Mind the gap between non-activated (non-aggressive) and activated (aggressive) indoor fungal testing: impact of pre-sampling environmental settings on indoor air readings

Dear Birgitte, 

We are very grateful for your useful feedback. To address the issues you raised we have proceeded with the following amendments. 

1.      We have changed the headings throughout the manuscript as per your suggestion. 

2.      We have corrected the words you pointed out  

• Abstract: deleted “enormous” 
• Introduction: Used the term “biota” instead of flora 
• Sec. 3.2: Written the meaning of RFU in this section 

3.      Regarding your comment on page 7 – this is indeed wall, and not floor, because the test was carried out horizontally in this instance. 

4.      In Sec. 2.2. we specified that the tests were performed in September 2022 

5.      In Sec. 2.2 we provided the room’s area and added several sentences in the 4^th paragraph and table 3 to clarify the conditions under which the case study tests happened as per your suggestions.  

6.      In Sec. 3.2 we made the figure larger so that the bars can be easily distinguished. 

7.      You are correct. We did not detect any Chaetomium or Trichoderma from our case study tests. 

8.      Thank you for pointing out the difference in spore sizes among different species. Different species have indeed different spore sizes and shapes and activation may able to lead to the capture of larger particles that could be undetectable if a non-activated protocol was implemented. We are now discussing these things in the last paragraph of sec 3.2 have added some sentences to showcase the manifestation of activation on the case study results as you suggested.   

9.      We understand that doing the analysis showcased in sec 3.2 might be redundant and as so we decided to remove this part in the revised version as you suggested. 

10.  Airing the house can indeed lead to the removal of fungal spores and humid air or be a source for species that are not present in the indoor spaces tested. We have added a sentence in 3.2 to underline the effect of the windows’ opening to the sampling readings  

11.  In the conclusions part we have now included our recommendations pertaining the blowing duration, and the sampling height. However, we believe that at this point our research cannot lead to the proposal of suggested waiting times before sampling, sampling volume and type of analysis. 

12.  You rightfully ask us to send our ms to Mycometer and Housetest. Thanks for this – we did and both companies were happy with the contents. 

Indoor fungal testing has been within the researchers’ scope of interest for more than a century. Various sampling and analysis techniques have been developed over the years, but no testing protocol has been yet standardised and widely accepted by the research and practitioner communities. The diversity in fungal taxa within buildings with varied biological properties and implications on the health and wellbeing of the occupants and the building fabric complicates the decision-making process for selecting an appropriate testing protocol. This study aims to present a critical review of non-activated and activated approaches to indoor testing, with an emphasis on the preparation of the indoor environment prior to sampling. The study demonstrates the differences in the outcomes of non-activated and activated testing through a set of laboratory experiments in idealised conditions and a case study. The findings suggest that larger particles are particularly sensitive to the sampling height and activation, and that non-activated protocols, despite dominating the current literature, can significantly underestimate the fungal biomass and species richness. Therefore, this paper calls for better-defined and activated protocols that can enhance robustness and reproducibility across the research domain focused on indoor fungal testing.