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Mold Growth on Imported Products

Mold growth on products is a major problem facing many manufacturers, vendor suppliers, warehouses, wholesalers, distributors, and retailers. Millions of dollars are lost every year to mold growth. The problem is that products of organic nature such as clothes, leather products, wooden products and many others are subject to damp conditions while in transit from overseas manufacturers.

Mold on Wooden Box
Mold on Wooden Box

The conditions during transit can cycle between hot and cold during the day and night respectively. During the day the high temperatures allow the air inside the container to absolve a lot of moisture from the sea and from the products and wooden pallets. As the temperature cools at night, this moisture condenses on the surface of the products.

Since the products often take several weeks (typically six to eight) to reach their destination the long duration in transit and the cycling between hot humid conditions and cooler conditions provide an ideal environment for mold growth. Products may also develop mold at their destination if stored in damp warehouses.

Products Prone to Mold Growth

Although mold will grow on a wide range of products including wooden pallets; leather products, textile, food products and paper goods are especially prone to mold growth. This is because these materials not only readily absolve the moisture but they are also a ready source of nutrients for mold growth.

Common Types of Mold on Imported Products

mold on shoes
Mold Growth on Shoes

The damp conditions in shipping containers generally favor the growth of those types of mold that do not require a lot of moisture to grow. These molds, referred to as xerophilic fungi, grow and thrive under conditions of low water activity (Aw). Depending on the material, a wide range of molds may grow. In our lab the most common types of molds we isolate from imported products, especially leather and textile products include Aspergillus restrictus, Aspergillus versicolor, Aspergillus sydowii, Eurotium herbariorum (Aspergillus glaucus), species of Penicillium and many others. On wooden products you may find species of Cladosporium, Aspergillus and Penicillium.

Control of Mold Growth on Products

Mold growth on products can be prevented by controlling moisture and eliminating practices that contribute directly or indirectly to mold growth. Such practices are:

1. Use of contaminated raw materials;
2. Improper storage of raw materials and finished products in warehouses
3. Improper packaging and shipping of the raw materials and/or finished products
4. Faulty manufacturing processes that facilitate mold growth.

Determining if the Moldy Products can be Salvaged

While some mold may be visible on a product, it does not necessarily mean that the product has been damaged. Most products can be cleaned and restored to original condition. Mold & Bacteria Consulting Laboratories (MBL) can help you in the risk assessment and mitigation process by definitively determining the type of mold responsible for the damage, supporting the formulation of a clean-up plan, and developing testing criteria to determine safe-for-sale. MBL has a team of microbiologists with expertise on testing biological contaminants on imported products, evaluating clean-up strategies and assessing the suitability of the cleaned product for sale.

Water Activity Requirements For Mold Growth

Definition of Water Activity

Water activity (aw) refers to “free” water available for microbial growth. For microbial growth to occur, moisture must be freely available. The water activity of a material determines which types of mold would grow on that material.

Technically, the water activity is defined as the ratio of the vapour pressure exerted by the water in the material to the vapour pressure of pure water at the same temperature and pressure.

Mold growth due to high water activity
Mold growth due to high water activity

Types of Mold based on Water Activity

Indoor molds vary in their water activity requirements and this ranges from 0.7 to >0.9. Higher aw materials tend to support the growth of more microorganisms. Unlike molds, bacteria usually require a water activity of at least 0.91 aw. Molds can be grouped according to their moisture requirements as follows:

  • Extremely to moderately xerophilic. These molds are the primary colonisers and they are capable of growth below 0.8 aw. Examples include Aspergillus and Penicillium
  • Slightly xerophilic. These molds are the secondary or intermediate colonisers. They are capable of growth between 0.8 and 0.9 aw. Examples of molds in this category include Altemaria and Cladosporium
  • Hydrophilic. Molds in this category are tertiary colonisers and require at least 0.9 aw for growth. Such high water activity can only be achieved through water intrusion and to a lesser extent high humidity and condensation. Examples of hydrophilic molds include Stachybotrys and Chaetomium

Note:

  1. Xerophilic means “dry loving.” Thus, xerophilic molds are those that can or prefer to grow in “dry” environments. “Dry” is used here in relative sense since no mold can grow in a completely dry environment.
  2. Hydrophilic means “water loving.” Thus, hydrophilic molds are those molds that require very high levels of moisture to grow.

Indicators of Water Damage

Water damage may occur over many months, mainly through roof leakage, but also via rising damp and defective plumbing, which result in mold growth. Indoor molds as well as bacteria are usually saprophytic, meaning that they obtain nutrients from dead organic matter. The nutrients are from the breakdown of simple to complex sugars such as starches, cellulose and pectin. The materials most susceptible to mold growth are organic materials containing cellulose (i.e. jute, wallpaper, cardboard and wooden materials). The tertiary colonisers are used as the indicators of moisture damage. The table below shows some of the most common indicators of water damage in buildings.

Indicators of excessive moisture or chronic condensation

Molds indicative of moisture or chronic condensation in a building
Alternaria alternata Phialophora sp
Aspergillus fumigatus Fusarium sp.
Chaetomium spp Ulocladium sp.
Trichoderma (some species) Yeasts (Rhodotorula spp.)
Exophiala sp. Memnoniella echinata
Stachybotrys chartarum
(synonym S. atra)

What is Aspergillus fumigatus?

Aspergillus fumigatus is a thermotolerant/thermophilic fungus capable of growing over a wide temperature range (12°C–53°C). It has a worldwide distribution. Being thermotolerant, it is a significant component of compost microflora.

Health effects associated with Aspergillus fumigatus

The spores of Aspergillus fumigatus are very small. Having a diameter of 2–3.5 µm, they are easily inhaled deep into the lungs. It is estimated that people inhale at least several hundreds of spores of Aspergillus fumigatus per day without harm. However, Aspergillus fumigatus is an opportunist pathogen (Hazard Risk Group 2) and can cause a lung infection (aspergillosis), in people with weak immune system. Most of the cases of aspergillosis are caused by this fungus. Because of its potential role as an opportunist pathogen, there is concern about high concentrations of Aspergillus fumigatus spores in the vicinity of hospitals, where those with compromised immune system such as organ transplant and cancer patients, may be at increased risk of infection. Therefore, monitoring for airborne spores in hospitals especially during renovations is a good practice.

Aspergillus fumigatus
Aspergillus fumigatus

Occurrence of Aspergillus fumigatus

Aspergillus fumigatus naturally occurs in decaying organic material, but because it is a thermotolerant fungus it grows well at raised temperatures experienced during the composting process. Within the indoor environment, A. fumigatus belongs to the group of indicator microorganisms typical of moisture-damaged buildings such Stachybotrys, Chaetomium, Fusarium and Ulocladium. The following characteristics make A. fumigatus a ubiquitous opportunistic pathogen: 1) ability to survive and grow in a wide range of environmental conditions, 2) effective dispersal in the air, 3) physical characteristics that allow spores to reach deep into the respiratory system, and 4) ability to swiftly adapt to the host environment.

Air Sampling for Aspergillus fumigatus

In hospital environment, air sampling may be conducted to monitor air quality during construction, to verify filter efficiency, or to commission new space prior to occupancy. Because aspergillosis cases have occurred when airborne fungal spore concentrations ranged as low as 0.9–2.2 colony-forming units per cubic meter (CFU/m3) of air, it is suggested that an air volume of at least 1000 L (1 m3) should be considered when sampling highly filtered areas.

Sampling media and incubation temperature for Aspergillus fumigatus

Malt extract agar (MEA) can be used to sample for Aspergillus fumigatus. For selective isolation of A. fumigatus, a high incubation temperature of 37 to 40 is then used as this inhibits growth of other saprophytic fungi.

References

  1. Kwon-Chung, K. J., & Sugui, J. A., 2013. Aspergillus fumigatus—What Makes the Species a Ubiquitous Human Fungal Pathogen? PLoS Pathogens, 9(12), e1003743. http://doi.org/10.1371/journal.ppat.1003743
  2. Sehulster, L. et al., 2003. Guidelines for environmental infection control in health-care facilities. Morbidity and Mortality Weekly Report Recommendations and Reports RR, 52(10).
  3. Swan, J. et al., 2003. Occupational and environmental exposure to bioaerosols from composts and potential health effects: a critical review of published data, HSE Books.
  4. Streifel, A. et al., 1983. Aspergillus fumigatus and other thermotolerant fungi generated by hospital building demolition. Applied and environmental microbiology, 46(2), pp.375–378.

How to improve spore trap results

Spore trap data should be reliable if results are to be used in assessing indoor spore types and concentrations and ultimately to evaluate the validity of exposure potential or remedial success.  Spore trap results are primarily used in indoor mold investigations to:

  • assess the airborne spore concentrations and the potential for human mold exposure
  • justify remedial recommendations and implementation
  • evaluate quality control and post-remedial assessments of mold remediation projects.
spore trap results
Non-viable Air Sampling

In an earlier article, Understanding Spore Trap Results, we discussed the factors that influence the reliability of spore trap results. These factors include:

  • The amount of air sampled
  • The non-uniform distribution of spores in the air
  • Related to the non-uniform distribution of spores in the air, is the distance from the source to the air sampling point. For actively growing mold, the spore concentration is higher closer to the moldy structures/materials
  • Changes in fungal spore types and concentrations within the day. The release of spores from moldy materials is not continuous and is dependent on ambient conditions such as temperature, air movement and relative humidity (rh). Ambient conditions also influence the types of spores released. For example, at very high humidity  fewer spores of Aspergillus/Penicillium are released than at low rh
  • The experience of the analyst.

How to improve spore trap results

Bearing in mind the factors that influence the reliability of spore trap results, how can we then improve the spore trap results? Below are some suggestions on how we could minimize the effect of the above factors. Professional judgement is required as some of the some of the suggestions may not be applicable in every situation. Since most of the suggestions involve increasing the number of samples collected, it is important to evaluate whether the added cost and sampling time are worth it.

Amount of air sampled

By increasing the amount of air sampled we can improve the spore trap results. As was discussed in article, Understanding Spore Trap Results, by collecting 75L of air we’re sampling only 7.5% of 1000L, the ideal amount of air that should be sampled. Unfortunately increasing the amount of air especially in a dusty environment, would lender the spore traps difficult if not impossible to analyze. We therefore recommend collecting at least 150L or more whenever the environmental conditions allow. If sampling a dust free environment, collecting more than 150L would be recommended. If using the 15L per minute pumps, it would take slightly more than an hour to collect a sample which, to some extent, takes care of the changes of spore concentration with time.

Non-uniform distribution of spores in the air

Since the spores are not uniformly distributed in the air, the only way to minimize the effect of this factor is to take more than 1 sample at different locations within the same room. For example if collecting air samples in a room, you could take a sample from each corner of the room and one in the middle, that means a total of 5 samples. If dealing with huge open spaces, the number of samples to collect would be more. Also, if dealing with very tiny rooms, you could collect only 2 samples.

The spatial and temporal variability of spore concentration in the air

The spatial (relating to space) and temporal (relating to time) variability of spore concentration in the air can be minimized by taking samples at different locations and at different times within the day. For examples we can decide to take some samples at same locations early in the morning, at noon and in the evening.

Conclusion

The important thing is first to define the purpose for spore trap air sampling. Then design a sampling strategy that is practical and that would give you data good enough for your defined purpose.

References

Michael Oberle, Markus Reichmuth, Reto Laffer, Cornelia Ottiger, Hans Fankhauser and Thomas Bregenzer (2015). Non-Seasonal Variation of Airborne Aspergillus Spore Concentration in a Hospital Building. Int. J. Environ. Res. Public Health 2015, 12, 13730-13738.

Robertson, L.D and Robert Brandys (2010). A multi-laboratory comparative study of spore trap analyses. Mycologia January/February 2011 vol. 103 no. 1 226-231.

Hyvärinen A; Vahteristo M; Meklin T; Jantunen M; Nevalainen A; Moschandreas D (2001). Temporal and spatial variation of fungal concentrations in indoor air. Aerosol Science and Technology 35, 688 – 695.

Understanding Spore Trap Analyses and Results

When enumerating airborne fungal spores, most labs, for practical reasons, analyze a percentage of the sample trace (often 15-25%) instead of 100%. A few labs claim that they analyze 100% of the sample trace. Obviously analyzing 100% of the sample trace sounds better than analyzing say 15 or 25%. In fact, some people argue that analyzing less than 100% of the sample trace is incorrect, inaccurate and gives skewed results. However, it is important to note that the accuracy of spore traps is affected by so many factors such that the contribution to inaccuracy/accuracy by the percentage of the sample trace analyzed in the lab is highly likely insignificant if at least 15% of the sample is analyzed. Even if 100% of the sample trace is analyzed, the results of spore traps would still be skewed in one way or another. Perhaps the most important of these factors is the sample volume collected. The volume of air collected for analysis is in most cases 75L or 150L. However, the final spore count is reported as the number of spores per cubic meter of air. That means ideally we should collect and analyze 1000L of air because by collecting 75 or 150L of air, the samples are only 7.5% and 15% respectively of what should have been analyzed. Unfortunately the spore traps (Air-O-Cell, Allergenco, etc.,) are not designed to collect large volumes of air especially in a dusty environment. In a dusty environment spore traps get overloaded with dust making it difficult for the analyst to see the spores.

The other factor that skews results is the fact that fungal spores are not uniformly distributed in the air.  That means if the spores are not uniformly distributed in the air, even the position and the height at which we place the sampler would skew the results.

Another factor that skew results is the fact that the spore concentration in a building varies not only with the season but also with the day and time of the day. In one study it was found that the spores of Aspergillus fumigatus can increase temporary to a significantly high concentration and then decline to “normal” level within 30 min only. This means the 5 or 10 minutes samples that are collected using spore traps are only a measure of spore concentrations at the time of sampling.

Spore trap- notice the non-unform distribution of Asp/Pen spores
Asp/Pen- notice the non-uniform distribution of spores

Is Analyzing 25% of Spore Trap Sample Trace Incorrect and Inaccurate?

The simple answer to this question is NO. First, spore traps are not meant to determine the actual spore count in the air. They are meant to give an idea of the types of spores present in the air and the concentrations of these spores relative to each other. Analyzing spore traps by direct microscopy is a complex process and there are limitations. Depending on the experience of the analyst there are counting and identification errors. Because of eye fatigue, counting errors are likely to be higher for a sample trace analyzed at 100% than one analyzed at 25%.

At MBL we use the ASTM Standard “Standard Test Method for Categorization and Quantification of Airborne Fungal Structures in an Inertial Impaction Sample by Optical Microscopy (Designation: D 7391 – 17 e1)“. This is the only standard currently available for spore trap analysis. The standard does not specify the minimum percentage of the sample trace to count and for good reasons. Practically, not every sample can be analyzed at 100%. The density of particulates (spores and other particulates) on the sample trace and the size of particulates determine how easy and fast the sample can be analyzed. Analyzing 100% of a highly loaded sample is not only difficult but could take one to several hours and will have little or no added value to the results because counting errors will be made during the analysis.

When Is It Possible to Analyze 100% of Sample Trace

The only time it is practically possible to analyze 100% of sample trace is when you have relatively few scattered spores and other particulates on the slide. With heavily loaded samples it’s very difficult for the analyst to keep track of what they have and haven’t counted under the microscope. As mentioned earlier, the analyst is likely to be fatigued and count many spores twice or miss counting some of them altogether. Compare the 2 images below which are showing spores and other particulates in just a single field of view of a microscope. Again, even for samples with low particle density, does analyzing 100% of sample trace add any value to the results? We would like to hear from you…

Spore trap- mixture of spores
Heavily loaded sample with tiny spores- Difficult to count 100%
Spore trap- Pithomyces spores
Big scattered spores- Easy to count 100%

Fungal Spore Identification Course

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