the Creative Commons Attribution 4.0 License.
the Creative Commons Attribution 4.0 License.
| 14 Nov 2025
Impact of burial conditions on NO3--N source apportionment in groundwater: Insights from PCA-APCS-MLR and MixSIAR methods
Abstract. NO3--N contamination in groundwater poses a significant threat to drinking water safety and ecosystem health, with accurate source identification being crucial for effective pollution control. Previous studies on NO3--N source apportionment in groundwater have largely neglected aquifer burial conditions. In this study, groundwater samples from aquifers with varying burial conditions were collected and analyzed using an integrated approach combining hydrochemical analysis (PCA-APCS-MLR) and stable isotope mixing modeling (MixSIAR) to identify and quantify NO3--N pollution sources. The results demonstrate that NO3--N concentrations in 75 % of the groundwater samples exceeded the WHO drinking water standard. PCA-APCS-MLR analysis revealed that the dominant NO3--N sources in unconfined groundwater and confined groundwater were chemical fertilizers (52.5 %) and manure & sewage (53.9 %), respectively. The MixSIAR model further identified soil nitrogen (58 %) and manure & sewage (37.9 %) as the primary contributors to NO3--N in unconfined and confined groundwater, respectively. These findings suggest that unconfined groundwater in regions with high soil nitrogen reserves is at persistent risk of NO3--N contamination. In addition, neglecting aquifer burial conditions would introduce absolute errors of 22 %–24 % in source apportionment results obtained from both PCA-APCS-MLR and MixSIAR approaches. This study highlights that aquifer confinement must be rigorously considered as a critical factor in NO3--N source identification and pollution control strategies to enhance the accuracy of source apportionment and the effectiveness of management measures.
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Status: final response (author comments only)
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RC1: 'Comment on egusphere-2025-5482', Anonymous Referee #1, 07 Dec 2025
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AC1: 'Reply on RC1', Tianyuan Zheng, 10 Jan 2026
General comments:
Response:
Thank you very much for your thorough and constructive review of our manuscript, and we sincerely appreciate the time and effort you have invested in evaluating our work. We are truly grateful for your valuable and professional comments, which will be of great help in enhancing the quality of our work. We have refined our manuscript carefully to bring our work to the high standards expected by the journal. Subsequently, a point-by-point response will be provided to each of the comments raised.
Detailed comments:
1. The term "burial conditions"is not appropriate in this context. Please consider using more precise hydrogeological terminology, such as "groundwater occurrence conditions", to better reflect the intended meaning.
Response:
Thank you for this helpful comment. As suggested, we have replaced “burial conditions” with the more precise hydrogeological term “groundwater occurrence conditions” throughout the revised manuscript.
2. The manuscript uses several abbreviations without introducing their full forms upon first use. The full form of each abbreviation should be provided at its first occurrence (e.g., Nitrate-N (NO3--N)).
Response:
Thank you for highlighting this oversight. We have carefully reviewed the manuscript and have now introduced the full form for each abbreviation at its first occurrence (e.g., "Nitrate-N (NO3--N)").
3. Sample-testing and correlation methods lack procedural detail and citations. Please add detailed information.
Response:
Thank you for this important comment. We acknowledge that the description of the sample-testing and correlation methods was insufficient. In the revised manuscript, we have added detailed procedural steps for both the experimental assays and the statistical correlation analysis. Furthermore, we have included appropriate citations to support the chosen methodologies.
4. L. 235: Add citation to the original work by Thurston and Spengler (1985) (https://doi.org/10.1016/0004-6981(85)90132-5) who proposed the APCS-MLR method.
Response:
Thank you for your valuable comment. We have added the suggested citation to the original work by Thurston and Spengler (1985). The reference has been included in the revised manuscript.
5. L. 256: Add citation to Moore and Semmens (2008) (DOI: 10.1111/j.1461-0248.2008.01163.x) and Stock and Semmens (2016) (doi:10.5281/zenodo.1209993.)for the MixSIAR method and R code.
Response:
As requested, we have added the following citations on line 256 (and in the reference list) of the revised manuscript.
6. What do you mean by "aquifer burial conditions"? Does that term refer to the differentiation of "single-layer" and "double-layer" aquifers (but sometimes called "single-aquifer layer" and "double-aquifer layer" instead)?
Response:
Thank you for your question regarding the term “aquifer burial conditions”. You are correct in your understanding – it does refer to the differentiation between “single-layer” and “double-layer” aquifers. And We adopted this specific terminology because it is commonly used in relevant hydrological literature to describe aquifer architecture (Baudron et al., 2013; Qian et al., 2020). In addition, we have revised the manuscript to replace "burial conditions" with the more standard term "occurrence conditions" to avoid any potential ambiguity.
7. Add an explanation on why atmospheric deposition (4-5 %) was kept in the model even though its contribution is minor, this justifies its inclusion for completeness.
Response:
Thank you for this helpful suggestion. Although atmospheric deposition contributes only 4-5% to the total NO₃⁻-N in the studied aquifers, it was retained in the MixSIAR model for three main reasons:
(1) Completeness of source apportionment: Atmospheric deposition is a known natural input of nitrate in groundwater systems, and its inclusion ensures that the model accounts for all potential sources, thereby reducing structural uncertainty in the source apportionment.
(2) Isotopic distinction: Atmospheric nitrate exhibits distinct δ¹⁵N and δ¹⁸O signatures (typically low δ¹⁵N and high δ¹⁸O), which help to better constrain the mixing space and improve the discrimination among the other major sources (e.g., chemical fertilizers, manure & sewage, and soil nitrogen).
(3) Model consistency and comparability: Retaining all recognized sources aligns with established practices in isotopic mixing modeling and facilitates comparison with other regional or global studies where atmospheric deposition may play a more significant role.Omitting this minor source could artificially inflate the contributions of the remaining sources and affect the robustness of the proportional estimates, even if its absolute contribution is small.
8. The discussion section remains descriptive and does not yet place the new findings in the wider context of existing literature. Please systematically compare your findings with previous studies.
Response:
Thank you for your careful review . We fully agree with your suggestion. In response, we have significantly revised and expanded the Discussion section to enhance its scientific depth.
9. I suggest putting forward more specific policy recommendations and summarizing the limitations of the research and its future directions in the discussion.
Response:
We thank the reviewer for this constructive suggestion. In the revised manuscript, we have added the dedicated content that outlines more specific policy recommendations based on groundwater occurrence conditions, such as promoting optimized irrigation and slow‑release fertilizers in unconfined aquifers and strengthening manure‑and‑sewage source control in confined aquifers, and also summarises the limitations of this work (e.g., spatiotemporal sampling constraints and uncertainties in isotopic signatures) together with future research directions, notably the integration of reactive‑transport modelling with isotopic mixing models to better elucidate nitrogen transformation processes. These additions strengthen the policy relevance and scholarly rigour of the study.
Citation: https://doi.org/10.5194/egusphere-2025-5482-AC1
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AC1: 'Reply on RC1', Tianyuan Zheng, 10 Jan 2026
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RC2: 'Comment on egusphere-2025-5482', Anonymous Referee #2, 07 Dec 2025
This manuscript investigates the sources and quantification of nitrate in groundwater, with emphasis on the role of burial conditions. PCA-APCS-MLR and MixSIAR are combined to apportion nitrate origins in groundwater samples collected from different layers of aquifers. The results are interesting, highlighting innovative concepts and methodologies in source apportionment and groundwater pollution control strategies, which finally could merit publication.
I have a number of comments:
- The term "NO3--N" is used extensively in the introduction. While it is likely that readers familiar with groundwater contamination will understand this abbreviation, it would be helpful to define it explicitly at its first appearance in the text. For example, you could write "NO3--N (nitrate-N)" the first time it appears.
- The objectives of the study are listed clearly at the end of the introduction, but transition from the discussion of existing methods and challenges to the specific aims of your study is somewhat abrupt. I recommend adding a brief paragraph to explicitly link the identified gaps in current research to the specific goals of your study.
- The introduction provides a comprehensive background on the problem of NO3--N contamination and the methods used for source apportionment. However, it lacks a clear statement of the hypothesis. Pleaseclearly state the hypothesis in the introduction.
- In the materials and methods section, the descriptions of sample testing methods are brief and there are no references provided to support these methods. Please provide detailed descriptions of the sample testing methods and cite relevant literature.
- The materials and methods section on data analysis briefly mentions the use of Pearson correlation tests but lacks details on the specific procedures used for these analyses.
- In the results section, the terms "generalized single-aquifer layer" and "actual double-aquifer layer" are used to describe different scenarios, but they are not clearly clarified. Define these terms explicitly at the beginning of the results section.
- In the discussion section, please include a more detailed comparison with previous studies on NO3--N pollution and source apportionment. Discuss how your results align with or differ from those of other studies and provide possible explanations for these differences.
Citation: https://doi.org/10.5194/egusphere-2025-5482-RC2 -
AC2: 'Reply on RC2', Tianyuan Zheng, 10 Jan 2026
General comments:
Response:
Thank you very much for your thoughtful feedback on our manuscript. We sincerely appreciate your recognition of the novelty and significance of our work, as well as your positive assessment of the methodological approach and its potential contribution to the field of groundwater pollution control. We have carefully revised the manuscript accordingly to enhance its quality. Subsequently, a point-by-point response will be provided to each of the comments raised.
Detailed comments:
1. The term "NO3--N" is used extensively in the introduction. While it is likely that readers familiar with groundwater contamination will understand this abbreviation, it would be helpful to define it explicitly at its first appearance in the text. For example, you could write "NO3--N (nitrate-N)" the first time it appears.
Response:
Thank you for pointing this out. We have revised the manuscript to explicitly define “NO3--N” as “nitrate-N” upon its first appearance in the text.
2. The objectives of the study are listed clearly at the end of the introduction, but transition from the discussion of existing methods and challenges to the specific aims of your study is somewhat abrupt. I recommend adding a brief paragraph to explicitly link the identified gaps in current research to the specific goals of your study.
Response:
We agree that a smoother transition would improve the logical flow of the Introduction. As recommended, we have added a bridging paragraph before the statement of objectives to explicitly link the identified research gaps to the specific aims of our study.
3. The introduction provides a comprehensive background on the problem of NO3--N contamination and the methods used for source apportionment. However, it lacks a clear statement of the hypothesis. Please clearly state the hypothesis in the introduction.
Response:
We appreciate your suggestion to strengthen the clarity and structure of the introduction. As recommended, we have revised the introduction to include a clear statement of the hypothesis.
4. In the materials and methods section, the descriptions of sample testing methods are brief and there are no references provided to support these methods. Please provide detailed descriptions of the sample testing methods and cite relevant literature.
Response:
Thank you for your constructive comment. We have accordingly revised the manuscript to include more detailed methodological descriptions and relevant citations.
5. The materials and methods section on data analysis briefly mentions the use of Pearson correlation tests but lacks details on the specific procedures used for these analyses.
Response:
Thank you for your thoughtful comment. We have revised the manuscript accordingly.
6. In the results section, the terms "generalized single-aquifer layer" and "actual double-aquifer layer" are used to describe different scenarios, but they are not clearly clarified. Define these terms explicitly at the beginning of the results section.
Response:
We agree that the terms "generalized single-aquifer layer" and "actual double-aquifer layer" should be explicitly defined to improve clarity. We have added a brief clarification at the beginning of the Results section.
7. In the discussion section, please include a more detailed comparison with previous studies on NO3--N pollution and source apportionment. Discuss how your results align with or differ from those of other studies and provide possible explanations for these differences.
Response:
Thank you for your careful review . We fully agree with your suggestion. In response, we have significantly revised and expanded the Discussion section to enhance its scientific depth.
Citation: https://doi.org/10.5194/egusphere-2025-5482-AC2
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RC3: 'Comment on egusphere-2025-5482', Anonymous Referee #3, 11 Dec 2025
This study investigated nitrate sources in groundwater by collecting and analyzing samples from aquifers with different burial conditions. The authors employed an integrated approach combining PCA-APCS-MLR and MixSIAR for source identification and quantification. The results were compared between confined and unconfined aquifers, highlighting distinct dominant pollution sources for each setting, which enhanced the accuracy of pollution source identification and the effectiveness of protection strategies. It is an interesting work. The approach and results in this study seem acceptable, but some minor revisions are needed. My specific comments are as follows.
(1) Title: The term “burial conditions” is non-standard. Replace it with a precise hydrogeological expression such as “aquifer confinement conditions” or “aquifer occurrence conditions”.
(2) L. 56: Add the WHO safe limit of 11.3 mg L⁻¹ NO₃⁻-N in drinking water for readers to see the exact threshold behind the health risk.
(3) L. 58: Clarify how groundwater NO₃⁻ reaches surface water; without this pathway the link to aquatic eutrophication appears as a logical leap.
(4) L. 61: NO₃⁻-N instead of this contaminant.
(5) L. 77: Add citations.
(6) L.93-94: Delete.
(7) L.132: Explain why both hydrochemical tracers and MixSIAR are needed together instead of using just one of them.
(8) L. 144: Add research hypotheses.
(9) L.228, L.257: Add citations.
(10) L.275: Add detailed information of the Pearson test.
(11) L. 313: “Generalized single-aquifer layer” and “actual double-aquifer layer” are used without definition. Clarify at the beginning of the section.
(12) L.405: How did the authors derive their fractionation data? What consequences would disregarding fractionation have for the MixSIAR results?
(13) L. 470: Add citations.
(14) L. 498-499: Without a statistical analysis, reporting absolute errors is inappropriate. Replace these percentages with a qualitative statement.
(15) To translate research findings into actionable groundwater protection strategies, the authors should explicitly outline how their results can guide targeted management practices.
Citation: https://doi.org/10.5194/egusphere-2025-5482-RC3 -
AC3: 'Reply on RC3', Tianyuan Zheng, 10 Jan 2026
General comments:
Response:
Thank you very much for taking time to review our manuscript. We sincerely appreciate your positive feedback and valuable comments, which have helped us improve the quality of our work. All revisions have been carefully considered and incorporated into the revised manuscript.
Detailed comments:
(1) Title: The term “burial conditions” is non-standard. Replace it with a precise hydrogeological expression such as “aquifer confinement conditions” or “aquifer occurrence conditions”.
Response:
Thank you for your valuable and feedback. To enhance the clarity and precision of our manuscript, we have replaced “burial conditions” with the more precise hydrogeological term “occurrence conditions” throughout the text, as you recommended.
(2) L. 56: Add the WHO safe limit of 11.3 mg L⁻¹ for NO₃⁻-N in drinking water for readers to see the exact threshold behind the health risk.
Response:
You rightly pointed out the need to explicitly state the WHO safe limit for NO3--N in drinking water, which provides readers with a clear health risk threshold. We have revised the manuscript accordingly.
(3) L. 58: Clarify how groundwater NO₃⁻ reaches surface water; without this pathway the link to aquatic eutrophication appears as a logical leap.
Response:
We appreciate this valuable suggestion. NO3--N can migrate from aquifers to surface waters through several key pathways, which vary depending on aquifer type and local hydrogeology. In unconfined aquifers, NO3--N-enriched groundwater often discharges directly into streams, lakes, or coastal zones via baseflow, especially in gaining stream systems. In confined aquifers, NO3--N may reach surface water through upward leakage where aquitards are discontinuous, or via pumping and irrigation return flows. In response to your comment, we have revised the relevant section in the manuscript.
(4) L. 61: NO₃⁻-N instead of this contaminant.
Response:
Thank you for your careful reading and precise suggestion. In response to your comment, we have revised the sentence in the manuscript.
(5) L. 77: Add citations.
Response:
Thank you for your comment. We have added the relevant citation(s) to L. 77. The revision has been made in the manuscript accordingly.
(6) L.93-94: Delete.
Response:
We have removed the content at L. 93-94 as recommended.
(7) L.132: Explain why both hydrochemical tracers and MixSIAR are needed together instead of using just one of them.
Response:
Thank you for raising this important point. The combined use of hydrochemical tracers (e.g., PCA-APCS-MLR) and the stable isotope mixing model (MixSIAR) is essential for NO3--N source apportionment, as each approach has unique strengths and limitations that, when integrated, provide a more accurate and reliable assessment than either method alone.
1) Complementary strengths and limitations
Hydrochemical tracers (PCA-APCS-MLR) are effective in identifying pollution sources based on ion correlations and concentration patterns. They can quantify contributions from sources with distinct chemical fingerprints (e.g., fertilizers, sewage) but may struggle to distinguish between sources with similar chemical profiles (e.g., soil nitrogen and atmospheric deposition) and cannot directly account for isotopic fractionation processes. MixSIAR, based on δ15N and δ18O isotopes, excels at differentiating between sources with overlapping chemical signals but distinct isotopic signatures (e.g., manure vs. soil nitrogen).
2) Enhanced accuracy and reliability
By using both methods, we cross-validate the results. For instance, PCA-APCS-MLR identified chemical fertilizers as a major source in the unconfined aquifer, while MixSIAR highlighted soil nitrogen as the dominant contributor. This discrepancy prompted a deeper investigation, revealing that soil nitrogen — a legacy source with a chemical signature similar to fertilizers — was being overlooked by the chemical method alone. The integration thus provided a more nuanced understanding: soil nitrogen is a critical, persistent source that must be managed alongside current fertilizer inputs.
In summary, the integration of hydrochemical and isotopic methods provides a more comprehensive and validated apportionment of NO3--N sources in groundwater, enhancing the scientific rigor and practical relevance of our findings.
(8) L. 144: Add research hypotheses.
Response:
Thank you for your constructive suggestion. We have added research hypotheses to the revised manuscript in the Introduction section.
(9) L.228, L.257: Add citations.
Response:
We have added relevant citations in the revised manuscript accordingly.
(10) L.275: Add detailed information of the Pearson test.
Response:
We acknowledge that the detailed information of the Pearson test was insufficient. In the revised manuscript, we have added detailed procedural steps for the statistical correlation analysis. Furthermore, we have included appropriate citations to support the chosen methodologies.
(11) L. 313: “Generalized single-aquifer layer” and “actual double-aquifer layer” are used without definition. Clarify at the beginning of the section.
Response:
We apologize for this oversight. We agree that the terms "generalized single-aquifer layer" and "actual double-aquifer layer" should be explicitly defined. We have added a brief clarification at the beginning of the Results section.
Response:
We appreciate the opportunity to clarify this key methodological aspect.
1) Determination of fractionation data
● Ammonification: The isotopic effect during ammonification is generally very small (< 2‰) and difficult to quantify precisely in complex environmental matrices. Given its minor magnitude relative to other processes, and consistent with approaches in similar studies, the fractionation associated with ammonification was neglected in our calculations.
● Nitrification: Nitrification is a major process affecting the isotopic signature of nitrate derived from ammonium-based sources (e.g., fertilizer, mineralized soil organic nitrogen). The fractionation factor for this process is known to vary widely (-35‰ to -5‰) depending on soil type and environmental conditions. Following common practice to obtain a representative value, we used the average of the range: -20.0 ± 2.7‰.
● Ammonia Volatilization: Manure and sewage are characterized by high ammonium content, making them susceptible to ammonia volatilization. Based on the previous study (which investigated volatilization in open systems relevant to manure management), the fractionation coefficient range of ammonia volatilization is 25-45‰. We adopted a fractionation factor of 24.1 ± 2.8‰ for this process.
To determine the nitrogen isotope fractionation factors of different pollution sources, it is necessary to consider the nitrogen transformation processes and their isotope fractionation effects. For atmospheric deposition, this study directly tests the nitrogen and oxygen isotope characteristic values of nitrate in precipitation. Therefore, there is no need to consider the isotope fractionation effect of atmospheric deposition pollution sources. For soil nitrogen and chemical fertilizer pollution sources, the fractionation effect of nitrification needs to be considered. Therefore, the fractionation factors for soil nitrogen and chemical fertilizer sources are both -20.0 ± 2.7‰. For manure sources, both isotope fractionation during ammonia volatilization and nitrification processes need to be considered. Therefore, the calculated isotope fractionation factor for manure sources is 16.8 ± 2.8‰.
2) Consequences of disregarding fractionation in MixSIAR results
Disregarding isotopic fractionation would critically compromise the validity of MixSIAR results. Without accounting for process-driven isotopic shifts, the model would misinterpret measured groundwater δ15N and δ18O values, leading to systematic bias in source attribution. For example, nitrate derived from soil or fertilizer that has undergone nitrification (∼20‰ depletion in δ15N) could be incorrectly assigned to atmospheric deposition or other light-isotope sources, thereby underestimating agricultural contributions. Similarly, neglecting the enrichment effect of ammonia volatilization in manure systems would cause the model to undervalue the role of manure and sewage. Ultimately, omitting fractionation would treat nitrate transport as a conservative mixing process, ignoring the transformative biogeochemical history of nitrogen in the aquifer. This would not only reduce the quantitative accuracy of source apportionment but also obscure key nitrogen cycling pathways, weakening the reliability of conclusions and the effectiveness of any resulting management recommendations.
(13) L. 470: Add citations.
Response:
Thank you for your comment. We have added the relevant citation(s) in the revised manuscript accordingly.
(14) L. 498-499: Without a statistical analysis, reporting absolute errors is inappropriate. Replace these percentages with a qualitative statement.
Response:
Thank you for pointing this out. We agree that reporting absolute errors without accompanying statistical analysis may be misleading. In the revised manuscript, we have removed the specific numerical error ranges (4%-20%, 5%-24%, etc.) from L. 498-499 and replaced them with an appropriate qualitative statement.
(15) To translate research findings into actionable groundwater protection strategies, the authors should explicitly outline how their results can guide targeted management practices.
Response:
We agree that translating scientific findings into actionable management guidance is crucial. To address this, we have expanded the Conclusion section to explicitly outline how our results can inform targeted groundwater protection strategies.
Citation: https://doi.org/10.5194/egusphere-2025-5482-AC3
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AC3: 'Reply on RC3', Tianyuan Zheng, 10 Jan 2026
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General comments:
Contamination of groundwater systems by high nitrogen emissions from agricultural land use are one of the most common and serious problems of water resources management and research at the global scale. Thus the paper addresses a highly relevant problem. The manuscript combines hydrochemical analysis (PCA-APCS-MLR) and stable isotope mixing modeling (MixSIAR) to identify and quantify NO3--N pollution sources in groundwater for unconfined and confined aquifers. It compares the result of computing the apportionment, assuming the two aquifers as a single water body, or as two water bodies. The argument for such comparison is that previous studies on NO3- -N source apportionment have largely neglected aquifer type. I tend to accept it after appropriate revisions.
Detailed comments: