the re-analysis values of stored samples against the freshly analysed soils?
Line 101: sampling time or storage duration?
Results
Line 116: archived sample sets would better explain the setup of the experiment
In my opinion, section 3.2 and 2.2 should clearly state when archived samples where reanalysed, mentioning the storage time elapsed after sampling, in a scheme. I found that the experimental design is not fully explaining if the samples from long term experiments were collected in year X and reanalysed for this study. In this case, can the authors compare the WEOC data from the first time they were analysed and the reanalysis here? The increasing or decreasing trends can be driven by the land use over years, not only storage.
line 139: why post-rewetting studies are cited to justify WEN decrease during storage?
Discussion
Lines 147-150: as I said above, a quick mention to doubts about POX-C methods and “lability” should be mentioned here.
Line 163: Again, the use of “temporal trends” could refer to trends driven by the agronomic practices inducing shifts in soil indicators or by the storage time at room temperature that is causing re-arrangements of C pools. I suggest to use different wording to avoid this confusion.
Line 181: “controlled conditions” does not equal to optimized condition to minimize soil microbial activity. WEOC can be analysed in stored samples if the moisture is kept very low and the storage temperature is low (but not on frozen samples). But I agree that measuring WEOC as soon as possible is the best option.
Conclusion
Line 190: see my comment about POX-C. I think it has potential to became a soil health indicator, but since the last 15 years the method has marginally improved and actually is a worst indicator than SOC, for example.