Indoor air quality and early detection of mould growth in residential buildings: a case study

Dear Editor

We would like to express our gratitude to the reviewers for their labour and their consideration paid to our research. We thank them for the positive feedback on the manuscript and for the constructive comments. We have accordingly revised our manuscript, including:

• Amendment of oversights and spelling errors as suggested by the reviewers;
• Revision of the graphs to improve readability;
• A detailed description of the sampling procedure followed, with information regarding the devices used;
• Revision of the conclusion section to minimize generalization of the findings.
• We believe that this improved version meets the quality standard of the journal.

We look forward to hearing from you

Best regards

On behalf of all Authors

Arianna Brambilla

Please find below our revision notes. Reviewers’ comments are reported in bold.

Reviewer #1

General remarks: 

• 2.2 Monitoring campaign
• Line 4-5 With the scope of identifying
• Line 5-7 Can you please provide more information about the sampling procedure?

Thank you for detecting the first two oversights, we have modified the text accordingly. Further, we included a sub-section under the 2.2 Monitoring Campaign to detail the sampling procedure used for both the swabs and the air sampling. We note that the sampling protocol followed the provisions reported in the manufacturer's instruction on the devices employed, which were benchmarked and compared against [26]. The modified methods section is as follows:

During July, several surfaces and air samples have been taken around the house with the scope of identifying potential fungal strains [26]. For this research, off-the-shelf instruments for mould sampling were used, following the standard protocols described in the device’s instruction manuals, which were compared against the literature [26] to ensure consistency.  The surface samplings were collected using Zefon adhesive Bio-Tape slides, which consist of a plastic slide with a pre-adhesive area of 4 cm2 that must be gently pressed on the designated surface before returning the slide to its individual protective case. This bio-tape allows determining the presence of microbial, bio-aerosol and inorganic dust, identifying the mould strains and quantifying the degree of contamination [26]. The sampling locations were selected after the inspection, as per [26] and the manufacturer’s instructions. Air sampling used Air-O-Cell cassettes, which are based on inertial impaction (the cassette is provided with a tapered inlet that directs particulate-laden air towards a slide with the collection media). This cassette falls under the sampler type “impaction onto coated surface” [26], where the collecting media is a cellulose acetate–coated glass. The sampling location was determined by the site inspection, as, following the standard protocol, it was performed near the centre of each area of the building affected by moisture intrusion, water damage, apparent mould growth, musty odours, and conditions conducive to mould growth. Following the manufacturer's instructions, the cassettes were operated with a flow rate of 15 l/m and activated for 10 minutes. The sampling devices were characterized by a detection limit of 38 FS/m3 and of 4 FS/m3, respectively for air and surface sampling. All samples were then analysed by a certified laboratory, following the standard protocols and the manufacturer’s instructions, staining the sample with lactophenol cotton blue and a professional magnifier for microscopic examinations.

For the swab testing

1^st) Can you please provide the rationale behind the selection of these particular locations (kitchen bench, hallway cupboard and bedroom) for wiping i.e. where they potential fungal reserves, where they dusty indicating potential storage of fungal particles etc?

Thank you for this comment. Following the standard testing protocol, the selection of the particular locations was driven by the initial site inspection, which is consistent with the guidelines in [26]: “An environmental consultant who is familiar with the appearance of these types of discolorations should be able to discern areas of fungal contamination on a surface, minimizing the number of surface samples needed.” During the site inspection, we noticed visible water staining on the bedroom wall that backs directly onto the bathroom downstairs, as well as a damp odour within the bedroom. Further, there was old damp and damaged furniture/items laid against this wall. Hence, we selected the bedroom wall and the hallway cupboard based on the visible stains. Further, the kitchen bench was chosen as a control sample, indicated by the dwellers as the location where the health symptoms would manifest. In the manuscript, we modified the 3.1 section as follows:  

This situation was aggravated by a leaky wall cavity from the shower, indicated by the damp odour originating from the bathroom, visible water staining on the adjacent walls, and damped-stained furniture laid against the leaky wall.  All these details indicated that indoor mould was likely to be found in this area of the dwelling. Based on the inspection, the surface samples were taken from the visible stains (bedroom wall and hallway cupboard), as well as from the kitchen bench as a control sample for an area not visibly affected.

2^ND) what kind of swabs did you use i.e. premoistened or dry swab, solvent used etc.

3^rd) was there a specific protocol followed i.e. wiping 10cm² of the surface under examination

The surface sampling used a tape lift sampling, which is the most common device for this type of application [26]. In particular, we employed a Zeflon bio-tape (https://www.kenelec.com.au/products/zefon-bio-tape-surface-sampler/), which consists of a flexible plastic microscope slide with a pre-defined adhesive area. This device allows to confirm the presence of fungi, identify the genera, and quantify the amount of each taxa.  The device comes with detailed instructions on the protocols that must be used for the sampling, which, in this case, consists in pressing the adhesive on the selected surface. We modified the section 2.2 to include a brief explanation of the device:

For this research, off-the-shelf instruments for mould sampling were used, following the standard protocols described in the device’s instruction manuals, which were compared against the literature [26] to ensure consistency. […] The surface samplings were collected using Zefon adhesive Bio-Tape slides, which consist of a plastic slide with a pre-adhesive area of 4 cm2 that must be gently pressed on the designated surface before returning the slide to its individual protective case. This bio-tape allows determining the presence of microbial, bio-aerosol and inorganic dust, identifying the mould strains and quantifying the degree of contamination [26]. The sampling locations were selected after the inspection, as per [26] and the manufacturer’s instructions.

For the air sampling

1^st) Can you please provide details about the sampling protocol followed i.e. sampling duration, airflow velocity if a pump or other mechanical mean was used, how many litres of air were sampled per room

Thank you for the comment. As we noted for the surface sampling, we followed the sampling protocols reported in the device instructions, which was consistent with the literature. In particular, the sampling was performed at the center of each room, where the cassettes were operated with a flow rate of 15 l/m and activated for 10 minutes. We modified section 2.2 to explain the protocol as follows:

The sampling location was determined by the site inspection, as, following the standard protocol, it was performed near the centre of each area of the building affected by moisture intrusion, water damage, apparent mould growth, musty odours, and conditions conducive to mould growth. Following the manufacturer's instructions, the cassettes were operated with a flow rate of 15 l/m and activated for 10 minutes.

2^nd) Were the occupants allowed to carry out any activities prior to sampling i.e. vacuum, exercise, open the windows, etc? If not for how many hours were they not permitted to carry out any activities.

This study was prompted by the adverse health symptoms with no clinical explanation shown by one occupant. For this reason, the sampling was undertaken without restrictions on the occupants, with the scope of assessing the everyday indoor conditions that the occupants were exposed to. We included the following sentence in section 2.2:

It is worth mentioning that occupants were asked to conduct their normal activities during the monitoring phase, in order to gather information representative of their everyday environmental conditions.

3^rd) Can you please give some specifications of the air sampling cassettes?

The air sampling device used were Zefon Air-O-Cell (https://www.zefon.com/airocell-user-manual), which are specifically designed for the rapid collection of a wide range of airborne aerosols including mould spores and pollen. The cassettes can be used with any standard off-the-shelf area sampling pump capable of drawing 15 l/m open flow. We included the description of the device in section 2.2:

Air sampling used Air-O-Cell cassettes, which are based on inertial impaction (the cassette is provided with a tapered inlet that directs particulate-laden air towards a slide with the collection media). This cassette falls under the sampler type “impaction onto coated surface” [26], where the collecting media is a cellulose acetate–coated glass. The sampling location was determined by the site inspection, as, following the standard protocol, it was performed near the centre of each area of the building affected by moisture intrusion, water damage, apparent mould growth, musty odours, and conditions conducive to mould growth. Following the manufacturer's instructions, the cassettes were operated with a flow rate of 15 l/m and activated for 10 minutes.

For both swab and air sampling

1^st) What methods were used for the analysis of the samples?

Thank you for this question. All samples were sent to a certified laboratory that followed the standard protocols as suggested by the manufacturer’s instruction. As we were not involved in this phase, we included the following sentence:

All samples were then analysed by a certified laboratory, following the standard protocols and the manufacturer’s instructions, staining the sample with lactophenol cotton blue and a professional magnifier for microscopic examinations.

2^nd) How do the surface and air sampling results complement each other?

Thank you for giving us the opportunity to better explain the rationale of our study. The methodology follows the protocol described in [26] and aims at holistically understanding the indoor environmental quality of the specific case study. Hence, the surface and the air samplings contribute to providing an insightful snapshot of the IEQ. As discussed in the paper, the surface samplings did not identify high volumes of mould, albeit present and visible. On the contrary, the air sampling resulted in extremely high values. Thanks to these results, we have been able to identify the old carpet as a major source of bio-contaminants and suggest an immediate replacement. This finding would not have been possible to achieve without both types of data (surface and air).

3.1 Site Inspections

Could you please specify whether the fungal readings originate from?

Table 3 reports the following information: type of sampling (air or surface), location of the sampling, total detected fungal structure, genera. Hence, we believe that this table reports all information needed to understand the data.

2^nd Paragraph Line 6: I think there is a spelling mistake (raspatory or respiratory)

Thank you for detecting this oversight, we have modified the text accordingly.

2^nd Paragraph Line 9-10: Could the elevated levels in the kitchen be associated to the fact that in kitchens the fungal readings might be capturing the existence of mould particles that originate from fruit, vegetables or food remnants that have infested by mould and have released spores or fungal particles over the previous days?

Thank you for this valuable comment. In general, the readings in the kitchen may suffer from contamination from other sources, such as food remnants. However, during the inspection, no such situation was observed, nor identified as a potential risk from the building occupants. Hence, we excluded this possibility. We modified the manuscript accordingly and added the following sentence:

The concentration found in the kitchen may be originated from other organic sources, such as food remnants developing mould, however, during the sampling, no such condition was observed and the occupants, enquired about this possibility, confirmed the low probability of this option.

Figure 2: Could you please change the line for TVOCs so that the line becomes clearer in the figure?

Figure 3: Can you please delete the zero values for the 31/08 so that there are no vertical lines in the graph?

Thank you for these suggestions. We modified the graphs as suggested and we believe that the readability improved accordingly.

The CO2 levels seem to be lower than the threshold values you reported. The peak values might be a result of larger occupancy density and since it has not risen above the threshold levels at any time wouldn’t that mean that the property is sufficiently ventilated?

This comment is highly relevant, and we thank the reviewer for the opportunity to clarify this point. Although this assumption is very valid, we note that the case study is a four-bedroom building split into three different levels and with generous-sized rooms, currently inhabited by two occupants only. According to the occupants’ comments, the building was never overcrowded during the monitoring campaign, and, therefore, this possibility can be excluded. Admittedly, the owners never ventilated the bedrooms, not in use. It is therefore plausible to conclude that the CO₂ peaks are the result of the underventilation of spaces. We included a brief sentence to express the occupancy status of the building (section 3.2):

Considering that the four-bedroom house was occupied by two residents only, excluding risks of overcrowding, and the observation made during the inspection, this might suggest that the space was significantly under-ventilated and that these parameters may be considered good indicators for early detection of high moisture-related risk probability.

Reviewer #2

My major comment is that I think the authors need to be a bit more careful with their conclusions and generalising the results. For  example, in the conclusions they state “Buildings with a high concentration of fungal spores are also more likely to present poor IAQ, high concentrations of particulate matter (PM10 and PM2.5), as well as a high level of CO2.” This was certainly the case in the studied building, but I am not sure you can generalise this to all buildings.

Thank you for the valuable comment on the manuscript. We agree with the reviewer about the risk of generalizing the results, especially if originated from the observation of a specific case study, albeit quite representative of a larger cohort of buildings. After internal discussions among authors, we decided to modify the conclusions section to contextualize the findings to our specific case.

Considering that the four-bedroom house was occupied by two residents only, excluding risks of overcrowding, and the observation made during the inspection, this might suggest that the space was significantly under-ventilated and that these parameters may be considered good indicators for early detection of high moisture-related risk probability.

More minor comments: I appreciate what you mean by occupant habits being a risk factor. But, something like having a cold home – and increasing the risk of damp – may not necessarily be a habit or choice, but a result of fuel poverty.

Thank you for raising this point. We appreciate the reviewer's comment and we agree. We, therefore, changed habits with living conditions, which seems to better represent a variety of situations that may or may not be directly correlated with the occupants’ own choices.

section 3.1

• were any mechanical ventilation systems present?

Thank you for giving us the opportunity to better explain the context of our research. As commonly found in Australian homes, the building was not provided with any mechanical ventilation, heating or cooling systems. We included a brief sentence to better contextualize it. Section 2.1 describing the case study:

As commonly found in average Australian homes, no mechanical ventilation, heating or conditioning system was present.

• raspatory should be respiratory

Thank you for detecting this oversight, we have modified the text accordingly

section 3.2

• Can you give more information on the air sampling? What equipment, what is FS, how was it counted? Did you follow any standard monitoring methods?

Thank you for this comment. According to the reviewers' requests for more details on the sampling methodology and devices, we modified section 2.2 to include more details of the sampling procedure used for both the swabs and the air sampling. We note that the sampling protocol followed the provisions reported in the manufacturer's instruction on the devices employed, which were benchmarked and compared against [26]. The modified methods section is as follows:

During July, several surfaces and air samples have been taken around the house with the scope of identifying potential fungal strains [26]. For this research, off-the-shelf instruments for mould sampling were used, following the standard protocols described in the device’s instruction manuals, which were compared against the literature [26] to ensure consistency.  The surface samplings were collected using Zefon adhesive Bio-Tape slides, which consist of a plastic slide with a pre-adhesive area of 4 cm2 that must be gently pressed on the designated surface before returning the slide to its individual protective case. This bio-tape allows determining the presence of microbial, bio-aerosol and inorganic dust, identifying the mould strains and quantifying the degree of contamination [26]. The sampling locations were selected after the inspection, as per [26] and the manufacturer’s instructions. Air sampling used Air-O-Cell cassettes, which are based on inertial impaction (the cassette is provided with a tapered inlet that directs particulate-laden air towards a slide with the collection media). This cassette falls under the sampler type “impaction onto coated surface” [26], where the collecting media is a cellulose acetate–coated glass. The sampling location was determined by the site inspection, as, following the standard protocol, it was performed near the centre of each area of the building affected by moisture intrusion, water damage, apparent mould growth, musty odours, and conditions conducive to mould growth. Following the manufacturer's instructions, the cassettes were operated with a flow rate of 15 l/m and activated for 10 minutes. The sampling devices were characterized by a detection limit of 38 FS/m3 and of 4 FS/m3, respectively for air and surface sampling. All samples were then analysed by a certified laboratory, following the standard protocols and the manufacturer’s instructions, staining the sample with lactophenol cotton blue and a professional magnifier for microscopic examinations.

• in my experience, the kitchen can have higher levels of mould due to undetected leaks in the sink cabinet

Thank you for this valuable comment. In general, the readings in the kitchen may suffer from contamination from other sources, such as food remnants or undetected leaks. However, during the inspection, no such situation was observed, nor identified as a potential risk from the building occupants. Hence, we excluded this possibility.

• Figure 3 – I think it would be best to remove the measurements for 29/8-31/08. I initially interpreted it as CO2 dropping to zero, which is not the case.

Thank you for this suggestion. We modified the graphs as suggested and we believe that the readability improved accordingly.

Conclusions

I am not convinced that the evidence supports the conclusions. At least “Buildings with a high concentration of fungal spores are also more likely to present poor IAQ, high concentrations of particulate matter (PM10 and PM2.5), as well as a high level of CO2.” Yes, mould spores could be detected as PM, and CO2 is often used as a proxy for ventilation if there are indoor CO2 sources. But, with CO2 for example: there can be other causes of mould than just ventilation issues - for example damp and mould due to damage. So, I urge caution when in the conclusions, and would avoid generalising this based on the results of a case study investigation. Perhaps, though, the methods could be a easy low-threshold way of investigating possible problems prior to air sampling

Thank you for the valuable comment on the manuscript. We agree with the reviewer about the risk of generalizing the results, especially if originated from the observation of a specific case study, albeit quite representative of a larger cohort of buildings. After internal discussions among authors, we decided to modify the conclusions section to contextualize the findings to our specific case.

Considering that the four-bedroom house was occupied by two residents only, excluding risks of overcrowding, and the observation made during the inspection, this might suggest that the space was significantly under-ventilated and that these parameters may be considered good indicators for early detection of high moisture-related risk probability

Mould growth affects 1 in 3 homes, and it is the biggest cause for complaints and litigations filed to the relevant authorities in Australia, while significantly affecting the physical and psychological health of the building occupants. Indoor mould is caused by excessive dampness, resulting from poor architectural specification, construction and maintenance practices, as well as inappropriate occupants’ behaviour. The consequences range from early biodeterioration of building materials, requiring anticipated renovation works, to deterioration of the indoor environment, posing a serious threat to the building occupants. This study investigates indoor air quality and mould growth, providing a snapshot of the current indoor air quality of Australian residential buildings regarding air pollutants. It uses a case study representative of the typical Australian suburban home to investigate the effects of unnoticed mould growth. Results of the monitoring campaign indicate that buildings with a high concentration of fungal spores are also more likely to present poor indoor air quality levels, high concentrations of particulate matters (PM ₁₀ and PM _2.5) and CO ₂. This research suggests the need for the development of early detection strategies that could minimize the health hazard to people, thereby preventing the need for a major renovation.