Summary

This study measured the reactive oxygen species (ROS) and oxidation potential (OP) of two different types of SOA that was produced from the oxidation of naphthalene and beta-pinene. To mimic the urban environment, soot particles were used as preexisting organic matter. The authors measured ROS by using an online technique coupled with DCFH/HRP assay and compared it to the offline measurement using the same assay. They also compare the results to the measurement of OP by using DTT assay and the results from cell exposure. A significant amount of work was performed in this study to understand the toxicity of two different types of SOA and to evaluate the chemical assays for better representing cellular response. The authors concluded that naphthalene SOA, in general, has higher toxicity compared to beta-pinene SOA. They also concluded that ROS quantification could be a feasible method to represent aerosol toxicity. This manuscript is suitable to be published in ACP after minor revision by considering the comments as listed below.

Major comments:

1. Page 1, line 29, “The SOA mass was condensed onto soot particles …”. What is the major purpose to use soot particles? Will the soot particles also cause OP or cellular response? It would also be better to briefly introduce how soot particles are connected to SOA and OP in the abstract.
2. Page 5, line 134. It would be better to provide a simple explanation to calculate the carbon oxidation state of aerosol. The brief description of the post-process of AMS data would also be helpful.  
3. L163: The different precursor produces the different ROS which can have varying physicochemical properties. Naphthalene SOA and b-pinene SOA may have different solubility in water. Can Milli-Q water thoroughly extract the organic products on the filter?
4. Page 9, line 254. In order to show that the ROS is caused mainly by the SOA coated on the soot particle but not the soot only, it would be valuable to add the ROS control for soot particle only in Figure 2.
5. Page 9, line 263-265. Is there any evidence during the experiments about hygroscopic aerosol growth when RH increased from 40% to 70%? Both naphthalene SOA and beta-pinene SOA are relatively less polar in general (Chhabra et al., 2010; Chen et al., 2015). If water is less partitioned onto aerosols, the impact of humidity on the aerosol phase reaction will be little, and humidity will have a small impact on aerosols. However, for other types of SOA, such as isoprene oxygenated products, it is relatively polar, and their SOA formation, as well as OP, could be potentially impacted by RH. Can the authors conclude the the humidity effect on the SOA by using less polar organic matter ?
6. Page 11, line 313-319. Figure 3 shows that the carbon oxidation state for terpene SOA is different between the experiments under 40% RH and that under 70% RH (the highest number is 3 times different). Please provide the explanation for the higher carbon oxidation at the higher RH ? Is it due to the high OH radical concentration at the higher RH ? (high ozone production at the high RH). However, their observed ROS is very similar. It seems that the correlation between ROS and carbon oxidation state is only valid within the type of precursor but it is not sensitive to experimental conditions within the same precursor (i.e., SOA from different RH). This needs to be explained.  
7. Page 12, line 333-line343. The filter samples were stored at -20^oC for about 6 months. Can the short-lived ROS decay during this period and cause the uncertainties in the analysis? If SOA products are semi-volatile compounds, how can the one understand whether these species are reduced due to decay or by evaporation? There is no clear definition between short-live and long-live species in this manuscript. What is the expected lifetime of short-live species? Is it in second or minutes magnitude? The comparison of the samples between real-time samples and samples after 6 months is too much long time gap.
8. Page 15, line 378. When were OP determined by suing the filter samples? Were these samples measured right after sampling or stored for 6 months before analysis?
9. Page 15, line 382. What is the possible reason for the higher OP at 40% RH than that at 70% RH? Does this observation happen in NAP SOA but not in terpene SOA?
10. Section 3.4. The cell studies were performed with the filter extractions. Then, were the most ROS species used for cell studies long-live products? Can the cell studies using filter extraction be same with the results of online ROS measurement?
11.  Page 23, line 527. Will the ROS products slowly decay in the cell medium in the absence of cell cultures? It may also be useful to test how SOA products decay in the cell culture buffer without cells within 24 hrs.
12. Table 1: Author increased the concentration of both soot particles and VOC, and Table 1 shows the changes in the oxidative characteristics of particles generated form the different concentration of soot particles and VOC. If there is a change in the concentration of VOC or soot particle only, are those oxidative characteristics influenced? Which one mainly cause this difference in oxidative characteristics?
13. Table 1: Carbon oxidation state of particle from SOA_bpin-SP are negative values. What does the negative values of carbon oxidation state of particle mean?
14. Is there possible impact of vapor-wall loss on the oxidative characteristics of particles produced in the reactor or sampling lines?
15. QC/QA: How many data points were used for the QC/QA? This information can improve the reliability of the QC/QA.
16. Figure 2, Figure 4, Figure 5, Figure 6, Figure 8, and Figure 9: Please explain the calculation of the error bars?

Minor Comments:

1. Page 9, line 276. What is “photothermal aging”? Should it be “photochemical aging”?
2. Page 9, line 277, “highest ROS formation” -> “the highest ROS formation”.
3. Figure 3 and 7. It would be better to add order number (e.g., a, b, c, and d) for each sub figures.
4. It would be also useful for readers to organize the experimental conditions in a Table (in main content or SI) for the experiments described in Section 3.1-3.3.

Reference

Chen, Qi, et al. "Elemental composition of organic aerosol: The gap between ambient and laboratory measurements." Geophysical Research Letters 42.10 (2015): 4182-4189.

Chhabra, P. S., R. C. Flagan, and J. H. Seinfeld. "Elemental analysis of chamber organic aerosol using an aerodyne high-resolution aerosol mass spectrometer." Atmospheric Chemistry and Physics 10.9 (2010): 4111-4131.

The paper from Zhang et al. discusses the suitability of ROS to predict the toxicity of carbonaceous particles. Several measures of ROS such as particle-bound ROS, cellular ROS and acellular ROS were used and the results were compared with the cellular toxicity (i.e. viability of the cells). Two SOA precursors, i.e. biogenic and anthropogenic precursors were used and the results essentially revealed that photochemical aging of both SOA increases both the particle-bound ROS as well as the OP, which correlates with the cellular toxicity. This is a nicely written manuscript and shows important results.  I recommend its publication in the journal ACP. However, I have some comments as below:

1. Page 11, line 303: The authors attributed the differences in the content of organic peroxides vs. total ROS in naphthalene vs. pinene-derived SOA to the oxidation regimes. My 1^st question: isn’t there much more in the total ROS than simply the organic peroxide? And, if so, is this comparison valid? My 2^nd question: if it is attributed to the oxidation conditions, then which regime is more atmospherically relevant (photo-oxidation vs. ozonolysis)?
2. Page 12, Line 333: Do you really have to store the filters for 6 months? It is kind of expected that most of the particle-bound ROS will be lost in that time-frame. A more relevant experiment could have been analyzing the filters after couple of days (which is equivalent to ambient filter sampling for days), so that the effect of the integrated filter sampling, could have been better captured.
3. Section 3.4: I think the relevant discussion of this section actually starts from line 444. The discussion above that line does not fit under the heading of this section. Some rearrangement is warranted in this section.
4. Lines 450: The insignificant toxicity of fresh or aged-soot particles is surprising and inconsistent with the previous studies. I think it is related with water-insolubility of the soot particles. Did the authors make sure that soot particles remained suspended and are not lost?
5. The trend of carbon oxidation state vs. ROS content does not match in Table 1 vs. Figure 3. Figure 3 shows an increase in the ROS content with the carbon oxidation state while Table 1 shows the reverse trend (see top two rows). Can the authors provide an explanation?
6. Line 533: Since the authors didn’t measure different ROS components (and just hypothesized), I don’t think this sentence is well supported from the authors’ results.

The work by Zhang et al. used an online instrument based on the DCFH/HRP assay to measure particle-bound ROS in SOA generated from representative biogenic and anthropogenic precursors. They then collected the same types of SOA on filters and quantified the ROS formation using the same DCFH assay as well as OP using the DTT assay on the filter extracts. They also investigated cytotoxicity via cell viability and cellular ROS production using a DCFH-DA assay from A549 cells exposed to buffer-extracts of these SOA. They found that most of the acellular ROS are short-lived and photochemical aging enhances ROS production and DTT activity. Compared to biogenic SOA, the anthropogenic SOA has a higher acellular and cellular ROS production and higher cytotoxicity. They also concluded that acellular particle-bound ROS could be a suitable metric to predict aerosol particle toxicity and health effects of aerosol given its strong correlation with cellular ROS.  Overall, the results are interesting and the paper provides good discussion on related work from literatures and limitations of the study. Below are my comments:

Major comments:

1. Please specify what ROS species can be detected by the DCFH/HRP assay? OH, O2- etc?

also organic peroxides can react with the assay but organic peroxides are not ROS.

1. The authors find that naphthalene SOA has a higher ROS content than the biogenic SOA, and it is likely due to quinones and semiquinones in naphthalene SOA that forms superoxide radicals. Is DCFH assay known to be sensitive to superoxide? Semiquinone radical can oxidize DCF radical to form DCF which yields superoxide (Rota et al., provided below) and superoxide forms H2O2. How do the authors tell whether the ROS signal is from quinones-DCF chemistry or SOA aqueous chemistry?

Rota C, Chignell CF, Mason RP. Evidence for free radical formation during the oxidation of 2'-7'-dichlorofluorescin to the fluorescent dye 2'-7'-dichlorofluorescein by horseradish peroxidase: possible implications for oxidative stress measurements. Free Radic Biol Med. 1999 Oct;27(7-8):873-81. doi: 10.1016/s0891-5849(99)00137-9. PMID: 10515592.

1. The analytical methods used in the work (DCFH/HRP, DTT, and cellular DCFH assay) are known to be sensitive to H2O2 and/or organic peroxides. Is it possible that peroxides are essentially what the authors are measuring which explains the strong correlations between acellular and cellular ROS?
2. section 2.2, details are in Fuller et al. (2014) and Wragg et al., (2016) but it would be useful to briefly discuss what the differences are between online and offline ROS measurements? Are the online extracts filtered? How did you quantify losses inside the denuders? Some descriptions about the DCFH/HRP methods are mentioned in section 2.3, if the online system uses the same method, maybe should move the related method description up to section 2.2.
3. line 327, “…ROS components react with HRP seconds after the particles enter the instrument.” Some ROS have lifetimes in a range of ns. It would be useful to specify what ROS can the authors capture with the method.
4. I am also confused by the authors’ use of “particle-bound” and “Short-lived ROS” to describe ROS formation from SOA. My understanding of the online system is that SOA are collected into liquid and then mix with the DCFH probe. Some ROS lifetime can be very short that by the time the samples react with DCFH probe, they might be gone. Particle-bound ROS refer to ROS on the SOA particle. However, the method not only captures the particle-bound ROS with a lifetime longer than the time it takes to travel from PAM to mix with the probe, but also the ROS formed through SOA aqueous chemistry.

Minor comments:

1. Offer et al. (2021) are cited many times throughout the manuscript, but according to the reference list, it is a paper under review. Please specify in the main text.
2. line 125, could you explain why O3 are removed prior to online measurement and filter collection?
3. line 387, “To the best of our knowledge, the OP of SOAβPIN-SP from this study is the first reported in the literature.” Tong et al., EST 2018 paper has provided OP of SOAβPIN.
4. line 404, “Compared to naphthalene-derived SOA, β-pinene SOA are expected to contain a negligible amount of quinones but peroxides are suggested to contribute significantly to the OP of β-pinene SOA.” The authors cited Wang et al., 2018 and Jiang and Jing, 2018, but these two studies did not use β-pinene SOA. Please correct.