the Creative Commons Attribution 4.0 License.
the Creative Commons Attribution 4.0 License.
| 30 Jul 2026
Harmonizing measurements of oxidative potential of PM: an interlaboratory comparison of the ascorbic acid assay
Abstract. Oxidative potential (OP) of atmospheric particulate matter (PM) is a metric of increasing scientific interest because it potentially links chemical particle properties to particle health effects. OP has been recently introduced as a recommended monitoring metric in the European Air Quality Directive. However, inconsistent protocols in the existing literature make it difficult to compare results across studies. Following a 2023 inter-laboratory comparison that focused on PM OP measured using the dithiothreitol (DTT) assay, this paper presents the findings and lessons learnt from a second inter-laboratory study focused on the ascorbic acid assay (OP-AA). In this study, twenty-six laboratories worldwide quantified OP of four PM filter samples and of one chemical compound to evaluate the entire analytical chain, including the extraction step, using a simplified OP-AA protocol. While most laboratories produced repeatable internal results when applying the simplified protocol, significant discrepancies between participants highlight the need for each laboratory to carefully evaluate deviations from the simplified OP-AA protocol. Over half of the 26 participants achieved satisfactory results, suggesting that the protocol is suitable for large-scale implementation. Beyond assessing performance, this work investigates technical, analytical, and mathematical refinements to measurement protocols. Building on the first DTT assay study, this second inter-laboratory comparison represents a significant step toward harmonizing OP assays, and provides specific recommendations to ensure consistent future measurements, ready to be applied in the new air quality directive EU 2024/2881.
Competing interests: At least one of the (co-)authors is a member of the editorial board of Atmospheric Measurement Techniques.
Publisher's note: Copernicus Publications remains neutral with regard to jurisdictional claims made in the text, published maps, institutional affiliations, or any other geographical representation in this paper. While Copernicus Publications makes every effort to include appropriate place names, the final responsibility lies with the authors. Views expressed in the text are those of the authors and do not necessarily reflect the views of the publisher.- Preprint
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Status: open (until 12 Sep 2026)
- RC1: 'Comment on egusphere-2026-3918', Anonymous Referee #1, 27 Aug 2026 reply
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RC2: 'Comment on egusphere-2026-3918', Anil Patel, 06 Sep 2026
reply
Overall assessment:
This manuscript presents a timely and valuable interlaboratory comparison of the ascorbic acid (AA) assay for measuring the oxidative potential (OP) of particulate matter (OP-AA). I found the overall effort impressive, particularly the scale of the exercise involving 26 laboratories. The harmonization of the analytical procedure, including the extraction step, and the detailed assessment of factors contributing to interlaboratory variability are particularly valuable and well justified. The assessment of variability across repeatability, extraction, and between-laboratory components is informative, while the comparison of different analytical approaches provides useful insights for further harmonization of OP-AA measurements.
The study is especially relevant given the continuing challenges in obtaining comparable OP measurements across laboratories and studies. In particular, factors such as PM mass loading and initial AA concentration can substantially influence the reported OP if not appropriately considered. The present effort therefore provides a useful contribution toward improving the consistency and interpretation of OP-AA measurements.
Overall, I consider the study potentially suitable for publication in Atmospheric Measurement Techniques. However, several aspects of the statistical treatment, analytical evaluation, and interpretation would benefit from further clarification before publication. I therefore encourage the authors to address the following points.
Comments:
- Lines 270-280 and 395-400: The definition of the proficiency standard deviation and the z-score categories need clarification:
The manuscript states that the standard deviation for proficiency assessment was fixed at 20% of the assigned value, and the z-score is calculated as:
z = (xi - X) / (0.2X)
However, the manuscript subsequently describes ±20% as the “acceptable range,” while defining z within ±2 as satisfactory.
With the equation used here, a ±20% deviation corresponds to z = ±1, whereas z = ±2 corresponds to ±40% deviation from the assigned value. Thus, “20% acceptable range” and “satisfactory at z within ±2 ” are not equivalent concepts.
I recommend that the authors clearly distinguish between:
- the standard deviation used for proficiency assessment (20% of X), and
- the limits used to classify z-scores as satisfactory, warning or action.
- Lines 395-425 and 610-615: The reported assigned values, particularly EF2, need additional contextualization:
The assigned values reported for samples A, C, D and EF2 are 0.1210, 0.4674, 0.1047 and 78.49 nmol min-1 µg-1, respectively. For EF2, which was introduced specifically as a potential chemical reference, I think it would be useful to provide more information on how the assigned value compares with previously published OP-AA measurements of CuSO4.5H2O under comparable conditions.
More importantly, the manuscript reports a CV of 40% among the 26 participants and concludes that this is still too high to establish an OP-AA reference value. Please clarify exactly how this 40% CV was calculated. This clarification is particularly important because EF2 is being considered as a potential future reference material.
- Lines 445-455 and 500-520: Signal-to-background and analytical sensitivity.
The large variability observed for the lower-OP samples deserves further consideration in relation to the signal-to-background conditions of the measurements. Samples A and D show the largest z-score ranges, while their assigned OP values are substantially lower than that of sample C. At the same time, the blank-filter experiment shows substantial differences in estimated LOD among laboratories, particularly between cuvette- and plate-reader users. A larger fraction of cuvette users also reported samples A and C as below their estimated LOD.
I therefore suggest that the authors examine whether the observed laboratory-to-laboratory variability, particularly for the lower-OP samples, is related to signal-to-background ratio or laboratory-specific analytical sensitivity. Since laboratory-wise z-scores and LODs are already available (Figs. 3 and 5), an analysis of the relationship between these parameters could be informative. Reporting the approximate signal-to-background characteristics of the different samples, where possible, would also help clarify whether the larger z-score ranges for the lower-OP samples partly reflect measurements being closer to the analytical detection limit. This could provide a more quantitative basis for interpreting the observed instrument-related differences.
- Lines 445-455; Figure 5: The LOD calculation deserves further methodological clarification:
The theoretical LOD is calculated as 3σB, where σB is obtained from nine blank-filter measurements, after allowing up to three outliers to be removed while retaining at least six results. I understand the rationale for using the blank filter as a procedural and instrumental noise. However, removing up to one-third of the observations before estimating the standard deviation could substantially reduce the estimated σB, and therefore produce an optimistic LOD. The authors should clarify the exact criterion used to identify the up-to-three outliers, and whether the LODs change substantially if all nine measurements are retained. This is important because the LOD is subsequently used to interpret differences between cuvette and plate-reader performance.
- Lines 475 and throughout Sections 3.4.3 and 3.5: The calibration performance should be examined more explicitly:
The manuscript identifies calibration accuracy as a possible source of variability but does not appear to provide sufficient quantitative information on calibration performance among laboratories. Since the calibration curve is directly used to calculate OP, calibration quality could be a major contributor to between-laboratory differences. I suggest providing, if available, information on the calibration slopes, intercepts, and linearity. Comparison of calibration quality within each instrument configuration, together with its relationship to laboratory z-scores, could be very informative.
- Lines 485-520: The relationship between [AA]0 and laboratory performance is potentially one of the strongest findings, but the interpretation could be strengthened:
The manuscript reports differences in performance between laboratories whose measured [AA]0 was within versus outside the theoretical range of 120-146 µM. Please clarify the basis for the proposed 120-146 µM range (10%) and whether this range can be recommended as a practical QC criterion for identifying potential analytical problems.
- Section 3.5, Lines 525-545: clarify the terminology and comparison of the two OP calculation approaches:
This section is scientifically interesting, but the terminology used for the two calculation approaches is somewhat difficult to follow; e.g., AA calibration curve, mathematical determination of OP, and concentration-based methods (L525-528).
I suggest defining the two approaches explicitly at the beginning of the section and using consistent terminology throughout, for example:
- calibration-curve-based OP calculation, and
- concentration-based OP calculation using measured [AA]0
- Lines 630-635: The recommendation concerning methanol pre-wetting of Teflon filters needs stronger support:
The manuscript recommends using a drop of methanol to wet Teflon filters before extraction because of their hydrophobic properties. I think this recommendation requires some additional justification, as the methanol pre-wetting step could influence the recovery of certain hydrophobic or redox-active constituents during a subsequent aqueous extraction. This is distinct from performing an extraction using methanol, but the authors should nevertheless provide evidence or literature support demonstrating that the proposed pre-wetting step does not introduce a systematic change in measured OP-AA. If the recommendation is retained, I suggest specifying approximately how much methanol is intended and the time between pre-wetting and addition of the aqueous extraction solution.
- Section 2.3, lines 230–240:
I would appreciate some clarification regarding the preparation and distribution of the filter punches. Samples A-D were collected on individual ambient filters, while the ILC involved 26 laboratories and multiple punches and extractions per sample. The manuscript states that sufficient punches were provided according to the participants’ declared number of extractions, but it would be useful to explain how the required number of punches were prepared from each original filter and how these punches were distributed among the participating laboratories. This is relevant for understanding how consistency of the test material was maintained across all participants.
Citation: https://doi.org/10.5194/egusphere-2026-3918-RC2
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- 1
Air pollution is of great concern for the whole humanity for the obvious reasons. In addition to PM2.5 mass, their oxidative potential (OP) is getting more and more attention in recent years as OP is a metric that potentially links chemical species in aerosols to health effects. This manuscript (MS) summarizes the findings and lessons learnt from an inter-laboratory comparison (ILC) campaign focused on the ascorbic acid assay for OP measurements (OP-AA). The ILC evaluates the capacity of the scientific OP community to implement the protocol and identifies the key parameters that must be strictly controlled to improve reproducibility and comparability. By assessing both intra- and inter-laboratory variability, using a common protocol on identical samples, this work provides valuable insights for the global standardization of OP measurements. Twenty-six laboratories worldwide quantified OP of four PM filter samples and of one chemical compound to evaluate the entire analytical chain, including the extraction step, using a simplified OP-AA protocol. It is a very important study, which is well designed and nicely executed.
I recommend the publication of this MS. However, the following points needs attention for possible betterment and future ILC campaigns.
L134: Give a reference that PAHs has no intrinsic OP
L135: Define “Latent OP” for readers, as it is not a usual terminology
L165-170: It would be relevant to make a case here that why multiple assays are important to assess PM OP. This discussion shall also include limitations of DTT-OP and how AA-OP will reduce the limitations.
L225-227: This step can introduce a considerable uncertainty in the measurements as well as in OP estimation. How the mass of PM used in the measurements was calculated? As the PM extracts were not filtered (as per L211-213), some initially undissolved species may contribute differently because different chemical species may have different dissolution rates. Some very fine particles can also affect absorbance measurements via physical absorbance of light by these particles. How was this issue addressed?
Fig. 2: This information can be given in tabular form with greater details including names of institutes/countries, unless participants requested for anonymity. Further, please include a reason for including labs mostly from Europe some from USA, and a few only from other countries. There are many other countries from where OP studies are reported. Including the maximum possible labs with different environmental/ meteorological conditions would have been beneficial for the whole scientific communities.
L445-446: Uniform area of filter punch should have been used by all the labs because here the purpose was also to check the contributions from filters to OP-AA.
Fig. 3: Here, it would be nice to include a comparison of reagent blanks for all labs. Reagent blank means everything is same as samples during the analytical procedure but instead of sample or blank filter extracts, only deionized water was added. From the description given in the methodology section, it is not clear whether 'negative control' itself is reagent blank or it was just the absorbance correction due to buffer solution. Comparison of reagent blanks and filter blanks would help in assessing the steps introducing variability among different labs. Add this discussion in section 3.4.3.
L474-475: If the measurements are precise, these factors can cause a systematic deviation and not random deviations.
L475-477: Use of average slope of the negative controls could be another significant source of uncertainty, unless it is very similar on different days. Reagent blank must be run along with every batch of analysis, and respective blanks shall be used for corrections. It has been observed that this reagent blank vary considerably from day-to-day. If reagent blanks were done everyday, the suggested way can be checked for better comparison among labs.
L631-633: Why glass fibre or other types of filters cannot be used? There was no description on the limitation of filter media for OP-AA analysis?
L696-700: It would be nice to include reagent blank in each batch of analyses, which shall be suitably subtracted from sample's OP.
L701-706: For future ILC, I would recommend to use multiple real samples collected from regions dominated by different types of sources e.g., megacities, semi-arid/arid regions, biomass burning dominated regions, pristine environment like Arctic/Antartic or remote mountains, marine or island sites, etc. This will help in assessing the effect of different matrix extractions in different labs. In this section, please add that all lab shall periodically run internal standard (CuSO4), and make sure that they are getting an acceptable blank corrected slope (define it). It will help in generation of quality data across the labs, and values can be used/compared for broader scientific purposes.