From the journal Environmental Science: Atmospheres Peer review history

20-Dec-2023

Dear Mr Hakkarainen:

Manuscript ID: EA-ART-09-2023-000136
TITLE: Fuel aromatic content and cold operating temperatures: Effect to emission toxicity in vitro

Thank you for your submission to Environmental Science: Atmospheres, published by the Royal Society of Chemistry. I sent your manuscript to reviewers and I have now received their reports which are copied below.

I have carefully evaluated your manuscript and the reviewers’ reports, and the reports indicate that major revisions are necessary.

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Reviewer 1

See attached file

Reviewer 2

Vehicle exhaust is a key contributor to poor air quality and adverse health outcomes. To investigate the health impacts of vehicle emissions, the authors employed a novel in vitro exposure method, air-liquid interface exposure, to assess the toxic effects of light-duty vehicle exhaust, including genetxicity and inflammatory responses. Furthermore, this manuscript meticulously examines the influence of fuel composition, operating temperature, and after-treatment devices on the exhaust toxicity outcomes of vehicles. This study provides a scientific foundation for future emission reduction policies in the context of safeguarding human health through motor vehicle regulations. This work represents a novel attempt to test toxic potency directly at the emission sources, which is commendable. However, the lack of comprehensive data evidence to support the conclusion is the major flaw of this study, requiring major revision before acceptance for publication in Environmental Science: Atmospheres. The specific suggestions and comments below should be addressed in the revised manuscript:
1. The clear result needs to be stated in the abstract. The authors need to clearly state that higher aromatic content in fuels enhances exhaust toxicity (Line 39). Additionally, it is recommended that the background of this study be briefly described in the abstract (Lines 29-34).
2. In the introduction sector, the author should supplement information about commonly used exhaust after-treatment devices in vehicles, despite having provided a detailed overview of the current state of research related to this study.
3. The structure of the Materials and Methods section is expected to be reorganized for clarity and coherence. For example, Sections 2.3 and 2.4 can be merged into a single section and then subdivided into subsections as needed. Furthermore, the uncertainty analysis of the Multivariate Mixed Model and quality control of the experiments need additional clarification.
4. In the discussion section, there should be a reasonable interpretation of the results and robust evidence for the conclusions. For example, the authors need to explain the inconsistency between genotoxicity and inflammatory responses, particularly concerning the "Eu6 Alkylate" group, beyond immunosuppression (Lines 472-474). Furthermore, the data in Fig. 2 and Fig. 3 may not be sufficient to demonstrate that “the cold operation temperature substantially increased the toxicity of the exhaust” (Line 483), given the inconsistency in the results.
5. The significant correlation between BC and toxicity is evident from Table 3. However, I did not find a clear explanation from the authors in the discussion regarding the impact of BC on toxicity. This point requires further elaboration and discussion from the authors.
6. Here are some suggestions just for authors to consider:
1) encourage adding a comparison with traditional cell exposure methods to underscore the advantages of air-liquid exposure;
2) determining other chemical components in the exhaust, such as metals (possibly due to the use of catalysts in the after-treatment system) and polycyclic aromatic hydrocarbons, would contribute to understanding the impact of fuel composition and after-treatment devices on toxicity;
3) choose contrasting colors when creating graphs to enhance visual clarity (Fig. 2 and Fig. 3);
4) ensure that the full terms corresponding to abbreviations are provided the first time they appear (Line 397: “Org”).

[This text has been copied from the PDF response to reviewers and does not include any figures, images or special characters.]

Reviewer 1 comments
This manuscript describes interesting work in which changes in fuel composition and operating temperature are shown to affect the toxicity of exhaust emissions from four modern light duty vehicles. The set of experiments conducted in this work is very impressive, requiring extensive instrumentation, adherence to rigid sampling and measurement protocols, and cooperation among various laboratories and personnel.
In many instances, differences in emissions (and their toxicity) were noted with changes in the fuels and testing temperatures that were used. The authors broadly attribute most of these different outcomes to changes in the aromatics content of the fuels. However, it should be acknowledged that many other fuel properties were also changed – not just aromatics content. (It would be useful to expand supplemental Table S2 to show more properties of all the fuels.) Because of this, it does not seem warranted to attribute the observed changes in emissions toxicity entirely to aromatics content.
Authors answer: We would like to express our gratitude to the reviewer for the great comments on the manuscript. We hope that we have addressed sufficiently the points suggested by the review. Our answers to the suggestions are below of each suggestion and modifications can be found in the track-changed manuscript version.
It is true that several other fuel properties also change between the fuels used. However, previous studies have speculated the important role of aromatics in the emissions from both gasoline and diesel fuels (Lu et al., 2012; Karavalakis et al., 2015; Yang et al., 2019; Yilmaz & Donaldson, 2022), therefore, the aromatics were considered to have the highest impact on the emissions and their toxicity. According to the reviewer's suggestion, we have added more information about the fuel differences to Supplementary Materials 1 Table 2 and Table 3 (the name of the table changed to S1 Table 2)
A small mention of the difference between the fuels and their potential role in the results is added to the discussion lines 414-419.
Lu T., Huang Z., Cheung C.S., Jing M.J. Size distribution of EC, OC and particle-phase PAHs emissions from a diesel engine fueled with three fuels. Sci. Total Environ., 438 (2012), pp. 33-41 doi: 10.1016/j.scitotenv.2012.08.026
Karavalakis G., Short D., Vu D., Russell R., Hajbabaei M., Asa-Awuku A., Durbin T.D. Evaluating the effects of aromatics content in gasoline on gaseous and particulate matter emissions from SI-PFI and SIDI vehicles. Environ. Sci. Technol., 49 (2015), pp. 7021-7031, doi: 10.1021/es5061726
Yang J., Roth P., Durbin T., Karavalakis G. Impacts of gasoline aromatic and ethanol levels on the emissions from GDI vehicles: Part 1. Influence on regulated and gaseous toxic pollutants. Fuel, Volume 252, 2019, Pages 799-811. doi: 10.1016/j.fuel.2019.04.143.
Yilmaz N. & Donaldson B. Combined effects of engine characteristics and fuel aromatic content on polycyclic aromatic hydrocarbons and toxicity. Energy Sources, 44 (4) (2022), pp. 9156-9171. doi: 10.1080/15567036.2022.2129880

Consequently, I suggest changing the title of the paper to: “Effects of fuel composition and vehicle operating temperature on in vitro toxicity of exhaust emissions.” In the case of the diesel fuels, aromatics content may be the most significant compositional difference between the petroleum-derived diesel and the “renewable diesel,” which was produced by hydrotreating of vegetable oil. However, there likely are also significant differences with regard to distillation properties, viscosity, ratio of branched/straight-chain paraffins, naphthene content, cetane number, and low temperature operability – all of which could contribute to the observed differences in emissions.
Authors answer: The title of the manuscript has been changed according to the reviewer's suggestion.
As regards the physical properties of fuels, these fuel properties change inevitably when fuel composition is altered. However, physical fuel properties, such as distillation and viscosity, were not particularly different between fuels in view of the exhaust emissions. Notably, renewable fuel here is available on the market and experienced as a suitable fuel for diesel combustion.
In the case of gasoline, differences between a conventional, commercial grade gasoline (such as EN228 used in this study) and an alkylate blend stock are probably even more significant. Besides different aromatic contents, these fuels vary greatly in terms of olefin content, naphthene content, distillation properties, vapor pressure, octane number, and energy density – all of which could contribute to the observed differences in emissions.
Authors answer: The differences between the gasoline fuels are now also displayed in detail in Supplementary Materials S1 Table 3.
The olefin content was another big difference between gasoline fuels, but it has not been associated with an increase in toxic exhaust components (Hajbabaei et al., 2013). Moreover, alkylate components typically consist of compounds having high octane numbers (Perander et al. 2001). Hence, alkylate can replace aromatics in gasoline while still maintaining fuel properties suitable for spark-ignition gasoline engines.

A small mention of the differences is added in the discussion lines 529-530.
Hajbabaei M., Karavalakis G., Miller J.W., Villela M., Huaying Xu K., Durbin T.D. Impact of olefin content on criteria and toxic emissions from modern gasoline vehicles. Fuel, Volume 107, 2013, Pages 671-679, doi: 10.1016/j.fuel.2012.12.031.
Perander, J., Rantanen, L., Pentikäinen, J., Aakko, P. et al., "No Major Backsliding in Air Quality when Replacing MTBE with Isooctane in CARB Gasoline," SAE Technical Paper 2001-01-3588, 2001, doi: 10.4271/2001-01-3588
A significant finding from this work was that the amount of emissions (and their toxicity) increased substantially during regeneration of the diesel particulate filter (DPF). Whether this also occurs during regeneration of a gasoline particulate filter (GPF) is a matter of great interest. This should be discussed.
Authors answer: The GPF as a system is quite different compared to the DPF and usually relies on passive regeneration during the operation on high loads, however, the regeneration process could still increase the emissions as seen in previous studies (Chan et al., 2016). The addition of the effect of GPF on the emissions concentrations has been included in the discussion on lines 522-524.
Chan, T.W., Saffaripour, M., Liu, F. et al. Characterization of Real-Time Particle Emissions from a Gasoline Direct Injection Vehicle Equipped with a Catalyzed Gasoline Particulate Filter During Filter Regeneration. Emiss. Control Sci. Technol. 2, 75–88 (2016). https://doi.org/10.1007/s40825-016-0033-3

The dilution of vehicle exhaust emissions prior to analysis and ALI exposure is a bit confusing. Is the same dilution gas used in the CVS system as in the sample stream going to the ALI. Was this dilution gas simply ambient air, or was it purified in some way prior to use? Also, was the ALI system exposed to dilution gas alone, without containing vehicle exhaust, to provide a baseline? Some clarification of these points would be useful.
Authors answer: Within the ALI system, clean air exposures were made with synthetic air number 4.0. This information has been added on line 204.
CVS and porous tube diluter (PTD) used different dilution air. Dilution air in the PTD and ejector diluter in the ALI line was compressed air passed through a scrubbing tower (CO2 and H2O removal) and a multi-stage filtration system (HEPA equivalent).
The manuscript has been updated to contain info on the dilution air, lines 168-170.

The bottom half of page 16 (lines 425-441) discusses reasons why vehicle emissions were higher under cold operating conditions. It is true that fuel atomization and volatilization are poorer at lower temperatures, thereby increasing emissions. But surely the main factor is the relative ineffectiveness of cold catalysts and other components within the emissions control system. This would be seen more clearly in higher emissions of hydrocarbons (HC) and carbon monoxide (CO) at low temperatures. Were HC and CO emissions measured in these experiments? Another important factor is that these modern vehicles are programmed to provide fuel enrichment (i.e., introduce excess fuel) upon cold start, to ensure satisfactory engine performance when cold. This would also increase emissions of HC, CO, and PM during the first couple minutes of operation. Finally, in the case of gasoline, it is important that a winter grade of fuel be used under cold temperature conditions. Was this done in these experiments?
Authors answer: HC and CO values are added in the supplementary materials S1 Table 4.
The gasoline used in the measurements had a vapour pressure of 65.8 kPa, and characteristics for E70, E100, E150 and VLI were 44.1, 52.3, 85.4 %(V/V) and 966.7, respectively. Hence, gasoline met the class E/E1 of EN228:2012+A1:2017, which is the winter grade quality.
Alkylate gasoline is not designed to meet the requirements of EN228:2012+A1:2017. However, the volatility of alkylate used in the measurements met the requirements for winter grade class D/D1, except for a slightly lower E100 value than required in EN228:2012+A1:2017 (Table 1.).
EN 228:2012 + A1:2017 Volatility requirements: “…Class A shall apply during summer, starting not later than 1 May and ending not before 30 September. In countries with low ambient summer temperatures, as defined in [3], Class B shall apply during summer, starting not later than 1 June and ending not before 31 August. Each country shall apply one or more volatility classes with VLI (Class C1, D1, E1, or F1) for the transition periods on either side of summer. Each transition period shall be a minimum of four weeks. When transition periods are deemed critical, the critical transition period(s) shall be a minimum of eight weeks. During the remaining period, one or more winter classes shall apply with or without VLI (Class C, C1, D, D1, E, E1, F or F1)….”
Table 1. Volatility classes in EN 228:2012 + A1:2017.

Results from the CNG vehicle are puzzling, It is explained on page 18 (lines 476-477) that gasoline injection is used at certain times, such as under cold start conditions. (This seems odd to me since the desired cold start fuel enrichment could easily be achieved by simply increasing injection of CNG.) Was it confirmed that dual fuel operation actually occurred during the experiments conducted here? If so, what was the ratio of CNG/gasoline that was used? The very high ammonia emission rates for the CNG vehicle are also surprising, perhaps suggesting that NOx was being catalytically reduced due to excessive enrichment of the combustion mixture. Can the authors offer an alternative explanation for this? Because of these and other questions about operation of the CNG vehicle, I suggest that the results from this vehicle not be included in the paper.
Authors answer: The cars used in the study were all unmodified vehicles from traffic. The dual operation is controlled by the software of the vehicle decided by the car manufacturer. The CNG car used in the measurements is on the market and represents the latest emission standard, Euro 6 d. The CNG car, including its fuel injection system and engine map, is designed by OEM and the user cannot affect these adjustments. The tank for gasoline is only below 10 litres, which reflects the design of the car to use CNG, and gasoline only in special conditions. Actually, this CNG car follows the bi-fuel and not the dual-fuel principle. A bi-fuel car can use two fuels separately depending on conditions. A dual-fuel engine would use two fuels simultaneously (e.g. a pilot fuel).
For the ammonia emissions, the authors have also concluded that the emissions are due to catalytic conversion. In addition to ammonia, the car emitted also unburned methane, which contributed to the relatively high total hydrocarbon content of the emissions. Ammonia emissions are typically high from the TWC-equipped spark-ignition cars, including those fuelled by CNG. Ammonia is formed as a by-product in the TWC catalysts depending on e.g. rich-to-lean transition. Literature and results related to this issue are presented for example by Aakko-Saksa et al. 2020.
The authors would like to keep the results from the CNG vehicle as well since it was an essential part of the project. In the European context, the CNG car that was used is highly representative of the new CNG cars in the fleet. Therefore, this result will give important insight into the emissions of these vehicles in operational conditions. For the toxicological experiment, this is also a highly interesting emission, since it provides quite different chemical composition for the study matrix. Therefore, we see significant points not to exclude this part from the manuscript.
Aakko-Saksa, P., Koponen, P., Roslund, P., Laurikko, J., Nylund, N. O., Karjalainen, P., Rönkkö, T., & Timonen, H. (2020). Comprehensive emission characterisation of exhaust from alternative fuelled cars. Atmospheric Environment, 236, Article 117643. https://doi.org/10.1016/j.atmosenv.2020.117643

Several other specific comments, questions, and suggestions for the authors’ consideration are given below.
• On lines 95-96 it is stated that “… the cost of air pollution management is shown to be outweighed by the benefits of clean air policies.” I don’t think this is universally true. It might be better to include a qualifying phrase: “In most cases, the cost of air pollution management …”
Authors answer: This is very true, and the sentence has been modified according to the suggestion.

• On lines 97-99, the authors comment that fuels have a potential to decrease vehicle emissions “… even in vehicles without any after-treatment systems.” I would say “particularly in vehicles without after-treatment systems.” Effective after-treatment systems generally act as equalizers – minimizing differences in emissions resulting from fuel compositional changes.
Authors answer: This is also a great suggestion and has been modified accordingly.

• The description of the vehicles in supplementary Table 1 is good, but could be expanded slightly. For example, based on the displacement, power, and torque specifications, it appears that the “Gasoline GPF” vehicle was turbocharged, while the “Gasoline” vehicle was not. Is this true? Also, the injection system abbreviations should be explained. Why not indicate the make and model of each vehicle?

Authors answer: Displacement, power, and torque information have been added to the table 1. To clarify the vehicles and their injection and ignition systems, the abbreviation names have been also changed and injection/ignition systems are indicated separately in Table 1. These changes are made in all the tables and figures. The gasoline car with GPF did include turbocharge, this is also now indicated in Table 1. The same information has been added in the text above Table 1, lines 194-197. The maker and model of the vehicles were intentionally not indicated.

• Near the bottom of page 4 (lines 140-143), it is explained that two consecutive WLTC cycles were run, constituting a 1-hour total test duration. However, only a single cold-start was included in this test sequence. It is quite likely that the amount of emissions occurring during the first 1-2 minutes of cold start exceeded the amount of emissions occurring throughout the rest of the hour. The significance of this with respect to the in vitro toxicity tests should be discussed. Would the results be different if an emissions stream of constant composition were used rather than this short burst of high emissions followed by a much longer period of low emissions?
Authors answer: It is correct that the emissions during the cold start are higher, but obviously both consecutive cycles could not include cold starts. One of the points was to include as good as possible real-life relevance to toxicological study. The cold start emissions are probably also those that people are most exposed to. Therefore, we see that the inclusion of the cold start at the beginning of the cycle was the right decision. Even though the cold start is the most different point during the cycle, it is noteworthy that in the cycle even with the warm engine, the particle size and chemistry somewhat change due to different engine loads.
A small mention of the increase in emissions at the start of the cycle is added in the discussion on lines 501-502.

• Although stated in lines 152-153 that fuel properties are shown in supplementary material S1 Table 2, only aromatics content is included here. As suggested above, this table should be expanded to include other fuel properties.
Authors answers: The S1 Table 2 has been expanded to include other parameters of the diesel fuels. Additionally, S1 Table 3 added for the details of gasoline fuel properties.

• The vehicle emissions results given in Table 2 (and in the supplementary material) are expressed in concentration units (i.e., µg/m3 ). Normally, vehicle emissions are expressed (and regulated) on the basis of mass per distance traveled (i.e., mg/km). If possible, mass-based emissions results should be given.
Authors answer: Indeed, the distance travelled units are usually expressed when concerning traffic emissions. However, as this manuscript is mainly focusing on the toxicity of the emissions, we decided to use the concentration units here instead. The authors will publish a second paper on the measurements, which is focused in very detail on the chemistry of the emissions, also including the regeneration event of DPF.

• At the bottom of page 18 (lines 506-507), it is mentioned that “non-aromatic fuels are made from more sustainable source materials.” This is true for renewable diesel fuel, which is produced from fats, oils, and greases (FOGs), but not for alkylate, which is a petrochemical product that is produced in a petroleum refinery.

Authors answer: This has now been clarified that in the present study, this statement is only relevant to the non-aromatic diesel fuel. Line 527.

• The overall quality of the English language throughout the manuscript is excellent. However, there are a few typos, word changes, and other editorial issues that should be addressed.

Authors answer: The authors like to thank the reviewer for these excellent points to improve the language of the manuscript. All the suggested modifications below have been added to the revised manuscript.
These are identified below for the authors’ attention.
o Lines 127-128: This is an incomplete sentence.
o Line 138: Change “till” to “until”
o Line 142: Change “WLTP” to “WLTC”
o Line 143: “The instrumental setup is shown in supplementary materials S1 Figure 1.”
o Line 156: Change “Shortly” to “Briefly”
o Line 354: Change “lean air” to “clean air”
o Line 421: “… and lower vapour pressure.”
o Line 449: “… NOx and NO2 also displayed a statistically significant increase …”
o Line 457: Change “displayed” to “shown”
o Line 474: Delete this line.
o Line 492: Delete the word “similarly”
o Line 505: “The utilization of non-aromatic fuels in traffic is a possible method to
decrease …”
o Line 523: “… fuel components such as aromatics affect exhaust emissions…”
o Line 524: Delete the word “increase”



Reviewer 2 comments
Vehicle exhaust is a key contributor to poor air quality and adverse health outcomes. To investigate the health impacts of vehicle emissions, the authors employed a novel in vitro exposure method, air-liquid interface exposure, to assess the toxic effects of light-duty vehicle exhaust, including genetxicity and inflammatory responses. Furthermore, this manuscript meticulously examines the influence of fuel composition, operating temperature, and after-treatment devices on the exhaust toxicity outcomes of vehicles. This study provides a scientific foundation for future emission reduction policies in the context of safeguarding human health through motor vehicle regulations. This work represents a novel attempt to test toxic potency directly at the emission sources, which is commendable. However, the lack of comprehensive data evidence to support the conclusion is the major flaw of this study, requiring major revision before acceptance for publication in Environmental Science: Atmospheres. The specific suggestions and comments below should be addressed in the revised manuscript:
Authors answer: The authors would like to thank the reviewer for the positive comments, and we hope that we have addressed sufficiently the points suggested by the review. Our answers to the suggestions are below of each suggestion and modifications can be found in the track-changed manuscript version.
1. The clear result needs to be stated in the abstract. The authors need to clearly state that higher aromatic content in fuels enhances exhaust toxicity (Line 39). Additionally, it is recommended that the background of this study be briefly described in the abstract (Lines 29-34).
Authors answer: To state the role of aromatic more clearly, the word “indicate” has been changed to the word “demonstrate” on line 41. One line to link the emission increases due to aromatics and cold temperature to the toxicity of the emissions has been added as background for the study on lines 36-37.

2. In the introduction sector, the author should supplement information about commonly used exhaust after-treatment devices in vehicles, despite having provided a detailed overview of the current state of research related to this study.
Authors answer: A couple of lines of information have been added from the three-way catalyst and the diesel particulate filter on lines 95-99.

3. The structure of the Materials and Methods section is expected to be reorganized for clarity and coherence. For example, Sections 2.3 and 2.4 can be merged into a single section and then subdivided into subsections as needed. Furthermore, the uncertainty analysis of the Multivariate Mixed Model and quality control of the experiments need additional clarification.
Authors answer: The sections have been combined as one section 2.3 Exposures. The 2.3.1 ALI exposures were left as a subsection in this section.
Multivariate Mixed Models are robust for distributional deviations (see e.g. Schielzeth et al., 2020). As the purpose of MMM here was to find pollutants associated with toxicological endpoints, including their potential mediation in two- or three-predictor models, and not build predictive models, no heavy uncertainty analyses were needed. The estimates and their sensitivity to mediation by other pollutants were examined with the standard errors and the confidence intervals.
Schielzeth H, Dingemanse NJ, Nakagawa S, et al. Robustness of linear mixed-effects models to violations of distributional assumptions. Methods Ecol Evol. 2020; 11: 1141–1152. https://doi.org/10.1111/2041-210X.13434
4. In the discussion section, there should be a reasonable interpretation of the results and robust evidence for the conclusions. For example, the authors need to explain the inconsistency between genotoxicity and inflammatory responses, particularly concerning the "Eu6 Alkylate" group, beyond immunosuppression (Lines 472-474). Furthermore, the data in Fig. 2 and Fig. 3 may not be sufficient to demonstrate that “the cold operation temperature substantially increased the toxicity of the exhaust” (Line 483), given the inconsistency in the results.
Authors answer: As there was an observed increase in cytokine levels in the Alkylate group, with the addition of genotoxicity, the genotoxicity is likely a secondary genotoxicity, where the oxidative damage-inducing pathways result in inflammation and genotoxicity. Whereas the primary genotoxicity is induced in the absence of inflammation (Schins and Knaapen, 2007).
A small addition of this is added on the discussion lines 465-468.
The Cold temperature sentence was modified, substantially removed and genotoxicity emphasised, as the cold temperature displayed a more consistent increase in genotoxicity. Line 477 and 499.
Schins RP, Knaapen AM. Genotoxicity of poorly soluble particles. Inhal Toxicol. 2007;19 Suppl 1:189-98. doi: 10.1080/08958370701496202
5. The significant correlation between BC and toxicity is evident from Table 3. However, I did not find a clear explanation from the authors in the discussion regarding the impact of BC on toxicity. This point requires further elaboration and discussion from the authors.
Authors answer: Because there were high BC concentrations in emissions of the gasoline vehicle without the GPF, and as that exposure resulted in a significant decrease in cytokine levels, the correlation of BC and toxicity rises likely from that. However, some studies have estimated BC to have a quite high role in PM-mixture (Janssen et al., 2011). Several lines (483-487) have now been added to the discussion about this subject.
Janssen NA, Hoek G, Simic-Lawson M, Fischer P, van Bree L, ten Brink H, Keuken M, Atkinson RW, Anderson HR, Brunekreef B, Cassee FR. Black carbon as an additional indicator of the adverse health effects of airborne particles compared with PM10 and PM2.5. Environ Health Perspect. 2011 Dec;119(12):1691-9. doi: 10.1289/ehp.1003369
6. Here are some suggestions just for authors to consider:
1) encourage adding a comparison with traditional cell exposure methods to underscore the advantages of air-liquid exposure;
Authors answer: A small addition from this subject has been added to the discussion on lines about the advantages of the system on lines 537-540.
2) determining other chemical components in the exhaust, such as metals (possibly due to the use of catalysts in the after-treatment system) and polycyclic aromatic hydrocarbons, would contribute to understanding the impact of fuel composition and after-treatment devices on toxicity;
Authors answer: We fully agree with the reviewer that such aerosol data would be a great addition to the manuscript. Sadly, PAH compounds were not measured in the campaign. However, in our last article, Hakkarainen et al., 2023 data from PAH compounds and how they differ from non-aromatic and aromatic diesel fuels is included. A small addition in discussion is added concerning this, on lines 465-468.
3) choose contrasting colors when creating graphs to enhance visual clarity (Fig. 2 and Fig. 3);
Authors answer: The colors of Fig. 2 and 3 have been changed to enhance the visual clarity.
4) ensure that the full terms corresponding to abbreviations are provided the first time they appear (Line 397: “Org”).
Authors answer: This has been now fixed as Org indicates the organic matter in Table 3.

19-Feb-2024

Dear Mr Hakkarainen:

Manuscript ID: EA-ART-09-2023-000136.R1
TITLE: Effects of fuel composition and vehicle operating temperature on in vitro toxicity of exhaust emissions

Thank you for your submission to Environmental Science: Atmospheres, published by the Royal Society of Chemistry. I sent your manuscript to reviewers and I have now received their reports which are copied below.

After careful evaluation of your manuscript and the reviewers’ reports, I will be pleased to accept your manuscript for publication after revisions.

Please revise your manuscript to fully address the reviewers’ comments. When you submit your revised manuscript please include a point by point response to the reviewers’ comments and highlight the changes you have made. Full details of the files you need to submit are listed at the end of this email.

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Please also encourage your co-authors to sign up for their own ORCID account and associate it with their account on our manuscript submission system. For further information see: https://www.rsc.org/journals-books-databases/journal-authors-reviewers/processes-policies/#attribution-id

Environmental Science: Atmospheres strongly encourages authors of research articles to include an ‘Author contributions’ section in their manuscript, for publication in the final article. This should appear immediately above the ‘Conflict of interest’ and ‘Acknowledgement’ sections. I strongly recommend you use CRediT (the Contributor Roles Taxonomy, https://credit.niso.org/) for standardised contribution descriptions. All authors should have agreed to their individual contributions ahead of submission and these should accurately reflect contributions to the work. Please refer to our general author guidelines https://www.rsc.org/journals-books-databases/author-and-reviewer-hub/authors-information/responsibilities/ for more information.

I look forward to receiving your revised manuscript.

Yours sincerely,
Dr Tzung-May Fu
Associate Editor
Environmental Science: Atmospheres
Royal Society of Chemistry

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Reviewer 2

My concernings were basically addressed. No further comment.

Reviewer 1

The authors have satisfactorily addressed most of my comments on the previous draft. This revised version is much improved in terms of clarity and thoroughness.

I'm still a bit uneasy about including results from the CNG vehicle. The authors have now clarified that this vehicle utilizes a bi-fuel approach (using either CNG or gasoline) rather than a dual-fuel approach (using a mixture of CNG and gasoline). However, it is not clear which fuel was actually being used during the sampling periods of this experimental study. The discussion on page 18 (lines 491-502) mentions the possibility that increased PM and toxicity could be due to increased gasoline combustion. In my opinion, failure to know what fuel is being used at each time period greatly diminishes the usefulness of these results. If the CNG results are not eliminated altogether, the authors should emphasize that there is considerable uncertainty about the fuel composition being used.

We are glad to hear that our answers did meet the reviewers’ expectations. The addition required by reviewer 1 concerning the limitation of the CNG vehicle is now included in the revised manuscript on lines 539 to 541. Note that one funding project was added to the manuscript.

Best regards
Henri Hakkarainen & co-authors

08-Mar-2024

Dear Mr Hakkarainen:

Manuscript ID: EA-ART-09-2023-000136.R2
TITLE: Effects of fuel composition and vehicle operating temperature on in vitro toxicity of exhaust emissions

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Reviewer 1

Thank you for clarifying the limitations of the CNG vehicle.