Experimental evaluation of the growth rate of mould on finishes for indoor housing environments: Effects of the 2002/91/EC directive (original) (raw)

Experimental evaluation of the growth rate of mould on finishes for indoor housing environments: Effects of the 2002/91/EC directive

Marco D’Orazio a, { }^{\text {a, }}, Marco Palladini a { }^{\text {a }}, Lucia Aquilanti b { }^{\text {b }}, Francesca Clementi b { }^{\text {b }}
a{ }^{a} Department of Architecture Construction and Structures - DACS, Università Politecnica delle Marche, Via Brecce Bianche, 60131 Ancona, Italy
b{ }^{\mathrm{b}} Department of Food Science, Agricultural-Engineering, Physics, Agricultural Economics and Landscape Science - SAIFET, Università Politecnica delle Marche, Via Brecce Bianche, 60131 Ancona, Italy

A R T I C L E I N F O

Article history:
Received 15 May 2008
Received in revised form
3 November 2008
Accepted 5 November 2008

Keywords:
Mould
Plaster
Paint
Biocontamination

A B S T R A C T

We report the results of a study to evaluate the growth rate of three species of mould on plasters, finishes and paints typically used in structures with heavy weight building envelopes. The aim was to determine the influence of the chemical composition (in terms of organic fraction of the materials) on the growth rate of moulds. The study was carried out in the following steps: - characterization of materials; inoculation of mould spores ( 3 species) on 7 types of material ( 2 plasters, 3 finishes, 2 paints); - growth in a climatic chamber (23∘C\left(23^{\circ} \mathrm{C}\right. and 90%RH90 \% \mathrm{RH} ); - analysis of the mould growth rate using various experimental techniques (fluorescence microscopy analysis, thermogravimetric analysis, etc.). Results show a clear correlation between the organic substances contained in paints, plasters and finishes and the growth rate of the mould.
This study is part of a more general research program which addresses the effects on indoor environment air quality based on the European directive 2002/91/EC. This directive specifically indicates that energy consumption in buildings should be limited and sets threshold values for the thermal resistance of the building walls and windows. As a consequence window manufacturers are improving the thermal property of windows by reducing the air permeability, which may increase the indoor and surface relative humidity percentage (RH%) and lead to the development of mould in the indoor environments.
© 2008 Elsevier Ltd. All rights reserved.

1. Introduction

The tendency in industrialized countries to reduce energy consumption has been accelerated in the EC member states by the introduction of the 2002/91/EC directive [1].

In fact, this directive fixed the minimum requirements which must be respected when building new structures and when renovating existing buildings so as to have a more effective reduction in energy consumption for the air-conditioning of indoor environments.

This strategy has, however, led to some undesired effects. Windows manufacturers are currently producing systems with low permeability (class A4 UNI EN 12207, 2000) in order to obtain better thermal performance.

A reduction in permeability, in buildings which are already characterized by a limited amount of air exchange, can increase the indoor and surface RH%\mathrm{RH} \% in winter [16]. Consequently there may be an increased growth of mould species on some parts of the walls.

Numerous studies have been conducted in recent years to address the environmental aspects that favour the growth and the

[1]subsequent sporulation of fungi species. Rousseau [2] and Hud [3], indicate the following factors: oxygen availability; a suitable temperature range; a substrate of deposit that acts as nourishment; and a certain degree of humidity [4,34,36][4,34,36]. Krus et al. [5] have studied these conditions, together with a series of other specific factors that can influence the growth of fungi: pH value and the roughness of the substrate on which the mould grows, the light, the biotic interaction and the exposure time. Baughman and Arens [6] have underlined that, although moulds can grow at temperatures between 0∘C0^{\circ} \mathrm{C} and 40∘C40^{\circ} \mathrm{C}, a range going from 22∘C22^{\circ} \mathrm{C} to 35∘C35^{\circ} \mathrm{C} can be considered optimal for the species that are most frequently found inside buildings. On the contrary, the level of airborne spores in the indoor environment, which is one of the conditions able to influence the development of moulds, is dependent on seasonal changes in the external environment [7]. Adan [7] has verified that there are a considerable number of buildings that offer favourable temperature conditions for the germination and the growth of mould on construction material and indoor environment finishes.

Sedlbauer [8-11] has recently completed an important research and development study of models which are able to estimate mould growth inside buildings, classifying the various materials.

These different authors appear to be in agreement, however, in indicating that different kinds of fungi require minimum levels of

1. • Corresponding author. Tel.: +39 712204587; fax: +39 712204783.
     E-mail address: m.dorazio@univpm.it (M. D’Orazio). ↩︎

humidity in the support surface material in order to proliferate and these values vary for different species [6], [12], [35]. Ayerst [12] has conducted experiments in order to characterize the ideal conditions and the minimum values of humidity required in the substrate for mildew formation. These experiments indicate a range between 71%71 \% and 94%94 \%, according to the fungi species.

Moulds (typically present on construction materials) can be separated into three groups on the basis of their water activity ( awa_{w} ) [13]: primer colonizer or storage moulds, capable of growing at aw<0.8a_{w}<0.8; secondary colonizer or phyllophane fungi, requiring a minimum awa_{w} between 0.8 and 0.9 ; and tertiary colonizer or water-damage moulds, needing aw>0.9a_{w}>0.9. The last class includes the most toxic mould species for human health.

Although each species has a preferential humidity for growth, the International Energy Agency [33] indicates an average RH% of 80%80 \% as the critical threshold for mould development. A reduction in moisture content in building materials to below 80%RH%80 \% \mathrm{RH} \% is, therefore, the way to reduce mould growth.

Only a limited amount of information is available about the influence of the composition of the finish materials on the germination and development, in terms of percentage, of moulds.

Ritschkoff et al. [14] have conducted experiments on materials made of wood (tables in wood fibre and plywood), compound materials (gypsum and concrete panels) and insulation materials (rockwool and fibre glass) in various temperature and RH%\mathrm{RH} \% conditions. The results showed that all the materials in a building can contribute to the growth of moulds if the RH% reaches 90%90 \%. Shirakawa et al. [15] have conducted studies on gypsum panels.

Finally, on the basis of previous studies, Sedlbauer [8] has divided building construction materials into four classes according to the type of substrate with respect to the nourishment that the material is able to provide for the fungi.

Nevertheless this method of classification tends to excessively homogenize materials which have different types of binder and different quantities of organic substance content. The latter aspect is, however, fundamental in determining the very different growth rate for the mould on the surfaces.

The narrow number of specific studies on materials indicates the need to extend research to different materials, trying to establish a relationship between the organic substance content of the materials and the growth rate of the mould.

2. Steps, materials, methods

2.1. Steps

The study was conducted in 4 steps.
1 Characterization of 2 plasters, 3 finishes and 2 paints for indoor environments and subsequent experimental evaluation, in
a climatic room, of the growth rate of 3 fungi species on these materials;
2 evaluation, through analytical methods [UNI EN ISO 13788], of the increase in RH%\mathrm{RH} \% in different housing environments following the reduction in air permeability of the windows; these data were then compared with the vapour production classes indicated in the studies carried out as part of “annex 24” of the IEA [17];
3 evaluation by means of WUFI software of the temperature and water content conditions of various types of plasters and finishes for indoor environments following variations in environmental conditions;
4 application of the WUFI-BIO calculation model to estimate the probability of growth of some species on the analyzed support surfaces.

We report the results of the first research step which is preliminary to the subsequent modelling activities.

2.2. Materials

Materials were selected in order to contemplate the most common types of finishes in indoor housing environments. Table 1 shows the data regarding the composition of the materials used for the evaluation of growth in the different fungi species. We selected two types of plaster (A,B), three finishes (C,D,E) and two paints (F,G) so as to consider different types of support belonging to the classes indicated by Sedlbauer [8]. Plasters were selected in order to have materials with different porosity. Plaster type A is a commercial plaster (low-medium porosity) with a hydraulic binder (hydraulic lime), siliceous sands normally used in building’s interiors.

Plaster type B is a commercial plaster (high porosity) with a hydraulic binder (hydraulic lime), siliceous sands, perlite and additives useful to increase open porosity.

Finishes were selected in order to have sample with different binders. Type C is a finish with a hydraulic (pozzolanic) binder, type D is a finish made with a hydraulic binder (natural hydraulic lime). Type E is a finish made with pozzolanic cement as binder.

Paints were selected in order to have sample with different binders. Type F is a commercial paint named “tempera” with a mix of organic and inorganic binders. Type G is a commercial paint with a silossanic binder (Table 1).

The three different fungi species were chosen on the basis of their toxicity. The species analyzed are those that have the greatest probability of developing and regenerating in indoor housing environments. Specifically the following species were selected: Aspergillus versicolor; Penicillium chrysogenum; Stachybotrys chartarum.
A. versicolor is a species that belongs to the group indicated as “primer colonizer” [13] Fig. 1.

Table 1
Composition of support surfaces and classification according to Ref. [8].

Fig. 1. Aspergillus versicolor, sporulation.

For the germination of the spores a temperature between 8∘C8^{\circ} \mathrm{C} and 42∘C42^{\circ} \mathrm{C} is required, although 30∘C30^{\circ} \mathrm{C} is optimal, while for the growth of mycelia the temperature range is between 4∘C4^{\circ} \mathrm{C} and 40∘C40^{\circ} \mathrm{C} with an ideal temperature of 30∘C30^{\circ} \mathrm{C}. As regards the RH%\mathrm{RH} \% of the substrate, the lowest limits are 74%74 \% for germination and 75%75 \% for growth of mycelia, the ideal values being 91%91 \% and 94%94 \%, respectively [8].
A. versicolor is the species most commonly found inside buildings owing to its particular characteristic of surviving on material that is not a great source of nourishment, such as concrete or plaster.

This species is usually able to produce a great amount of potentially carcinogenic mycotoxins, sterigmatocystins [18,19], and may represent up to 1%1 \% of the biomass. On water-saturated materials, A. versicolor produces sterigmatocystin in quantities of up to 20μ g/cm220 \mu \mathrm{~g} / \mathrm{cm}^{2} [20]. Sterigmatocystin is not very cytotoxic in itself, but becomes carcinogenic after activation in the liver of the cytochrome P450 mono-oxidase. [21]. In addition to these toxic properties, sterigmatocystin also acts as a strong inhibitor of tracheal ciliary movement [22].
P. chrysogenum is a species which is also included in the primer colonizer group and, like A. versicolor, it is one of the most commonly found species in indoor environments (Fig. 2). For the development of mycelia a temperature between −4∘C-4^{\circ} \mathrm{C} and 38∘C38^{\circ} \mathrm{C} is required, the ideal value being 28∘C28^{\circ} \mathrm{C}.

Fig. 2. Penicillium chrysogenum, sporulation.

As regards the RH%\mathrm{RH} \% of the substrate, the lowest limits are 78%78 \% for germination and 79%79 \% for the development of mycelia, with optimal values of 79%79 \% and 98%98 \%, respectively [8].

Secalonic acid D is the only toxin generated by this species and it is not commonly found in indoor environments. Other products which derive from the metabolism of this species are more easily found, such as ω\omega-hydroxyemodine, pyrovoylaminobenzamides, chrysogine, meleagrin, and xanthocillin X [23].

On the basis of the studies carried out by Grant et al. [13], S. chartarum belongs to the “tertiary colonizer” group and is able to develop with am>0.9a_{m}>0.9 (Fig. 3). For spore germination a temperature between 5∘C5^{\circ} \mathrm{C} and 40∘C40^{\circ} \mathrm{C} is required, with an optimal value of 25∘C25^{\circ} \mathrm{C}, while for the growth of mycelia the temperature range is between 2∘C2^{\circ} \mathrm{C} and 37∘C37^{\circ} \mathrm{C}, with an ideal value of 23∘C23^{\circ} \mathrm{C}. As regards the relative humidity of the substrate, the lowest limits are 85%85 \% for germination and 89%89 \% for the growth of mycelia, the optimal values being 97%97 \% and 98%98 \%, respectively [8].
S. chartarum is one of the most toxic species that can be found inside buildings. Its name is often associated with IPH (Idiopathic pulmonary hemosiderosis) [24-26]. The high toxicity of this mould is caused by the release of biomass which contains higher quantities of secondary metabolites than other indoor moulds [27].

2.3. Methods

The characterization of the materials was carried out in order to obtain both compositional and hygrometric data. A predisposition for mould growth is consequent to the quantity of nourishment available and to adsorption/desorption phenomena. This study was also extended so as to have data to apply during the second phase using WUFI and WUFI-BIO software.

Concise information about composition was obtained by determining the loss in mass in the specimens after exposure to a temperature of 200∘C200^{\circ} \mathrm{C} in broth and by carrying out analysis in FT-IR and XRD. The specimens were pulverised, placed in crucibles and weighed progressively until constant mass was reached. At a temperature of 200∘C200^{\circ} \mathrm{C}, these materials lose all the unbound water and the organic substance content.

Having preliminarily desiccated the materials at 105∘C105^{\circ} \mathrm{C} so as to attain constant mass, it was possible to determine the weight loss connected with the loss of organic substances.

The hygrometric information was obtained in different ways according to the various types of materials. For plasters, mercury porosimetric analyses were carried out by means of Micromeritics Autopore II [28], measurements of vapour permeability according

Fig. 3. Stachybotrys chartarum, sporulation.

to [ASTM, E-98-2000] [29] and measurements of capillary absorption.

The specimens in the porosimetric analyses, 3 for each type of support surface, were dessicated and placed in a penetrometer for analyses at low and high pressures. On the basis of these analyses, it was possible to obtain the distribution of pore dimension, which is useful for finding information about the hygrometric behaviour of the specimens.

The vapour permeability measurements were conducted in a climatic room according to ASTM E-98-2000. In order to perform these measurements aluminium glasses containing deionised water were used. After placing the specimens on the containers and sealing them, the weight variation was measured in a climatic room set up at 23∘C23^{\circ} \mathrm{C} and 50%RH%50 \% \mathrm{RH} \%. The specimens were weighed at intervals of 24 h until a constant loss of mass was reached.

Capillary absorption measurements were conducted according to [UNI EN ISO 15148, 2003] [30]. After being placed in a dryer with silica gel, the specimens were placed on Wathmann filter paper n∘5n^{\circ} 5 in a dish containing deionised water in an environment with constant temperature and RH%\mathrm{RH} \%. Using an analytical balance the variation in weight was then determined at successive instants for an overall period of 8 h . The same tests performed on the plasters were also carried out on the finishes, with the exception of the porosimetric and vapour permeability measurements.

For the paints, vapour permeability data were taken from the certification data supplied by the manufacturers.

The recommendations contained in American regulations ASTM D 3273-2000 [31] were used to assess the susceptibility of the various substrates to mould growth. These test protocols describe a method for testing resistance to mould development on indoor surface using a climatic room (Fig. 4). More specifically, the guidelines indicated by Shirakawa et al. [15] were followed since these authors have carried out similar experiments on gypsum specimens.

The cultures used to determine the susceptibility to the mould growth of the various types of support surface were supplied by the DSMZ (Deutsche Samlung von Mikroorganismen und ZellKulturen, Braunschweig, Germany.). The first two were supplied as freezedried and it was, therefore, necessary to perform a preliminary subculturing in Malt Extract Broth (MEB, Oxoid), following the procedure indicated by Magnusson and Schnurer [32].

The revitalized cultures were then inoculated in Malt Extract Agar (MEA) slants, prepared as follows: 8 ml of MEA (agar-added

Fig. 4. Assessment of mould formation susceptibility on different types of plasters, finishes and paints - incubation of the specimens with the fungi culture in the climatic room.

MEB, 18 g L−118 \mathrm{~g} \mathrm{~L}^{-1} ), previously sterilized for 15 min in autoclave at 121∘C121^{\circ} \mathrm{C}, were dosed in sterile glass test tubes with a diameter of 22 mm . In the cooling phase, the tubes were tilted, so as to obtain a larger inoculation surface.
S. chartarum DSM12880 was supplied as an active culture in MEA. For this species, therefore, only a refreshment phase was necessary.

In both cases, the tubes were incubated at 25∘C25^{\circ} \mathrm{C} for 10 days to allow sporulation, and the consequent production of conidia.

On the 10th day, the conidia were collected in sterile peptone water (distilled water with the addition of 0.1%0.1 \% peptone), and were then inoculated onto the support surfaces of the materials being tested.

Five millilitres of peptone solution were inoculated in each of the three tubes containing moulds to test. The separation of conidia from hypha was carried out by shaking the tubes vigorously. Five microliters of the peptone solution containing the conidia were collected and subsequently placed on a Thoma counting chamber in order to count the spores. The concentration of the conidia was determined with optical microscopy carried out using a 40×40 \times enlargement. After counting, the suspensions were diluted in sterile peptone water, in order to reach a final concentration of 10610^{6} conidia mL−1\mathrm{mL}^{-1}.

Support surfaces were made as follows. For the two types of plasters the procedure indicated by Shirakawa et al. [15] was followed. Plaster powder was mixed with water. After 28 days plasters were dried at 150∘C(18 h)150^{\circ} \mathrm{C}(18 \mathrm{~h}) and then mixed with sterile water ( 0.8 parts water for each part powder) and transferred to Petri caps. The abovementioned procedure was also followed for the three types of finishes. The different types of paint were mixed with sterile water in the due proportions and then poured into Petri caps in order to form a thin layer of paint (Fig. 5).

Two hundred microliters of each spore suspension, prepared as previously described, were inoculated onto the surface of the various test materials. All the inoculated caps were then incubated in a climatic room which can guarantee constant environmental conditions of 25∘C25^{\circ} \mathrm{C} temperature and 95%RH%95 \% \mathrm{RH} \%. All the tests were carried out as double trials.

After an incubation period of two weeks in the climatic room the development of moulds on the different materials tested was assessed. In addition to a naked eye comparison, a laser fluorescence microscope was used (Bio-Rad MRC 124, Nikon). In particular, a blue light with a wavelength of 488μ m488 \mu \mathrm{~m} was used to reveal the presence of the moulds which had developed on the surface of the specimens under study.

Fig. 5. Mould formation over the film of paint - specimen F (Stachybotrys chartarum).

3. Results

3.1. Preliminary characterization

Table 2 shows the overall results of the characterization activities carried out on the support surfaces used in the experimentation. The first remarkable finding concerns the capillary absorption of the various support surfaces. ama_{m}-Values for plasters and finishes are very low. This indicates the presence inside these materials of additives which reduce the absorption of water by the support surface. This is confirmed by the results obtained with the mass loss tests performed on the analyzed materials. As expected, low values of capillary absorption were found in the paints. This finding appears to be coherent with the measurements of mass loss. Finally, as expected, the data obtained through porosimetric analyses conducted on the plasters underline the substantial differences between plasters and finishes. The former show high porosity between 35 and 39%, typical of products with lightened inert. The latter have a much more limited porosity which is typical of the products belonging to this category.

3.2. Growth rate of the mould

The results of observations are summarized in Table 3, which, for each type of support surface, shows the mould growth index for each specific fungi species inoculated on the specimen.

The mould growth index refers to the growth value attributed to any fungi species that has developed on a particular support surface in function of the percentage of surface covered [14].

As can be observed, the mould index found for the various types of support surfaces, independent of the type of fungi species, can be referred to visible classes only through use of the microscope. Only in one case (specimen F - tempera paint) the growth was visible to the naked eye. The differences in mould index with respect to the fungi species appear to be limited. As expected, S. chartarum is the species that appears to grow most quickly, since the temperature and RHS values provide an ideal climate for its development.

Fig. 6 shows a set of images of the fungi species on the various types of support surface obtained using the microscope.

The laser microscope used is able to identify the presence of microorganisms taking advantage of the property of these organisms to emit “in the green” at a wavelength of 520μ m520 \mu \mathrm{~m} when stimulated by a blue light with a wavelength of 488μ m488 \mu \mathrm{~m}. It must be underlined that a study of the images of specimen A shows that the development of the fungi species is rather limited, above all if we

Table 2
Characterization activity results. The symbol (-) indicates that the determination was not carried out on the specimens.

Table 3
Mould index and surface of area covered by moulds on the different types of support surface after exposure in the climatic room. The growth index is defined as follows: 0 = percent covered equal to 0%;1=0 \% ; 1= percent covered <1%;2=<1 \% ; 2= percent covered <5%<5 \%; 3 = percent covered <10%;4=<10 \% ; 4= percent covered <20%;5=<20 \% ; 5= percent covered <40%<40 \%; 6 = percent covered <60%;7=<60 \% ; 7= percent covered <80%;8=<80 \% ; 8= percent covered >80%>80 \%; and 9 = percent covered <100%<100 \%. Classes 1−51-5 are visible only with the microscope. Classes from 6 to 9 are visible to the naked eye.

consider that the images refer to the area in which the inoculation was performed where the initial concentration of spores was great. In this specimen the most developed species is P. chrysogenum, while S. chartarum is the species that had the most difficulty in adapting to the substrate.

The images of the B specimens show a more marked development of mould in comparison with the plaster A. A. versicolor had a less consistent development than the other two species. By comparing the two types of plaster it is possible to hypothesize that the latter has a higher content of organic nourishment than the former.

For finish C the development of A. versicolor was also very limited while in the zone inoculated with the species S. chartarum a clear and homogeneous growth can be seen over the whole area. The growth on finish D is similar for all three species. As in the previous cases, the growth of A. versicolor was less than that of P. chrysogenum and S. chartarum. The third image of the series is particularly interesting, since there is a clear borderline between the area in which the inoculation was performed and the non-infested area. The microscope images for support “E” show that the development of the three fungi species is very limited. It is possible to observe a more noticeable presence of A. versicolor and S. chartarum.

As far as the paints are concerned, the three images that refer to type F differ from the others because, unlike the previous ones, they were obtained using an optical microscope. This was possible because the development of the fungi species on the support was clearly visible even to the naked eye after only two weeks of incubation. The first two images were taken using a 5×5 \times objective, while the final image used 10×10 \times. The tempera paint proved to be the most “nourishing” support for the fungi species examined, since the growth is much greater, above all for S. chartarum and P. chrysogenum. For paint G, the laser microscope was used with 5×5 \times enlargement, as with the naked eye it was not possible to identify the presence of the mycelia. Significant results were obtained only for the first specie (A. versicolor) in which a “cobweb-like” development was observed corresponding to the inoculated area. For the other two fungi species it was not possible to identify the development of mycelia, since this type of support surface is not able to provide great quantities of organic substances for nourishment.

3.3. Correlation between the mould growth area and the content of organic substances

Fig. 7 shows the correlation between loss in mass at 200∘C200^{\circ} \mathrm{C} and percent of surface covered by mould. This calculation, which was carried out in order to identify the possible correlation between the rate of growth and the content of organic substances, allowed us to demonstrate that there is a noticeable linear correlation between rate of growth of the fungi species and the content of organic substances.

Fig. 6. Results of the analysis carried out with the fluorescence microscope, (5×)(5 \times).

Although there is a certain amount of data dispersion for the plasters and the finishes (characterized by a limited content of organic substances) it is clear that, in terms of ability to serve as nourishment for the fungi species, the type of substrate is the dominant factor for the development and growth of the species analyzed.

By observing the data divided by fungi species the following features can be noticed: the species S. chartarum on average had the greatest development on the various types of support surface used, followed by P. chrysogenum and A. versicolor. Of the species studied S. chartarum is also the most highly toxic for human health.

Fig. 7. Correlation between mass loss at 200∘C200^{\circ} \mathrm{C} and area covered by the fungi species after exposure in the climatic room.

It is interesting to notice that although the various types of plaster and finish belong to the same class of substrate (II) according to Sedlbauer’s classification [8], in reality there are sometimes quite remarkable differences in the results for the various substrates. This is also true for the paints.

4. Conclusions

This study has shown that:

• there is a clear correlation between the growth rate of the fungi species and the content of organic substances that the various types of support surfaces possess;
• we found very important differences regarding hygrometric properties of plasters, finish and paints analyzed, but they didn’t have effect on the mould growth rate;
• the most susceptible substrate for the development of mould is type F (tempera paint), since its particular composition allows it to provide great quantities of nourishment for the microorganisms that form colonies on the surface;
• substrates made with inorganic binders (finish and plasters) shown some remarkable differences in the results, although they belong to the same class of substrate (II) according to Sedlbauer’s classification;
• S. chartarum is the species that appears to grow most quickly, since the temperature and RHS values provide an ideal climate for its development. The risk involved is that the metabolic products of this species, which are mainly made up of toxins which are harmful for human health, may be dispersed in the air.

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