2.1 Characterization of Systems

Samples of standard gypsum boards were arranged for testing, which measured 75 x 100mm and were tested in accordance with ASTM D3273-16 (^{ASTM 2016}), with five coating types:

a) Water-based matte acrylic paint: three coats of water-based matte acrylic paint were applied over the gypsum boards with a soft bristle brush, with intervals of 24 hours between each coat. This paint was white-colored with 80% water dilution;

b) Smooth spackle: three coats of the product were applied with a plastic spatula, with intervals of 24 hours between coats;

c) Textured spackle: three coats of the product were applied with a texture foam paint roller, with intervals of 24 hours between coats;

d) Water-based epoxy paint: two coats with 10% water dilution were applied with a short pile wool paint roller under a 24-hour interval between coats;

e) Flexible waterproofing polymer: an asphaltic product was applied, which was diluted in water for priming. When its wet-dry state was reached (after approximate 30 minutes), five cross-coats of the waterproofing solution (product mixed with sieved CP-IV Portland cement in a 1:1 relation) were applied with a brush, with intervals of 90 minutes between coatings.

The application procedures and materials followed the specifications of its manufacturers.

2.2 Test for Resistance to Fungal Growth

The test for fungal growth resistance was based on ASTM D3273-16 (^{ASTM 2016}). The test cabinet comprises an isolated lid to minimize heat loss and the bars that support the apparatus are polymeric to avoid contamination. Under these bars there is a tray for storing the soil, which has a metallic mash at the bottom. The operation counted with an interface of temperature control that granted isothermal conditions within the limits determined by the standard. Lastly, its base has a polypropylene tank. The components are described as follows.

a) Soil: suitable for the propagation of plants, with 25% peat moss and pH range (5.5-7.0);

b) Fungi: presented in (Table 1)

Table 1 Fungi used in the test 

c) Test panels: among the types the standard allows, type II was selected minding panels of gypsum boards of 75 x 100 mm, with thickness ranging from 13 to 25 mm.

The sequence adopted was to deposit the soil with water presence, incubate the fungi for 14 days and then proceed with the morphological analysis to assess fungal growth. The test procedure undertook the following sequence:

a) The samples were arranged and conditioned at temperature of 23 ± 2°C and humidity of 50 ± 5% for four days before being deposited in the test cabinet;

b) Exposure: the samples were suspended vertically 75 mm above the surface of the soil deposited to allow free circulation of air, as depicted in (Figure 1).

Figure 1 Samples hanged over the test chamber 

c) Evaluation: The fungal growth samples was performed every week along ten weeks through a scale of rating that estimates the surface’s percentage of defacement ranging from 0 to 10, where 10 means no visual defacement and 0 total defacement. This scale is set by subtracting the percentage of tainted area from 100%. The procedures for measuring the damaged area were based on (^{Pacheco 2016}), with treatment by ArcMap 10.3 software program;

d) The evaluation of the damaged area relied on the use the ArcMap 10.3 software suite for analysis of images and georeferencing. The identification of various colored spots in the image allowed to distinguish the regions affected by fungi and was rendered by the aforementioned software suite, as depicted in (Figure 2).

Figure 2 Damaged sample and image analyzed by the ArcMap 10.3 software to identify damaged áreas 

e) Next, the area damaged by fungal growth was measured by the AutoCAD® software.

2.3 Microscopic Analysis

The microscopic analysis was performed after nine weeks of testing. The assessment was performed with the automated digital microscope model Smart Zoom 5, ZEISS, with magnifications ranging from 10x to 1010x, extended depth of field, high-resolution images, adjustable angles between -45° and +45°, and resolution of 1 μm. The samples analyzed were the most deteriorated to the naked eye.

2.4 Identification of Fungi in Culture

Two samples from each coating group were chosen based on the probability of fungi presence by spots visible to the naked eye. After, the material with fungi was scraped and cultured in petri plates on potato dextrose agar (PDA) medium (Figure 3ª), b at 28°C for 5 days.

The fungal strains were identified based on their reproductive structures already known and documented in specialized bibliography. Thus, a small fraction of these structures was treated with KOH 3M, stained with phloxine and covered with glass cover slip (Figure 3c).

After, the material was visualized under the microscope ZEISS Primo Star with the AxioCam ERc 5S connected, and the ZEN imaging software was set to the objective lens of 40x, generating a magnification of 400x.

Figure 3 Morphological analysis procedures 

The fungi developed reproductive structures after staying in the oven for 5 days, allowing the determination of the microorganism species. For that reason, plates were prepared with a small fraction of the material that was removed with a previously-flamed platinum inoculation loop and mixed to KOH 3M and phloxine coloring to assist the visualization.

This recognition was achieved through the comparison of reproductive structures found in the samples with structures already known and documented in bibliography. The microscope used for this recognition was the ZEISS Primo Star as described.

3.1 Visual analysis and software handling

The action of the fungi on the gypsum board for 4 and 10 weeks revealed not only visual defacement but also physical defacement from the dislocation of the cardboard within the gypsum board and cracking on some coverings. The use of metallic staples caused runoffs and rust stains on the edges of the samples, as well as on the lateral of one of the acrylic paint samples. Some of the images presented differences of reflections and shadows due to the local illumination, which were excluded from the measurements.

There were different ratings of degradation on the samples, as noted in (Table 2), wherein 10 is the best and 0 is the worst situation, according to above mentioned standard. During the 4th week only the smooth spackle and textured spackle samples had shown signs of defacement, whereas during the 10th week only the waterproofing polymer and epoxy paint samples had not undergone apparent damages from the attack of these microorganisms.

Table 2 Rating of samples (higher degree of staining) 

To emphasize visual aspect modifications, (Figure 4) presents the initial aspect measured during the 4th week and the final aspect during the 10th week for the most visually damaged samples.

Figure 4 Analysis for 4 and 10 weeks of the samples with varied coverings 

These results denote that the resistance to growth of fungi was higher for samples covered with waterproofing polymer and epoxy paint, since these coverings mitigate the superficial accumulation of water. The possibility of development of these microorganisms on these samples should not be discarded though, as it can still occur, albeit slowly. Regarding the analysis by the rating scale of standard, only smooth speckle sample 03 achieved rating below 9 during the 4th week, while only textured speckle sample 04 achieved rating above 8 during the 10th week, bearing in mind that two thirds of the samples did not reach rating 6.

(^{Silva 2011}) realizes that the boards that were exposed to a natural atmosphere had gotten contaminated by several fungi, among which appeared the ones used in this study. The author explains that the visual analysis is inaccurate, since many contaminations found in biologic tests had not been noticed in their analysis. (^{Gajaca & Brazolin 2012}) then complement by stating that physical modifications can occur without changing the color of the materials and their covering, which can be considered a false evidence of performance.

3.2 Microscopic analysis

The microscopic analysis revealed the presence of fungi on the samples (Figure 5), along with the change of material color. It also exposed the lack of visual evidences of the presence of microorganisms on epoxy paint sample 01, complying with the visual analysis, and evidences of contamination on the waterproofing polymer sample, contradicts the previous analysis, even though these conclusions still have to be confirmed with the morphological analysis.

For the rest of this analysis, the waterproofing polymer and epoxy paint samples was highly damaged when analyzed with the microscope, despite not having presented visual modifications. The layer of water maintained during the waterproofing polymer test may have affected its most external coat by generating intense physical damage even without the conditions for fungal development. This result indicates that the waterproofing material can reach higher critical values, as it did not reach moisture levels and temperature that would allow the fungi to grow (^{Johansson et al. 2013}). (^{Silva 2011}) also noted that maintaining a layer of water delayed and inhibited the attack by fungi in an outdoor area subjected to weather effects.

Figure 5 Images generated by microscopy 

3.3 Morphological analysis

The morphological analysis by microscope identified the fungi based on (^{Putzke & Putzke 2004}) and differentiated genus taxonomically in order to find the three types of fungi present in the test chamber, as specified in (Table 3).

Table 3 Fungi found in the samples 

(Figure 6) shows the images generated by this analysis, which depict the reproductive structures of three fungi identified by what is described in the literature and their taxonomic groups.

Figure 6 Images generated by the morphological analysis by microscope 

As noted, all samples showed fungi development, even those which not presented visual signs of contamination, hence acting in accordance with the evidences from the microscopic analysis and confirming the reproductive structure of the three genera used in the study. Even without signs of defacement by microorganisms in the aforementioned analyses, the epoxy paint and the acrylic paint samples presented the formation of fungal colonies of Aspergillus and Aureobasidium and Penicillium and Aspergillus respectively. Aureobasidium and Penicillium, are among the fungi present in paint films listed by (^{Allsopp et al. 2004}). Furthermore, the fungus of type Aureobasidium developed only in the acrylic paint sample, and Penicillium was found only on the epoxy paint samples. Aspergillus was spotted on every sample except for one of epoxy paint, which coincides with the results of (^{Bach & Rangel 2005}) when they identified the fungus of genus Aspergillus. According to the authors, these results can be explained by two reasons: either the paints had extents of inefficient biocides or the fungi acquired resistance to these agents.

(^{Mensah-Attipoe et al. 2016}) noticed that Aspergillus fungus are more sensitive to variations of humidity than those species of Cladosporium and Penicillium. Still regarding genus Aspergillus, these authors noted that from day 0 to week 1 there was a significant reduction in colony forming units followed by a significant increase until week 4. Since testing conditions required constant humidity, there were no obstacles to growth and maintenance of Aspergillus.

Still regarding to the genera Aspergillus, (^{Shiriwaka et al. 2002}) reported its presence on painted surfaces, but not in deteriorated paints, thus, the test range of time may affect this genera identification.

(^{Shirakawa et al. 2002}) reported that Aureobasidium was present on the surfaces at the beginning of their tests, but the numbers fell during the fourth week and recovered in the next weeks, hence suggesting that the presence and concentration of fungi undergo modifications as time passes. Also, as the genera was present early in the surfaces, may have its initial fixation encouraged by the hydrophobic interaction of fresh paint surface. Analyzing real houses, (^{Gi et al. 2005}) don´t perceive the presence of Aureobasidium, them, the results agreed with this study, since in the real exposure and degradation of the materials, this genre can reduce its appearance and survivor.

These authors also state that this fungus seems to be left aside by modifications that affect the painting, which presumably boost other fungi in a preferential way, despite this microorganism being able to recover itself when exposed to more severe weather effects and being found mainly on old surface coverings. The results of this article are analogous, considering that this type of fungus developed only on samples covered with acrylic paint and was intensified as weeks passed.

(^{Sharpe et al. 2016}) perceived an increase of contamination risks of surfaces by genera Aspergillus, Penicillium, and Cladosporium due to the condensation, while the moisture within the building’s tissue was associated only to the increased risk of Aspergillus and Penicillium.