Indoor mould contamination persists as a critical public health challenge, particularly following water damage events precipitated by climate extremes, structural defects, inadequate ventilation, poor building design, aging infrastructure, and deferred maintenance. This review extends on prior work evaluating environmental and clinical thresholds for airborne fungal contamination. We reassess the continued relevance of the 1000 spores/m³ (or CFU/m³) threshold as a practical and evidence-based benchmark for indoor air quality assessment, especially with regard to health implications. However, for evaluating building conditions and identifying likely point sources of contamination, the indoor/outdoor (I/O) spore concentration ratio and changes in species profile are more diagnostically significant. An I/O ratio exceeding 2:1 or a shift in the expected species distribution is a strong indicator of indoor fungal amplification, even if absolute concentrations are below the 1000 threshold. Drawing on studies published since 2023, we confirm that environments exceeding 1000 CFU/m³ often correlate with visible or hidden mould contamination and adverse health outcomes, while unaffected environments fall below this level. Despite growing empirical and regulatory support, recent updates to key industry standards - including ANSI/IICRC S520 (2024) and S590 (2023) - increasingly frame quantitative sampling as optional rather than essential, favouring discretionary visual or subjective assessments that may lack objective verification. This shift risks enabling substandard remediation or assessment practices, undermining scientific and legal defensibility. We argue that quantifiable, objective sampling remains essential at all stages of mould investigation and remediation. While subjective site information can supplement assessments, it must not displace validated techniques like spore trap analysis, tape lifts, and viable culture for air and surface. This review reinforces the central role of the 1000 spores/m³ threshold as a health-screening tool and advocates its continued use alongside comparative OA data and I/O ratios to ensure robust, accountable, and health-protective mould assessment protocols.

Keywords: mould, fungi, spores, CFU, indoor air quality, thresholds, 1000 spores/m³, health risks, water damage, mycotoxins

Indoor mould exposure is a persistent public health issue, necessitating reliable methods for risk assessment. Airborne fungal spore concentrations are commonly measured, but interpretation requires meaningful thresholds. A previous work explored the challenges in defining such thresholds, noting the debate surrounding various proposed levels and highlighting the importance of 1000 spores/m³ (or CFU/m³) as a practical action level, especially in water-damaged or non-complaint buildings.¹

Assessing mould levels following significant water damage events, such as the catastrophic flooding recently experienced along Australia’s sub-tropical coastline exacerbated by climate change, presents considerable practical challenges.² The sheer volume of affected dwellings, often in remote areas, can overwhelm assessment resources, necessitating generic remediation scopes. This situation often results in disputes among parties involved (e.g., occupants, owners, tenants, landlords, builders, insurers, remediators, and owner’s corporations or body corporate in apartments or offices) concerning the identification and severity of mould-related risks, the effectiveness of remediation efforts, and the implications for insurance claims, potential litigation, and negligence allegations. Independent validation processes, such as Post Remediation Verification (PRV) and final Clearance Testing (CLT), have become crucial components of insurance assurance programs in managing these complexities and providing confidence in the safety of reinstated buildings following such large-scale ‘CAT’ events. Recent Australian research further underscores the scale of this issue, highlighting the high prevalence of dampness and mould in housing, exacerbated by climate-related weather events, and documenting the broad range of persistent, multi-system health symptoms reported by occupants living in such conditions, prompting government inquiries into biotoxin-related illnesses.³

While a concentration of 1000 spores per cubic meter (spores/m³) of air is commonly cited as a threshold for potential adverse health effects in sensitized individuals - particularly in relation to total airborne fungi. It is essential to consider this threshold in the broader context of environmental variability and building dynamics. Outdoor air (OA) concentrations can frequently exceed 1000 spores/m³, especially in warmer months, during pollen seasons, after major flooding or in certain climatic zones where spore production naturally fluctuates. As such, reliance on the absolute value alone may lead to misinterpretation. This is why the Indoor/Outdoor (I/O) ratio that relies on the outdoor air OA is a critical diagnostic tool in mould assessments. The Australian Mould Guideline⁴ specifically defines thresholds for both absolute concentrations and relative ratios in Tables 1 and 2, with indoor spore levels that are more than two times higher than outdoor levels classified as "Hazardous." When outdoor levels are elevated, an indoor environment that mirrors or amplifies this pattern, particularly with altered fungal profiles, suggests internal amplification and may indicate compromised building health, even if the total counts are above or below 1000 spores/m³. Therefore, 1000 spores/m³ should be viewed as a threshold for human exposure to mould indoors, while the I/O ratio is more indicative of the underlying building condition and possible fungal colonization indoors. This dual approach aligns with best practice in environmental health investigations and is critical to accurate interpretation of sampling results.

Importantly, recent immunological findings further support the need for conservative indoor air thresholds. A 2025 review found that fungal disease susceptibility varies widely between individuals due to genetic and immune system differences even in the absence of immunosuppression.⁵ This reinforces the precautionary value of the 1000 CFU/m³ threshold in protecting vulnerable populations across diverse indoor environments.

This updated review specifically aims to collate and present evidence from literature published since the original paper¹ that supports the continued relevance and importance of the 1000 spores/m³ threshold. The focus is narrowed to identify recent publications that cite, reference, or otherwise use this specific threshold, providing an updated perspective on its standing within the scientific and indoor air quality (IAQ) communities.

The structure of this paper proceeds from a review of relevant literature supporting the 1000 spores/m³ threshold, followed by a critical discussion of its practical application within current mould assessment standards and guidelines.

A comprehensive literature search was conducted using the PubMed and Scite databases to identify relevant publications on airborne microorganism concentrations, specifically focusing on the terms “1000 CFU/m3”, “1000 spores/m3”, and “fungal structures/m3”. The search strategy employed a combination of exact phrase searching and Boolean operators (OR, AND) to capture variations in terminology and reporting. A targeted analysis was also conducted on the recent pre-print biomedical sciences literature related to indoor mould and air quality. The analysis specifically sought instances where:

1. The threshold of 1000 spores/m³ or 1000 CFU/m³ was explicitly referenced, either as a measured value, comparative point, or inferential range (e.g., below or exceeding this level).
2. This threshold was cited as a guideline, regulatory limit, risk indicator, or meaningful benchmark for evaluating microbial air quality in indoor environments.

The context of these mentions was noted to understand how the 1000 spores/m³ threshold is currently being discussed or supported in recent literature.

Sampling and analysis methodologies for assessing fungal contamination, as reviewed by Loukou et al.,⁶ typically follow a multi-phase process involving physical inspection, sample collection, detection/identification, and evaluation (Figure 2 in reference 6). Sample collection strategies include direct material/surface sampling (e.g., bulk samples, scrapings, swabs, contact plates, or tape lifts for direct microscopy), settled dust collection (representing longer-term deposition), and air sampling (providing a snapshot of airborne spores).⁷ Air sampling can be passive (settle plates) or active (volumetric), with active methods like impaction generally considered more efficient. Common active samplers include impactors (onto agar or adhesive slides), impingers (into liquid), and filter samplers. However, results are influenced by sampling strategy (e.g., activated vs. non-activated), volume, time, location, and the aerodynamic properties of different fungal spores, with smaller, dry spores (e.g., Aspergillus, Penicillium) being more readily captured in air samples than larger or slime-spored fungi (e.g., Stachybotrys, Chaetomium).

Both spore trap and viable culture methods employ standardized conversion procedures to express results in comparable units, with data typically converted to colony forming units (CFU) per cubic meter of air, allowing for consistent quantification and interpretation across different sampling techniques.

The analysis identified several recent publications that continue to reference the 1000 spores/m³ or CFU/m³ level, or provide data supporting its relevance, reinforcing its position as a significant benchmark in indoor air quality discussions, despite the acknowledged lack of universal official standards.

1. General safety threshold

A 2024 review discussing fungi as indicators of indoor air quality explicitly stated that “a concentration of 10^3 microorganisms/m³ [1000 microorganisms/m³] is commonly considered a general safety threshold for indoor air quality”. This positions the 1000 level as a widely recognized, albeit general, safety guideline within the field. Evidence suggests that integrating preventive and treatment strategies-such as controlling humidity, improving ventilation, and ongoing air quality monitoring-can lead to significant improvements in health outcomes and reduce the risk of illnesses associated with fungal exposure.⁸

2. Health risks and threshold complexity

A comprehensive 2023 review⁹ investigating the microbial contamination of indoor air highlights the diverse health risks including respiratory illnesses, allergies, infections, toxic effects, and even potential links to cancer through exposure to mycotoxins like aflatoxins and ochratoxin A produced by common indoor fungi. The authors describe mycotoxins as non-viral biological carcinogens. Despite these known risks, the review underscores the complexity of the indoor microflora and the significant challenge posed by the lack of uniform international standards for acceptable microbial levels. It notes that while “a study by the WHO expert group to assess the health risks of biological agents in indoor environments suggested that the total microbial concentration should not exceed 1,000 CFU/m³”, other recommendations exist, such as maximum limits of 300 CFU/m³ for fungi and 750 CFU/m³ for bacteria. Chawla et al. further emphasize that “the standards followed may vary from one country to another and from one kind of indoor environment to another,” reinforcing the difficulty in establishing universally applicable thresholds while acknowledging the historical and ongoing reference to the 1000 CFU/m³ level.

3. Historical WHO reference

A 2024 study [10] on microbiological indoor air quality in private clinics in Harar, Ethiopia, highlights critical thresholds for microbial contamination in indoor environments. The study adopted a 1000 CFU/m³ threshold for bacterial air quality, in line with World Health Organization (WHO) recommendations. Bacterial air quality was categorized as "good" if levels were below 1000 CFU/m³ and "poor" if levels exceeded this figure. According to the study, 120 (46.15%) of the treatment rooms met the "good" bacterial air quality standard, while 140 (53.85%) exceeded the threshold, falling into the "poor" category.

For fungal (mould) contamination, the study applied a more stringent threshold than that for bacteria. Citing WHO standards, the study defined fungal air quality as "good" when fungal levels were below 500 CFU/m³, and "poor" if above this level. The findings showed that 150 (57.7%) rooms had fungal levels within the "good" range, while 110 (42.3%) exceeded this limit, falling into the "poor" category.

The 500 CFU/m³ threshold for mould aligns with the Australian Mould Guideline⁴ recommendation for mould in mechanically ventilated spaces [Australian Mould Guideline, 2010]. This comparison raises a key point: the commonly used 1000 CFU/m³ threshold for fungal contamination may be too lenient, particularly in healthcare settings where maintaining a clean and safe environment is paramount.

Notably then, the authors¹⁰ adopt The European Commission’s fungal and bacterial contamination thresholds as follows: Very Low (<50 CFU/m³), Low (51-100 CFU/m³), Intermediate (101-500 CFU/m³), High (501-2000 CFU/m³), and Very High (>2000 CFU/m³), with identical ranges applied to fungal load contamination. The study’s results indicated that the mean bacterial load was 904 CFU/m³, which falls into the "high" category, while the mean fungal load was 401 CFU/m³, placing it in the "intermediate" category. This research supports the use of the 500 CFU/m³ threshold for fungal contamination as a more protective and reliable standard for indoor air quality assessments, particularly in healthcare environments where vulnerable populations are at risk.

4. Reference limit in assessment

A recent 2025 study investigating microbial contamination in university dormitories in China, provides valuable insights on fungal concentration thresholds in indoor environments.¹¹ When examining the laboratory validation section comparing AC ON versus AC OFF conditions, the box plots reveal that most measurements remain below the 1000 CFU/m³ mark, particularly when air conditioning is turned off. Although the median values in AC ON conditions stay below 1000 CFU/m³, the upper quartiles and whiskers occasionally extend beyond this threshold, especially at specific time intervals (3h, 5h-12h, and 24h measurements show higher concentrations). Notably, the 500 CFU/m³ threshold, which the paper explicitly identifies as the World Health Organization's recommendation for indoor fungal concentrations, is frequently exceeded in both AC ON and AC OFF conditions, with AC ON conditions generally showing higher fungal concentrations. The authors also reference the Chinese national standard (GB/T 18883–2022) that sets a bacterial limit of 1500 CFU/m³ for residential and office spaces, though this specifically applies to bacterial rather than fungal concentrations. While the controlled laboratory conditions shown in the box plots demonstrate that the 1000 CFU/m³ level isn't routinely exceeded, the WHO-recommended 500 CFU/m³ threshold is frequently surpassed, supporting the paper's overall finding that "indoor fungal levels consistently exceeded recommended thresholds" throughout their year-long study. The laboratory experiments confirm that air conditioners can serve as emission sources for fungi, with fungal concentrations peaking shortly after air conditioner startup before gradually decreasing. This analysis reinforces the importance of using the more stringent 500 CFU/m³ standard as recommended by WHO for evaluating indoor fungal air quality, even though the 1000 CFU/m³ level is less frequently exceeded in the controlled laboratory conditions depicted in the box plots. Its inclusion as a reference boundary value, even alongside other limits (e.g. no measurements for indoor fungi >2000CFU), signifies its continued recognition and use in practical assessment contexts.

5. Context from long-term monitoring

Further reinforcing the relevance of the 1000 spores/m³ (CFU/m³) level as a significant benchmark, a recent multi-year study (2019-2023) monitoring daily aerospora - defined as airborne pollen grains and fungal spores - in Pretoria, South Africa, provides valuable context.¹² While the study identified 'high-risk' days based on the 90th percentile value (>1109 aerospora grains/m³), analysis of their published data (Figure 2a) reveals that the mean daily aerospora concentration for every month throughout the study period remained below 900 grains/m³. This demonstrates that even in an environment experiencing occasional high peaks during the October-May "aerospora season," the average condition typically falls well below the 1000 mark. Such findings lend credence to using 1000 grains/m³ (CFU/m³) as a meaningful threshold distinguishing generally acceptable conditions from periods requiring closer attention or potential action by public health authorities.

6. Reinforcement from review on surface & air contamination

A comprehensive 2023 review by Al Hallak et al.,¹³ examining fungal contamination of building materials and subsequent aerosolization provides further strong context for the 1000 CFU/m³ threshold. The review explicitly highlights World Health Organization (WHO) guidelines which consider airborne fungal spore concentrations (AFSC) above 500 CFU/m³ as hazardous, and concentrations exceeding 1000 CFU/m³ as extremely hazardous. This direct reference to WHO guidelines associating the 1000 CFU/m³ level with extreme hazard significantly reinforces its importance as a critical benchmark. The paper clearly establishes normal versus abnormal levels: "normal" conditions are maintained at below or around 500 CFU/m³ (typically in dwellings with no visible mould and no respiratory health problems), while AFSC >1000 CFU/m³ is considered "abnormal" and requires professional intervention for remediation, as recommended by the French agency ANSES. The review supports this distinction through systematic comparisons across building types, showing dramatic variations: hospitals (~33 CFU/m³), normal dwellings (≤535 CFU/m³ in most studies), mouldy dwellings (>1000 CFU/m³ in 6/8 studies), and mouldy museums (3573 CFU/m³).

Furthermore, Al Hallak et al.,¹³ analysed studies comparing surface and air contamination, finding that the most frequently detected fungal species on material surfaces (including Aspergillus, Penicillium, Cladosporium, and Stachybotrys) were also the most common in indoor air samples within the same buildings. The review links high surface contamination (e.g., 860,000 CFU/m² in a mouldy museum) with very high airborne levels (3573 CFU/m³), concluding that higher surface contamination generally implies higher airborne concentrations. Importantly, the paper notes that most frequently identified indoor species are xerophilic and can develop at low water activity (about 0.85), meaning mild moistening of materials may be sufficient to reach hazardous levels. This reinforces the connection between material contamination and airborne risk, underpinning the significance of airborne thresholds like 1000 CFU/m³ as indicators of potentially hazardous conditions originating from fungal growth on surfaces.

7. WHO recommended limit citations

Multiple recent studies consistently cite the 1000 CFU/m³ level as a World Health Organization (WHO) recommended limit for indoor microbial concentrations.^14-16 This threshold serves as a crucial benchmark for assessing indoor air quality across various environments. For instance, a 2024 study on university campus bioaerosols explicitly refers to the 'World Health Organization's recommended value (1000 CFU/m³)' when discussing total bacterial counts,¹⁴ noting that concentrations exceeding this value may be hazardous to human health. The research found that total airborne bacterial counts exceeded this threshold in 33% of classroom samples during periods of few personnel activities (UP) and in 55% of canteen samples during periods of personnel activities (OP). Specifically, as shown in Figure 1(b) of Ref 14, the canteen displayed mean bacterial concentrations of 1124 CFU/m³ during occupied periods, compared to just 347 CFU/m³ during unoccupied times. In contrast, fungal concentrations remained well below the WHO threshold across all locations and time periods, ranging from 84-646 CFU/m³ during UP and 85-228 CFU/m³ during OP. Similarly, investigations in cave environments consistently reference this WHO recommendation,^15,16 using it to evaluate both ecological impacts and human health risks in speleotherapeutic settings. These studies apply the threshold to assess anthropogenic disturbance, with concentrations above 1000 CFU/m³ being associated with potential adverse health effects and environmental concerns. The widespread citation of this WHO value in diverse indoor environments from educational facilities to natural caves underscores its continued recognition as a key reference point for evaluating microbial air quality and determining when intervention measures may be necessary.

Figure 1 Summary guide for interpreting indoor airborne mould levels based on colony-forming units per cubic metre (CFU/m³), indoor/outdoor (I/O) spore ratios, and ventilation context. Levels below 500 CFU/m³ are considered low-risk, particularly in mechanically ventilated buildings. Values below 1000 CFU/m³ are generally acceptable for naturally ventilated structures unless shift in Genus/Species. Readings exceeding 1000 CFU/m³ may indicate contamination requiring further investigation. While visual cues, odour, and documentation provide useful context (“see, smell, document”), comprehensive testing to map indoor spore concentrations and Genus/Species diversity remains essential for accurate assessment.

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8. Use in classification systems

A Malaysian study from 2025 of four hospitals in Peninsular Malaysia demonstrates practical implementation of 1000 CFU/m³ as a regulatory threshold.¹⁷ The Industry Code of Practice (ICOP) on Indoor Air Quality, established by the Department of Occupational Safety and Health (DOSH) in 2010, uses this value as the national guideline for acceptable total fungus counts in indoor environments served by mechanical ventilation systems. The study found that concentrations ranged from 18 to 2597 CFU/m³ across the hospitals, with a minimum standard value of <1000 CFU/m³ considered necessary to “avoid discomfort and/or adverse health effects among occupants of an indoor or enclosed environment served by a mechanical ventilation and air conditioning system (MVAC) including an air-cooled split unit”. Two locations (the Oral Surgeon Clinic and Orthopaedic Specialist Clinic) in Hospital D exceeded the 1000 CFU/m³ threshold, demonstrating how this standard serves as a practical boundary for identifying problematic areas requiring intervention. This threshold application is particularly significant in sensitive hospital settings where immunocompromised patients are present, with higher risks even at lower concentrations (less than 1 CFU/m³) noted for invasive aspergillosis. The use of 1000 CFU/m³ as a formal boundary in the ICOP framework exemplifies its role in distinguishing between acceptable and unacceptable contamination levels in healthcare environments. While this threshold was developed specifically for environments with mechanical ventilation systems including air-cooled split units, the study's findings underscore its broader applicability as a benchmark for fungal remediation triggers in various indoor settings where occupant health and comfort are priorities.

The 1000 CFU/m³ level is also frequently employed as a critical threshold in classification systems assessing microbial contamination levels and potential impacts. Studies evaluating anthropogenic impact in cave environments utilize classification schemes where concentrations exceeding 1000 CFU/m³ signify ‘strong influence’ or potential ‘irreversible ecological disturbance’ (Class 4 or 5).^15,16 A study assessing fungal contamination in healthcare settings in Iran also adopted a classification where concentrations > 1000 CFU/m³ were categorized as ‘high’ contamination.¹⁷ The use of 1000 CFU/m³ as a boundary marker in these formal assessment frameworks highlights its practical application in distinguishing between normal, moderate and high levels of contamination or impact.

9. Observed exceedances and correlation with damage in specific environments

Recent research provides empirical data showing concentrations significantly exceeding 1000 CFU/m³ in various environments associated with contamination sources, structural compromise, or high occupancy. Notably, a comprehensive Russian study by Fedorova et al.,¹⁸ titled “Hygienic assessment of mycological contamination of the internal environment of residential and public buildings”, documented fungal levels in 139 residential apartments (87 with visible fungal damage) and other building types. Using robust methodology including visual inspection and air sampling, the study documented baseline fungal levels in environments without fungal damage, reporting ranges of 20–250 CFU/m³ for residential spaces, 30–130 CFU/m³ for office buildings, and 50–460 CFU/m³ for medical facilities. In stark contrast, significantly elevated fungal levels were found in contaminated environments; residential spaces with fungal damage showed concentrations ranging from 590 to 11,500 CFU/m³, and contaminated office buildings ranged from 950 to 9,700 CFU/m³. These levels in damaged buildings were reported to be "tens to hundreds of times higher than the fungal flora content in the air of 'healthy' premises."

Crucially, Fedorova et al.,¹⁸ established a direct correlation (r =0.85) between the percentage of wall area visibly affected by mould and the airborne fungal concentration. Specifically, homes with spot lesions affecting less than 5% of the wall area had average airborne levels of 850±140 CFU/m³ (range: 590–1,880 CFU/m³), already approaching or exceeding the 1000 CFU/m³ benchmark in some cases. In contrast, spaces where ≥25% of the walls were contaminated exhibited significantly higher levels, averaging 5,070±535 CFU/m³ (range: 4,260–10,500 CFU/m³). This quantitative link between the extent of visible damage and the magnitude of airborne fungal load strongly underscores the relevance of the 1000 CFU/m³ threshold as an indicator of potentially problematic indoor environments requiring assessment and remediation, despite the authors noting the lack of formal hygienic standards for indoor fungi in Russia at the time.

10. Fungal aerosol distribution patterns

The authors of a 2024 study from Wuhan, China, thoroughly examined fungal infection in hospital wards with air conditioning.¹⁹ They investigated fungal aerosol concentration, particle size distribution, and dominant genera across three departments (Cardiology, Paediatric, and Respiratory) during winter and summer seasons in 2023. Their results showed higher winter concentrations (ranging from 312-346 CFU/m³) compared to summer (152-209 CFU/m³), with the dominant genera being Penicillium (69-73%), Aspergillus, Cladosporium, and Alternaria. The particle size distribution exhibited a normal pattern with the highest proportions (70-83%) occurring in stages III-V (1.1-4.7 μm), which can penetrate deep into the respiratory system. Notably, the study found no significant correlation between fungal concentrations and disease type or personnel density, but rather identified temperature, humidity, and seasonal changes as main influencing factors. While the authors didn't define specific threshold values for contamination, they implicitly validated their findings by comparing to other regional studies showing similar concentration ranges (20-150 CFU/m³) in hospital environments (from Iran), suggesting their measured values represented typical hospital air conditions. Although hospitals are expected to maintain stricter hygiene standards with lower acceptable fungal levels compared to residential settings where 1000 CFU/m³ might be more appropriate, the study emphasizes that even these lower hospital concentrations warrant attention as increasing fungal CFU levels are directly linked to elevated mould presence and potential mycotoxin exposure, posing particular risks to immunocompromised patients.

11. The Australian mould guideline (AMG 2010)

The Australian Mould Guideline⁴ established fundamental protocols for mould assessment and remediation across Australia, and recent studies demonstrate the continued relevance of these principles in contemporary practice. In a Queensland flood recovery study from 2023, Neumeister-Kemp et al.,² directly applied the guideline's methodology, identifying Penicillium chrysogenum and Cladosporium cladosporioides as dominant airborne species while establishing specific associations between fungi and building substrates. This work validated the guideline's material-based risk assessment framework while providing Australian-specific data. The healthcare facility study by Meda et al.,²⁰ though not directly referencing the guideline, refined these approaches by introducing paired control/test settle plates to compensate for natural background variations in fungal concentrations- an innovative methodological advancement that could enhance future guideline revisions.

These recent studies confirm the practical effectiveness of remediation protocols outlined in the Australian Mould Guideline and elsewhere in the literature about including HEPA filtration with horsehair brush agitation, wet wiping with microfibre cloths, and the use of naturally brewed vinegar, alcohol, or bio-enzymes while avoiding bleach and chlorine-based products. These consistent results across residential flood recovery and specialised healthcare settings demonstrate the robust nature of the guideline's recommended practices. Significantly, the healthcare study departed from the guideline's CFU-based threshold recommendations, explicitly avoiding setting "safe levels of fungal spores per cubic metre" and instead using the ratio of test to control counts to determine remediation success. This novel approach revealed that fungal contamination required 38 weeks to return to control levels despite physical remediation completion at 3 weeks post-flooding, emphasising the importance of prolonged monitoring regardless of specific CFU thresholds and validating the guideline's emphasis on comprehensive assessment beyond visible mould remediation.

It is also important to acknowledge that the 1000 spores/m³ threshold is not an arbitrary figure. It is firmly embedded in Australia’s best practice assessment and remediation framework through the Australian Mould Guideline (AMG 2010). The AMG includes specific concentration-based categories that classify airborne fungal contamination and health risk, with 1000 spores/m³ serving as the lower boundary for "High" levels and the start of thresholds requiring professional remediation in naturally ventilated buildings; and discusses the utility of the 500 spores/m³ threshold for mechanically ventilated buildings. That so many recent international and peer-reviewed studies independently reference and support these thresholds further validates its continued use as a benchmark for mould assessment, particularly in known or suspect post-water-damage or mould-risk environments.

1. Strengths and limitations of the S520 as a cleaning standard

The ANSI/IICRC S520 (2024) is widely recognised as a leading international reference for mould remediation;²¹ while the S500 focusses on the water damage component.²² Its strong emphasis on source removal - the physical elimination of mould-contaminated materials is one of its core strengths. The standard outlines thorough procedures for containment, engineering controls, and cleaning practices that align with best practice and health-protective goals. However, it is important to acknowledge that the S520 is fundamentally a cleaning protocol, not a diagnostic or exposure-assessment standard. This distinction has significant consequences: decisions about the scale and sufficiency of remediation can be made in some situations without any requirement for objective, quantitative testing.

2. Visual inspection alone is inadequate

In real-world practice, it is common to encounter mould-affected properties that are prematurely cleared based solely on visual inspection. While visual and olfactory cues are useful for initial evidence collection at site, they cannot identify hidden reservoirs, quantify airborne contamination, or assess toxic or allergenic fragments invisible to the eye. Reliance on subjective evaluation, particularly where there is suspect hidden mould, or in high-stakes or heavily water-damaged environments, undermines legal defensibility and leaves occupants vulnerable to ongoing exposure.

3. Flexibility in S520, S590 and draft S530 can undermine objectivity

The 2024 S520 for mold remediation, the 2023 S590²³ for HVAC assessments, and the draft 2025 S530²⁴ for indoor environmental assessments introduce procedural flexibility that, while often necessary, can enable remediation or assessment without objective, verifiable data. For example, S520 permits remediators to proceed without input from an Indoor Environmental Professional (IEP) if the decision is documented under the "Limitations, Complexities, Complications, and Conflicts" (LCCC) clause, provided there is no timely objection from materially interested parties. In turn, S530 would allow contamination levels (e.g., Condition 2) to be assumed based on the professional judgment of the assessor, with disclosure and justification included in the report under LCCC provisions. Similarly, S590 allows HVAC assessment to rely primarily on visual and olfactory observations, and microbial sampling is not required unless specified in the project scope or if visible/suspected contamination is observed. In all cases, this flexibility may inadvertently allow circumvention of independently verifiable environmental data, which can compromise transparency, especially in contested or litigious settings. In all cases, this permissiveness while convenient, enables the circumvention of independently verifiable data.

4. Risk of biased or incomplete assessment

When quantitative testing is not performed, mould investigations and clearance decisions can potentially be steered to align with the interests of those funding the work- typically insurers, builders, landlords, or property managers or other key stakeholders with financial involvement for repair or remediation. This creates the potential for selective reporting, under-scoping, and even sampling fraud. In insurance disputes or health-related litigation, the absence of credible, independent data severely weakens the chain of evidence.

5. Defending proven methods in remediation science

While particle counting, molecular and biochemical tools such as PCR, qPCR, and metabolite assays may supplement investigations, they must not displace foundational tools such as spore traps, tape lifts, and viable CFU analysis for mould testing. These validated approaches are well-understood, reproducible, and defensible across stakeholder contexts. Any standard that permits these to be bypassed through discretionary language fails to uphold scientific integrity and introduces risk in contested or high-stakes settings.

This comprehensive review reaffirms the 1000 spores/m³ (or CFU/m³) threshold as a critical benchmark for assessing indoor mould contamination. Additionally, an I/O ratio exceeding 2:1 or a shift in the expected species distribution is a strong indicator of indoor fungal amplification, even if absolute concentrations are below the 1000 threshold.

As water damage becomes more frequent due to climate extremes, structural flaws, inadequate ventilation, substandard construction, aging infrastructure, and deferred maintenance, the need for science-based, objective standards is more urgent than ever to safeguard public health and ensure accountable remediation.

Without validated thresholds, post-water-damage evaluations become inconsistent, risking the misclassification of buildings. Pre-remediation quantification enables targeted and proportionate interventions, while reliance on visual inspection alone fails to detect hidden microbial reservoirs or quantify airborne exposure -placing vulnerable occupants at risk. The 1000 spores/m³ benchmark anchors assessments in measurable, reproducible, and unbiased criteria, moving remediation and clearance decisions beyond subjective interpretation.

Studies confirm that affected environments frequently exceed this threshold, while unaffected environments remain below it. Regulatory frameworks such as Malaysia’s Industry Code of Practice (ICOP) and France’s ANSES guidelines exemplify how the threshold has transitioned from academic discourse to actionable public health policy. Despite this, standards like the ANSI/IICRC S520/S590 often underrepresent the role of microbial exposure assessment, instead privileging structural evaluation, containment, and moisture control protocols. As highlighted by Haverinen-Shaughnessy et al.,²⁵ remediation efforts that appear structurally sound do not always correspond with reductions in microbial loads or health risk, reinforcing the need for objective post-remediation sampling.

In response to the problems we commonly see when there is an absence of objective data in mould cases, Gans, Palmer, and Roberts in Uniform Evidence (2025) make clear that the legal system depends on evidence that can be independently tested and verified. They stress that courts should be cautious when dealing with expert evidence that lacks a solid factual basis or relies too heavily on professional opinion without supporting data. This reinforces the need for objective, measurable information such as spore counts and surface sampling in disputes involving mould contamination. In legal settings, especially where competing interests exist, such data provides what science refers to as ground truth: a fixed, observable reference point that can be used to validate claims and test competing narratives. Without this, decisions may rest on subjective judgment, which can be influenced by bias, oversight, or conflicting interests. Using quantifiable environmental data like the 1000 spores/m³ threshold not only establishes scientific credibility but also ensures legal robustness, allowing courts to anchor their conclusions in verifiable fact rather than speculation.²⁶

Recent findings by Foreman et al.,²⁷ further demonstrate that, even when knowledge of mould-related health risks and protective practices is widespread, actual implementation of remediation best practices and sampling remains inconsistent. This illustrates a persistent gap between policy awareness and practical compliance one that is exacerbated by standards that frame objective sampling as optional rather than mandatory.

In light of these findings, the 1000 spores/m³ threshold remains a vital trigger for determining levels of mould exposure and for guiding remediation scope, informing risk communication, and enabling regulatory enforcement. Its preservation in global guidance documents is essential. Future revisions of mould assessment standards must move beyond permissive language and incorporate enforceable, objective metrics that ensure accountability, scientific integrity, and the protection of public health across diverse indoor environments.

Figure 1 summarizes this review confirming the ongoing relevance of the 1000 CFU/m³ threshold in identifying environments of potential health concern.

The interpretation of airborne fungal load remains an evolving field. Future research should prioritize standardized reporting that integrates both spore concentration thresholds and ecological indicators such as species dominance, indoor/outdoor (I/O) ratios, and fungal community diversity. This is particularly important as interpretation often hinges on both the absolute burden and the shift in airborne fungal ecology. There is a pressing need for regionally harmonized, enforceable guidelines to move beyond superficial or visual-only assessments and to support routine air and surface sampling especially in sensitive environments such as hospitals, aged care facilities, water-damaged residences, and disability accommodation.

Importantly, this need extends beyond acute clinical or high-dependency settings. Residential tenancy agreements, commercial leases, and NDIS-type (National Disability Insurance Scheme) community-care models including those housing people with intellectual or physical disabilities require similar recognition of mould risk. These populations are often unable to self-advocate or modify their living environments and may experience prolonged exposure to substandard indoor conditions without appropriate intervention.

As highlighted by Baker et al.,^28,29 the prevailing “good housing paradigm” in Australia has led to an under-recognition of health disparities arising from poor-quality housing. A significant policy-relevant cohort lives in dwellings with structural and environmental deficiencies that may be directly associated with respiratory and chronic illness, including dampness and mould exposure. The health effects of such exposures remain systematically under-addressed by current housing regulations.

Indeed, Brooks et al.,³⁰ found in a scoping review that the presence of damp and mould in homes is strongly associated with negative psychological outcomes, including stress, shame, fear about health, and embarrassment related to visible mould or odours. Most studies reviewed found a significant relationship between mould and mental health, suggesting that environmental stressors like mould exposure contribute meaningfully to psychological distress, especially in socioeconomically disadvantaged groups.

Going further, Clair et al.,³¹ provides evidence that challenging housing circumstances such as private renting, housing arrears, and exposure to environmental pollutants are associated with accelerated biological ageing, as measured by DNA methylation. Their findings suggest that housing stress contributes to physiological decline via epigenetic mechanisms, with private renting showing a stronger biological ageing effect than unemployment or smoking. Crucially, this biological ageing is not fixed, it is reversible. This presents an enormous public health opportunity: improving housing quality may restore biological resilience.

To protect vulnerable populations, regulatory bodies should adopt tiered frameworks that consider building use, ventilation type (mechanical vs natural), occupant vulnerability, and the potential for microbial amplification indoors. Future policy and research must be oriented towards prevention through proactive environmental testing, public health education, and remediation mandates anchored in evidence-based fungal thresholds.

The authors would like to thank the anonymous reviewer for their thoughtful and constructive suggestions, which have significantly improved the clarity, depth, and overall quality of this final version.

The authors declare that there are no conflicts of interest.

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