From the journal Environmental Science: Atmospheres Peer review history

11-Jul-2022

Dear Dr Adams:

Manuscript ID: EA-ART-07-2022-000078
TITLE: A Preliminary Investigation Comparing High-Volume and Low-Volume Air Samplers for Measurement of PAHs, NPAHs and Airborne Bacterial Communities in Atmospheric Particulate Matter.

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Dr Nønne Prisle
Associate Editor, Environmental Sciences: Atmospheres

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Reviewer 1

The authors present a well written manuscript that systematically study the diversity and concentration of PAHs, NPAHs, and bacterial communities in aerosols in Rwanda. The high-volume air sampler and low-volume air sampler were compared and discussed.
I find the topic and the results of this manuscript very interesting and useful for the community. Even with limited data points (7 samples from 7 consecutive days), the data was well discussed, and the analysis is comprehensive. Besides, considering little information can be found in Africa, this manuscript can benefit both researchers on the characterization of atmospheric PM and the atmospheric scientists on the selection of aerosol monitoring technique. Below are some minor comments to help the authors improve the manuscript before publication.
1. I suggest the authors deposit their sequencing data in a public database and provide the reference number in the manuscript.
2. Can you provide the exact sampling time for each day? Like 9am?
3. I suggest the authors provide the accurate meteorological data for each sampling day besides an average value, maybe in supplementary material for reference.
4. As a reader without intensive knowledge of PAHs, I found it’s difficult to understand the reason that the authors choose these specific compounds of PAHs and NPAHs. I found some info about the sources in the Discussion section. However, it would be more straightforward to summarize the general sources of each PAHs and NPAHs with references in Table 2, if possible. A general discussion about dominant compounds in Introduction part would be appreciated as well.
5. Do the authors think the selection of different porous size of the filters (0.8 µm vs. 1 µm) will cause some variation of the results? Can you comment on it?
6. The PCoA results surprise me as I didn’t expect a significant difference between two samples that were taken from a close location simultaneously. Besides, the authors do not provide much explanation regarding this figure (Figure 6). I encourage the authors add more discussions regarding this figure.
7. The failure of microbial community analysis due to the low density would be a concern, as the DNA concentration reported in Figure 4 is relatively constant. Besides, LVAS samples have higher DNA concentrations compared with that of HVAS while the LVAS samples failed more than HVAS. Would that be some disadvantages to use this technique for bioaerosol analysis?

Reviewer 2

Experimental conditions were designed for HVAS and LVAS so that the linear velocity (LV), defined by LA = filter are/flow rate of air, was equal. However, there were differences in PM10, PAHs, NPAHs, DNA by the two samplers.

1. In them, PM10 might be the key to solve reasons for the other differences. PM10 fraction contains not only soils and particulates from plants but also soot (particulates from combustion), and that particulates from combustion have smaller particle sizes. Moreover, sizes of fungus, bacteria and viruses are different. They are important for the discussion on the following points.
i) Were particle sizes collected the same between HVAS and LVAS?
ii) Concentrations of PAHs and NPAHs as well as microorganisms and DNA amounts may be affected by not only flow rate but also particle size.

2. Concentrations of several PAHs and NPAHs were different between HVAS and LVAS, which could not be explained by the difference of PM10 amounts. In the trace determination, not only GC-MS but also HPLC often show interferences caused by coexisting components. But it does not appear in this paper that the reliability of the methods including pretreatments were evaluated to obtain the best conditions.

Without these considerations, there would be no benefit to the reader, and the paper cannot be accepted.

Dear Dr Nønne Prisle,
Associate Editor, Environmental Sciences: Atmospheres
We thank the reviewer for the feedback. We have made some major revisions to address the concerns raised by the reviewer and we have made numerous amendments to the manuscript.
Below you will find our changes in response to each reviewer comment. We have included the line numbers where the changes occur in the updated manuscript.
Regards,
Matthew Adams

Referee: 1
Comments to the Author

The authors present a well written manuscript that systematically study the diversity and concentration of PAHs, NPAHs, and bacterial communities in aerosols in Rwanda. The high-volume air sampler and low-volume air sampler were compared and discussed. I find the topic and the results of this manuscript very interesting and useful for the community. Even with limited data points (7 samples from 7 consecutive days), the data was well discussed, and the analysis is comprehensive. Besides, considering little information can be found in Africa, this manuscript can benefit both researchers on the characterization of atmospheric PM and the atmospheric scientists on the selection of aerosol monitoring technique. Below are some minor comments to help the authors improve the manuscript before publication.

We thank the reviewer so much for the positive feedback and nice comments provided to improve our manuscript

We have addressed the suggest comments below:

• I suggest the authors deposit their sequencing data in a public database and provide the reference number in the manuscript.

o Line 201-203: The sequencing reads were deposited online in the EMBL-EBI European Nucleotide Archive with accession number PRJEB33617.

• Can you provide the exact sampling time for each day? Like 9am?

o We have added the exact sampling time

 Line 97: Our sampling periods were 24 hours for each day (from 12:00 am to 12:00 am of the following day, local time).
• I suggest the authors provide the accurate meteorological data for each sampling day besides an average value, maybe in supplementary material for reference.

o Line125-126: Table 1 summarizing the meteorological data for each sampling day was added in the manuscript.
• As a reader without intensive knowledge of PAHs, I found it's difficult to understand the reason that the authors choose these specific compounds of PAHs and NPAHs. I found some info about the sources in the Discussion section. However, it would be more straightforward to summarize the general sources of each PAHs and NPAHs with references in Table 2, if possible. A general discussion about dominant compounds in Introduction part would be appreciated as well.

o Reason we choose these specific compounds are and are highlighted in the introduction section
 USEPA classified PAHs analyzed as priority compounds given to their toxicity and wide distribution in Environment
 Information of these PAHs and NPAHs in Africa is little to non-existentexistent
 PAHs and NPAHs can cause negative health effect even at very low-concentrations
 These organic compounds are biodegraded by airborne bacteria.

o Line 43-54: We have added general discussion on source and dominant compounds of PAHs and NPAHs in introduction section.


 PAHs and NPAHs are ubiquitous environmental pollutants primarily generated during the incomplete combustion of organic materials 6. They are released into the urban atmosphere from anthropogenic sources (e.g., diesel vehicles, gasoline, industrial processes, cool and wood combustion) and natural sources (e.g., volcanic eruptions and forest fires) 7. In the atmosphere, PAHs (two or three rings) exist in the vapour phase, whereas in multi-ringed, PAHs (five rings or more) are found to be bound with particles 7. Four-ring PAHs may occur in vapour or particle phase. Given the toxicity of PAHs and their nitro-derivatives, the US Environmental Protection Agency (EPA) has identified 16 PAHs priority compounds 8. The PAHs compounds such as benzo (a) pyrene have received great attention in air pollution and epidemiological studies due to their carcinogenic and mutagenic effects 6. The PAHs (Fluoranthene) and NPAHs (1-nitropyrene, 2-nitropyrene, and 2-nitrofluoranthene) compounds have been reported to be the most dominant PAHs and NPAH in atmospheric particulate matter, and these compounds are mainly emitted by automobile exhaust 9. Studies indicated that the NPAHs such as 1-nitropyrene exhibit higher carcinogenicity and mutagenicity effects on humans than their parent PAHs 10.

o Line 298-307: In discussion section, a summary of general sources of each PAHs and NPAHs with references to our findings in the Table 3 were added.

 The source of PAHs and NPAHs in cities depends on the type of energy used since many cities in developing countries use wood, oil and coal for heating and cooking. In Rwanda, the primary source of PAHs and NPAHs are old diesel vehicle emissions and biomass burning36. The 2 and 3-ring PAHs (Nap, Ant, Phe, Fle, Phe) are mainly emitted from biomass burning (wood burning) while PAHs of 4 rings (Flu, Pyr,BaA, Chr,BbF) 5-rings (BbF,BkF,BaP) and 6 rings (DBA,BPe,IDP) are mainly emitted from diesel engines and they are used as marker of vehicular exhaust emissions 7. The PAHs (Phe, Flue and Pyr) have been associated with salt particles 7.The NPAHs detected in this study in Rwanda originated from different sources such as diesel exhaust (1-NP, 7-NBaA, and 1, 3-, 1,8-DNPs), secondary formation (2-NP and 2-NFR) and from both primary emissions (vehicle and wood-burning) and secondary formation (9-NA). In this study, we found discrepancies in the levels of these PAH and NPAH compounds detected in both samplers36
• Do the authors think the selection of different porous size of the filters (0.8 µm vs. 1 µm) will cause some variation of the results? Can you comment on it?


 PAHs, NPAHs as well as primary biological aerosol particles had a bimodal distribution and can range from several nanometers to a few hundred micrometres in aerodynamic diameter. The pore size distribution of the particles can affect the adsorption kinetics. For example, large pore showed faster adsorption than adsorbents with smaller pores. The Particle Sampling Efficiency will depend on pore size of the filter and filter type. Lower porosities and pore sizes generally result in higher sampling efficiencies. In our future experiment, we will investigate the uses the uniform pore size.
• The PCoA results surprise me as I didn't expect a significant difference between two samples that were taken from a close location simultaneously. Besides, the authors do not provide much explanation regarding this figure (Figure 6). I encourage the authors add more discussions regarding this figure.

o Line 384-403: Several discussions regarding the Figure 6 and explaining the significant variation between two samplers were added in the present manuscript
 Our study used PCoA to elucidate the differences in bacteria communities from PM10 fractions by sampler types. The results found in this study show a significant difference between the two samples despite being co-allocated in close locations. These discrepancies (distinct bacterial communities) in the HVAS and LVAS samples can be explained by different factors such as sampler type, flowrate, and filter type and pore size. HVAS is in a housing designed to face the prevailing winds. Thus, PM10 impaction inlet ensures wind-direction insensitivity compared to LVAS. Both samplers were from different manufacturers, and this difference in design may also affect collection efficiency, as demonstrated in a previous study 77. Additionally, meteorological factors such as wind can influence the diversity and richness of airborne microorganisms 78. The large filter area size for HVAS with a high-flow rate, which was more than 10 times that of LVAS, could lead to the collection of more soil and plant bacteria communities attached to PM than LVAS with a low-flow rate and small filter size. A large filter area size can lead to more formation of bacteria. In contrast, the small filter area size in LVAS could cause the possible reaction of collected microorganisms with other airborne pollutants, which could cause the degradation of bacteria. Further, the level of PAHs and NPAHs found in this study were higher in HVAS than in LVAS. According to 79, PM air pollution usually contains organic compounds, soot and toxic metals, which are harmful to bioaerosols. Other studies found that the large filter samples collected with high flowrate sampler result in the uneven distribution of biomass across the filter, further complicating DNA extractions and downstream analysis 80. A large filter area size can also allow the bacteria to quickly disperse in the atmosphere compared to a small filter area. Previous study showed that the flow rate and pore size could affect the collection efficiency of airborne microorganisms81. They showed that the relative survival of microbial communities became lower with a high flow rate and a filter with a smaller pore size usually has a higher resistance to a high flow rate.

• The failure of microbial community analysis due to the low density would be a concern, as the DNA concentration reported in Figure 4 is relatively constant. Besides, LVAS samples have higher DNA concentrations compared with that of HVAS while the LVAS samples failed more than HVAS. Would that be some disadvantages to use this technique for bioaerosol analysis?

o Early studies have employed cultivation approaches to analyze Bioaerosol. However, those studies have provided limited insight into airborne microorganisms associated with atmospheric particulate matter because only viable and culturable organisms can be identified through culture methods. In this study, we applied the most advanced technique of culture-independent (High-throughput DNA sequencing), and this technique can reflect the actual diversity of viable and non-viable microorganisms. High-throughput DNA sequencing is very sensitive, significantly quicker and can be applied to any biological sample containing nucleic acid. However, the disadvantage of our techniques is that we can not identify specific taxa of microorganisms compared to culture-dependent ones that identify specific taxa. In our future studies we will combine both technologies to characterize biological aerosols in air samples

o For the DNA concentration, there is no universal protocol available for DNA extraction in air samples, and due to low biomass, it is challenging to obtain quantifiable DNA. Additionally, the volume of air going through the filter does not necessarily reflect the diversity of bacteria communities; it depends on the type of microbial community composition and sensitivity of instruments used. The sources of PM are diverse, and their concentrations vary according to size, location and composition. HVAS (large volume) could lead to the collection of more generated dust-associated bacteria atmosphere compared to LVAS. Although, we found that airborne microorganisms in HVAS were more diverse than in LVAS. It was expected that the use of HVAS would provide sufficient DNA yields and quantifiable microorganisms. However, high concentrations of PM and its adsorbed chemical components might inhibit or promote the growth of some airborne microorganisms. Overall, both sampling instruments have potential benefits. Our intention is not to recommend the performance of samplers based on their performance but to investigate whether LVASs, which are more cost-efficient than HVAS, can be used for aerosol sampling composition.


Referee: 2

Comments to the Author

Experimental conditions were designed for HVAS and LVAS so that the linear velocity (LV), defined by LA = filter are/flow rate of air, was equal. However, there were differences in PM10, PAHs, NPAHs, DNA by the two samplers.

In them, PM10 might be the key to solve reasons for the other differences. PM10 fraction contains not only soils and particulates from plants but also soot (particulates from combustion), and that particulates from combustion have smaller particle sizes. Moreover, sizes of fungus, bacteria and viruses are different. They are important for the discussion on the following points.

We thanks the Reviewer for constructive feedback. We have address the comments as follow:


• Were particle sizes collected the same between HVAS and LVAS?

o Yes, both samplers collected particles less than 10 µm (PM10), although there was the difference in filter pore size. The pore size of the 47 mm filters is 0.8µm for LVAS while the pore- size was 1 µm for HVAS.

• Concentrations of PAHs and NPAHs as well as microorganisms and DNA amounts may be affected by not only flow rate but also particle size.

o We agree that PAHs, NPAHs, and primary biological aerosol particles have a bimodal distribution and can range from several nanometers to a few hundred micrometres in aerodynamic diameter. Therefore, their concentration can be affected by these particle sizes. However, in this study, we applied one particle size (PM10).

2. Concentrations of several PAHs and NPAHs were different between HVAS and LVAS, which could not be explained by the difference of PM10 amounts. In the trace determination, not only GC-MS but also HPLC often show interferences caused by coexisting components. But it does not appear in this paper that the reliability of the methods including pretreatments were evaluated to obtain the best conditions.Without these considerations, there would be no benefit to the reader, and the paper cannot be accepted.

o Our experiments used HPLC with fluorescence (FLD) to analyze PAHs because HPLC-FLD showed higher sensitivity to PAHs having six rings or more than GC/MS. We identify NPAHs using identified by HPLC with chemiluminescence detection. Concentrations of NPAHs are much lower than those of corresponding PAHs in environmental samples. GC/MS is not sensitive enough for determining trace levels of NPAHs. In our previous study, there were no interferences caused by coexisting components (Kalisa et al.2018 Environ. Sci. Technol. 52, 12179–12187). Our sample pretreatment showed that the surrogate recoveries ranged from 60% to 102%, and no targeted compounds were found in blank samples.

28-Jul-2022

Dear Dr Adams:

Manuscript ID: EA-ART-07-2022-000078.R1
TITLE: A Preliminary Investigation Comparing High-Volume and Low-Volume Air Samplers for Measurement of PAHs, NPAHs and Airborne Bacterial Communities in Atmospheric Particulate Matter.

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Reviewer 1

All of my comments have been addressed or answered. I appreciate the authors' work.