Application of environmental and biological frequency indicators to assess the Serpula lacrymans fungus in wooden dwellings

Citation: Espinoza Maldonado, R.; Bobadilla, A.; Rubio-Bellido, C. Application of Environmental and Biological Frequency Indicators to Assess the Serpula lacrymans Fungus in Wooden Dwellings. Buildings 2024, 14, 589. https://doi.org/10.3390/ buildings14030589 Academic Editor: Derek Clements-Croome Received: 9 January 2024 Revised: 15 February 2024 Accepted: 16 February 2024 Published: 22 February 2024 Copyright: © 2024 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). buildings Article Application of Environmental and Biological Frequency Indicators to Assess the Serpula lacrymans Fungus in Wooden Dwellings Rodrigo Espinoza Maldonado 1,2, Ariel Bobadilla 1and Carlos Rubio-Bellido 2,* 1Centre for Research in Construction Technologies, University of Bío-Bío, Collao Avenue #1202, Concepción 4030000, Chile; [email protected] (R.E.M.); [email protected] (A.B.) 2Department of Architectural Construction II, University of Seville, 41004 Sevilla, Spain *Correspondence: carlosr[email protected] Abstract: High moisture content, low ventilation levels, and changes in the hygrothermal design of wooden dwellings could generate environmental conditions favorable for developing the rot fungus Serpula lacrymans, which is known as the most destructive fungus of its kind. The purpose of this research was to develop an experimental methodology to determine the conditions of the appearance of the fungus S. lacrymans, through in situ measurement of biological and environmental frequency indicators for wooden dwellings in South Central Chile. The referential standards for the appearance of the fungus were determined based on specialized literature and measurements of dwellings with and without deterioration. The frequency indicators for the appearance of the fungus defined and studied were as follows: frequency of air temperature FATs (%), frequency of relative humidity FRMs (%), frequency of ventilation rate FVRs (%), and rot fungus spore concentrations (CFU/pp). The methodology was validated by being measured in various enclosures and spaces under the floor in wooden dwellings in the cities of Valdivia and Temuco. The results confirmed the strong relationship between environmental relative humidity frequencies, ventilation levels, and spore concentration regarding the appearance of the fungus, where the growth dynamics cannot be separately explained without a previous analysis of the variables. In general, the dwellings most affected by the presence of the fungus had the greatest moisture levels, lower ventilation levels, and greater spore concentration. This study is the basis to diagnose the phenomena of the appearance of the rot fungus in wooden dwellings in climates favorable for its development. Keywords: hygrothermal monitoring; Serpula lacrymans fungus; spore concentrations; wooden dwelling; environmental frequency indicators 1. Introduction Moisture in dwellings could be caused by several sources: external ones, such as accidental installations, rainfalls, snow melting or frost, underground water, surface current waters, and construction water derived from wet processes inside materials, and internal ones, such as the effect of condensation, which is strengthened by building occupancy [ 1 ]. Moreover, high relative moisture content inside dwellings could generate favorable environmental conditions and nutritive substrates for the appearance of different species of fungi, mold, and bacterial proliferation or corrosion [ 2 ]. However, the formation of these microorganisms also depends on other environmental physical parameters, such as material moisture content, enclosure ventilation, thermal insulation, an appropriate design of the layers of the perimeter envelope, and biodeterioration treatments [3–6]. To reduce risks, the hygrothermal design should limit the heat transit and vapor mass through the various layers of the envelope, a phenomenon known as HAM transport [ 7 ]. Designers should consider certain physical criteria or rules to define and organise layers, as well as to define minimum thicknesses. The properties of thermal conductivity, the Buildings 2024,14, 589. https://doi.org/10.3390/buildings14030589 https://www.mdpi.com/journal/buildings Buildings 2024,14, 589 2 of 15 permeability to the passage of water vapor, and the moisture absorption of each layer should be considered according to their location and function in the enclosure to reduce condensation, i.e., a phenomenon that affects energy performance, indoor environment quality, and the durability of all buildings, to acceptable limits [8,9]. In buildings including wood as structural or non-structural elements, efforts should be made to ensure that the temperature field, moisture content, and vapor pressure among layers do not coincide for a critical period within the hygrothermal conditions required by the fungus to start its destructive process. These conditions should be verified by the project professional in the design phase to make corrections, if necessary, to obtain and certify an appropriate bio-hygrothermal design [ 10 ]. However, poor designs could lead to a high risk of fungi growth, thus affecting building’s performance and occupants’ wellbeing [11,12]. Changes in the moisture of the lignocellulosic materials of a building, such as structures, cladding, floors, and unprotected plates, could favor both fungi growth [ 13 ] and the appearance of xylophagous insects [14] that affect the structure of these elements [15]. Biodeterioration is caused by the presence of microorganisms, such as bacteria and fungi, and is visible when materials change [ 16 ]. However, some basidiomycete fungi, which cause the brown or cubic rot, are among the most aggressive xylophages in buildings [ 17 , 18 ]. These fungi significantly damage the internal structure of wood, thus causing great mechanical resistance loss, which becomes important in structures supporting a building [19,20]. Within this category, the most important and harmful brown rot fungi in wooden buildings are Serpula lacrymans, Coniophora puteana, Antrodia vaillantii, and Lentinus lepideus, which can quickly degrade cellulose and hemicellulose and modify the lignin structurally [ 21 ]. In white rot, basidiomycetes and some ascomycetes feed on the three polymers of the wood cell wall, although structural loss is slower than with brown rot. Wood acquires a “whitish” colour due to both lignin degradation and the intact cellulose not used by these fungi. The representative fungi species are Trametes versicolor, T. hirsute, and Schizophyllum commune. On the other hand, the ascomycetes responsible for white rot (i.e., Chaetomium globosum, Monodictys putredinis, Hypocrea muroiana, Cryphonectria parasitica, and Fusarium oxysporum), whose hyphae are developed in the lumen and inside the secondary cell wall, mainly degrade cellulose, thus changing the structure of the lignin and softening the lignocellulosic material [20–22]. In a study conducted in Austria, wood-destroying fungi were found more often in old buildings (530/82.1%) than in new buildings (115/17.9%). The Serpula lacrymans fungus was found 351 times (88.4%) in old buildings, mainly in flats and cellar rooms. Its close relative S. himantioides was isolated twice, on a rotten floor of spruce wood in a farmhouse and on spruce flooring in a multistory building [23]. In the last decades, the total number of appearances of basidiomycete in buildings in Europe (Czech Republic, Poland, Germany (old East and West), Belgium, France, Norway, Denmark, Finland, Latvia, Estonia, Romania, and Albania) mainly corresponded to S. lacrymans and C. puteana, except in Norway, where Antrodia was the most frequent genus [ 24 , 25 ]. S. lacrymans is the most aggressive rot fungus (the first out of 10) in wooden buildings worldwide, followed by Coniophora puteana, Trametes versicolor, and Donkioporia expansa [ 26 , 27 ]. Economically, this is the most important fungus, which is considered as the cancer of buildings, causing damage costs of around USD 36 million annually in France [ 28 , 29 ], and around USD 2.2 million weekly in the United Kingdom [ 30 ]. The solution in these cases is invasive [ 31 ] and includes the opening of suspicious areas to detect the area affected. This is generally carried out when the fungus is widely developed, i.e., when there is a visual proof [32]. The great development of the S. lacrymans fungus mainly affects the mechanical capacity of dwellings by weakening their structural components [ 33 ]. This fungus develops much better in coniferous woods or wood-based products with a moisture content of about 20%, with temperatures between 5 and 26 ◦ C, and with a wide range of environmental moisture [34,35]. Buildings 2024,14, 589 3 of 15 The biological growth dynamics of the fungus varies and depends on several concomitant factors, such as temperature, lighting, ventilation, moisture excess, and the substrate on which the colonization process is settled and starts [36]. S. lacrymans and other rot fungi require environmental moisture around 90% for at least 20% of the time to start their development and colonization, together with wood and other lignocellulosic material with moisture content levels greater than 20% and lower than saturation moisture content levels [ 37 ], with optimal values found by German experiments to be between 30% and 40% [ 38 , 39 ]. Most authors have indicated the temperature range between 5 and 35 ◦C [40], with optimal growth values between 21 ◦C and 22 ◦C [41]. In 2004 onward in Chile, the Universidad Austral de Chile identified the first appearance of the Serpula lacrymans fungus in some dwellings in Temuco and Puerto Montt [ 42 ]. In 2018, the Housing and Urban Planning Service (SERVIU) in Los Ríos Region identified isolated cases of dwellings affected. There were also some isolated cases in Concepción, in the Biobío Region. Between 2019 and 2020, the Ministry of Housing and Urbanism of Chile (MINVU) recorded 17 cases of dwellings affected to a lesser or greater extent by this fungus in the city of Valdivia: 1 in Paillaco, 1 in the Union, 12 in Temuco, and 3 in Padre Las Casas, thus affecting the populations of Teniente Merino in Valdivia and of both Millaray and Las Quilas in Temuco, as well as others in various areas of these and other regions [ 43 ]. The problems were mainly found in ventilated floors and in the joints between walls and floors, consistent with the advance of the mycelial growth from the natural ground [ 44 ]. Figure 1shows the damage and progress of the fungus under the floor, on the floor and in the walls of four dwellings in the city of Valdivia. Buildings2024,14,xFORPEERREVIEW3of16  muchbetterinconiferouswoodsorwood-basedproductswithamoisturecontentof about20%,withtemperaturesbetween5and26°C,andwithawiderangeofenvironmentalmoisture[34,35]. Thebiologicalgrowthdynamicsofthefungusvariesanddependsonseveralconcomitantfactors,suchastemperature,lighting,ventilation,moistureexcess,andthesubstrateonwhichthecolonizationprocessissettledandstarts[36]. S.lacrymansandotherrotfungirequireenvironmentalmoisturearound90%forat least20%ofthetimetostarttheirdevelopmentandcolonization,togetherwithwoodand otherlignocellulosicmaterialwithmoisturecontentlevelsgreaterthan20%andlower thansaturationmoisturecontentlevels[37],withoptimalvaluesfoundbyGermanexperimentstobebetween30%and40%[38,39].Mostauthorshaveindicatedthetemperature rangebetween5and35°C[40],withoptimalgrowthvaluesbetween21°Cand22°C[41]. In2004onwardinChile,theUniversidadAustraldeChileidentifiedthefirstappearanceoftheSerpulalacrymansfungusinsomedwellingsinTemucoandPuertoMontt[42]. In2018,theHousingandUrbanPlanningService(SERVIU)inLosRíosRegionidentified isolatedcasesofdwellingsaffected.TherewerealsosomeisolatedcasesinConcepción, intheBiobíoRegion.Between2019and2020,theMinistryofHousingandUrbanismof Chile(MINVU)recorded17casesofdwellingsaffectedtoalesserorgreaterextentbythis fungusinthecityofValdivia:1inPaillaco,1intheUnion,12inTemuco,and3inPadre LasCasas,thusaffectingthepopulationsofTenienteMerinoinValdiviaandofbothMillarayandLasQuilasinTemuco,aswellasothersinvariousareasoftheseandotherregions[43].Theproblemsweremainlyfoundinventilatedfloorsandinthejointsbetween wallsandfloors,consistentwiththeadvanceofthemycelialgrowthfromthenatural ground[44].Figure1showsthedamageandprogressofthefungusunderthefloor,on thefloorandinthewallsoffourdwellingsinthecityofValdivia.  (a)(b)  (c)(d)  (e)(f) Figure 1. Picture (a) shows a wooden plank flooring with visible damage; (b) fungi can be seen at the joint of the slotted plywood plate of a wall with floating floor; (c) degradation of an original wooden floor; (d) deterioration in the joint of the slotted plywood wall and the newly constructed structural plywood floor; (e) fruiting bodies under the floor and outdoor cladding; (f) presence of active mycelium under the floor. Buildings 2024,14, 589 4 of 15 In the last two decades, attacks of this fungus in Chile have been increasingly reported. Its appearance has been strongly related to the erroneous implementation of measures to reduce thermal transmission and air tightness of buildings in energy improvement [ 45 ]. Within this scenario, the Supreme Decree Number 255 of the Ministry of Housing and Urbanism of Chile, corresponding to the Familiar Heritage Protection Programme, was created. Among other goals, it aims to thermically renovate the existing dwellings through grants given by the State to provide inhabitants with greater comfort [46]. To avoid the risk of the appearance of the fungus S. lacrymans and other rot fungi with similar germination conditions, calculation tools or simulation software are required that allow us to define appropriate bio-hygrothermal designs, and which link the critical parameters of germination with the environmental conditions of the homes and construction elements of the envelope. However, design alterations over the years create favorable conditions for the rot fungus to develop. In this case, it is necessary to define the causes of the appearance of the fungus, with the aim of determining solutions for the improvement and remediation of the attacked homes. For this, it is necessary to use validated experimental methodologies, which can directly or indirectly account for the development of the rot fungus. This study aims to develop an experimental methodology based on the measurement of hygrothermal and biological frequency indicators to verify the appearance of the Serpula lacrymans fungus in wooden homes in South Central Chile. The critical standards for the appearance of the fungus are defined, and they are validated with measurements carried out in situ in wooden dwellings in Valdivia and Temuco. 2. Methodology For this study, the frequency indicators of temperature and relative moisture were applied [ 32 ], as well as of ventilation and spore concentrations defined by exhaustively analyzing the environmental and biological variables from actual measurements. Temperature, relative moisture, ventilation, and spore concentrations of rot fungi were simultaneously measured in living rooms, main bedrooms, and spaces under the floor in wooden dwellings in the cities of Temuco and Valdivia, where the presence of Serpula lacrymans was reported [ 42 ]. A total of three dwellings affected by this fungus were studied in the two cities, as well as one control dwelling with no damage for comparison. The methodology is included below. 2.1. Definition of the Frequency Indicators For quantitative measurements, dwelling performance measures were compared with the standards, which were defined for a normal state or condition according to both the Referential Standard IICRC S520 [ 47 ] and the existing literature that set limits for the variables of temperature and relative moisture for the appearance of molds and rot fungi [ 32 , 37 ]. The standard reference of the ventilation frequency was calculated under the criterion of new air ventilation rates to limit relative moisture levels up to 70%, according to the following dilution ventilation rate: Q=Mv / 3. (Wi −We)(1) The equation of dilution ventilation is as follows: n=3.6 Qe /V(2) Air changes per hour where: •Qe: extraction volume, l/s; • Wi: absolute moisture of the place at an air temperature of 19 ◦ C and 70% of relative moisture, g/m3; • We: absolute moisture of the locality with the mean temperature and absolute moisture in July, g/m3; Buildings 2024,14, 589 5 of 15 •n: volume air changes in the place per hour, 1/h; •V: volume of the place, m3. The maximum spore concentration thresholds for the attack of the fungus were defined according to the collection of spores in the environment of the dwellings not affected by the rot fungi. The environmental frequencies of relative moisture (FRMs), air temperature (FATs), and ventilation rate (FVRs) were analyzed for a weekly measurement period. Referential standards or relative moisture thresholds in the time were defined for the mold activation ( ≥ 70%) as the first barrier and for S. lacrymans ( ≥ 90%). Temperature ranges between 5 ◦ C and 35 ◦ C were defined for the appearance of the rot fungus, as well as ventilation rates lower than 1.8 (1/h) in the living rooms and (1.5) 1/h in bedrooms and spaces under the floor. Likewise, the minimum spore concentration standard was established in 52 units that form colonies per Petri plate, which were measured for a maximum of 4 h (Table 1). Table 1. Environmental and biological frequencies for the appearance of fungi. Performance Indicators Referential Standard Frequency of weekly relative moisture FRMs (%) mold ≥5% weekly time about 70% Frequency of weekly relative moisture FRMs (%) Serpulla fungus ≥20% weekly time about 90% Frequency of weekly air temperature FATs (%) mold and Serpula 100% weekly time within the range (5–35 ◦C) Frequency of weekly ventilation rate FVRs (%) mold and Serpula 0% weekly time lower than 1.8 (1/h) in living rooms, and 1.5 (1/h) in bedrooms and spaces under the floor Rot fungus spore concentration ≥52 (CFU/pp) measured for 4 h as maximum 2.2. Dwellings A total of 18 dwellings belonging to the room set Teniente Merino in the city of Valdivia and to the room set Las Quilas and Millaray in the city of Temuco were studied because the Agricultural and Livestock Service (SAG) of Chile identified the presence of the Serpula lacrymans fungus in all of them. The dwellings were built between the 1960s and 1970s in a semi-detached floor with a surface area of 60 m 2 , with both ventilated floors in the dry places and concrete floors in bathrooms and kitchens. The original dwelling was structured on radiata pine wood and raulífloor beams. The original indoor and outdoor claddings were wooden boarding. The dwellings of Valdivia maintained the system of ventilated floors, whereas the dwellings in Temuco replaced their structure of ventilated floors with concrete floors (Figure 2). Buildings2024,14,xFORPEERREVIEW6of16   (a)(b)(c)(d)   (e)(f)(g)(h) Figure2.Picturesfrom(a–d)correspondtothedwellingsinTemuco,andpicturesfrom(e–h)correspondtothedwellingsinValdivia.Dwellings(d,g)correspondtowitnessdwellings,withno damage. Somechangesweremadeinthedwellingswiththepassageoftimebyownersthemselvesandbythegrantsgivenforthethermalimprovementoftheenvelopeelements (windows,walls,ceilings,andventilatedfloors).Themainchangesintheoriginaldesign wereasfollows:adjacentincreases,replacementofwoodenflooringwithathermalsandwichfloorsolution,obstructedsmallwindows,constructionofshedsincourtyards,and replacementofventilatedfloorswithconcretefloors,amongothers.Outofthe18dwellings,6unitswereexperimentallyevaluated,3inValdivia(DwellingsNo.1,2,and3)and 3inTemuco(DwellingsNo.5,6,and8).Likewise,thewitnessdwellingsofNo.4inValdiviaandNo.7inTemucowerealsoincludedbecausetherewasnopresenceoftheS. lacrymansfungus(Table2). Table2.Changesinthedwellings. ItemAddressLocalityCityChanges (1)LagoMalleco#560Tte.MerinoValdivia Increase,obstructedsmallwindows, replacementofventilatedfloorwith thermalsandwichfloor (2) LagoMalleco#570Tte.MerinoValdivia Increase,obstructedsmallwindows, replacementofventilatedfloorwith thermalsandwichfloor (3) LagoVillarrica#2780Tte.MerinoValdivia Shed,obstructedsmallwindows, replacementofventilatedfloorwith thermalsandwichfloor (4) LagoLlanquihue#3030Tte.MerinoValdiviaShed,thermalimprovementinwall, nochangesinthefloor (5) LosAromos#0184LasQuilasTemucoReplacementofventilatedfloorwith concretefloor (6) LosHelechos#1450 LasQuilasTemucoIncrease,replacementofventilated floorwithconcretefloor (7) LosNotros#1463LasQuilasTemucoShed,replacementofventilated floorwithconcretefloor (8) Palihue#968.MillarayTemucoNosignificantchanges (4)WitnessdwellinginValdivia.(7)WitnessdwellinginTemuco.   Figure 2. Pictures from (a–d) correspond to the dwellings in Temuco, and pictures from (e–h) correspond to the dwellings in Valdivia. Dwellings (d,g) correspond to witness dwellings, with no damage. Buildings 2024,14, 589 6 of 15 Figure 2shows the dwellings used for study. Some changes were made in the dwellings with the passage of time by owners themselves and by the grants given for the thermal improvement of the envelope elements (windows, walls, ceilings, and ventilated floors). The main changes in the original design were as follows: adjacent increases, replacement of wooden flooring with a thermal sandwich floor solution, obstructed small windows, construction of sheds in courtyards, and replacement of ventilated floors with concrete floors, among others. Out of the 18 dwellings, 6 units were experimentally evaluated, 3 in Valdivia (Dwellings No. 1, 2, and 3) and 3 in Temuco (Dwellings No. 5, 6, and 8). Likewise, the witness dwellings of No. 4 in Valdivia and No. 7 in Temuco were also included because there was no presence of the S. lacrymans fungus (Table 2). Table 2. Changes in the dwellings. Item Address Locality City Changes (1) Lago Malleco #560 Tte. Merino Valdivia Increase, obstructed small windows, replacement of ventilated floor with thermal sandwich floor (2) Lago Malleco #570 Tte. Merino Valdivia Increase, obstructed small windows, replacement of ventilated floor with thermal sandwich floor (3) Lago Villarrica #2780 Tte. Merino Valdivia Shed, obstructed small windows, replacement of ventilated floor with thermal sandwich floor (4) Lago Llanquihue #3030 Tte. Merino Valdivia Shed, thermal improvement in wall, no changes in the floor (5) Los Aromos #0184 Las Quilas Temuco Replacement of ventilated floor with concrete floor (6) Los Helechos #1450 Las Quilas Temuco Increase, replacement of ventilated floor with concrete floor (7) Los Notros #1463 Las Quilas Temuco Shed, replacement of ventilated floor with concrete floor (8) Palihue #968. Millaray Temuco No significant changes (4) Witness dwelling in Valdivia. (7) Witness dwelling in Temuco. 2.3. Monitoring and Determinations Measurements were simultaneously conducted for the two cities between 4 and 10 November. Environmental variables, such as air temperature, relative moisture, ventilation rates, and spore concentrations, were measured in the living rooms and main bedrooms. These variables were also measured under the ventilated floor in the 4 dwellings in Valdivia. Variables, such as indoor climate, relative moisture, air temperature, and CO 2 concentrations, were measured every 10 min for 7 days. A digital sensor kit (HOBO UX120-006M made by ONSET COMPUTER CORP., Bourne, MA, USA) with 4 channels for data acquisition was used to measure environmental relative moisture in percentage (%), air temperatures in Celsius degrees ( ◦ C), and CO 2 concentrations in parts per million (ppm). Outdoor climate, environmental relative moisture (%), and air temperature ( ◦ C) were determined by using two weather stations (Davis 6163 Vantage Pro 2 Plus made by DAVIS INSTRUMENTS, Hayward, CA, USA) simultaneously installed in each city. Buildings 2024,14, 589 7 of 15 Natural ventilation was determined according to the fall in dilution concentrations of the CO 2 levels measured in each place, according to the calculation principles set by the ASTM E741 standard [ 48 ] by the optional regression method and given by the following dilution equation: At = ln C(0) −ln C(t) (3) Ventilation equation due to CO2fall, where: •At: air change rate (1/h); •ln C(0): initial CO2concentration, (ppm); •ln C(t): CO2concentration in the time t1, (ppm). Spore concentration in the air was indirectly determined by collecting spores of the rot fungus because of rainfall in Petri capsules. These capsules were located under the floor and inside the four dwellings in Valdivia. The biological load was collected for 4 h in each plate, and it was then put in a benlate/agar/malt extract growth (BEMA). Concentration was determined by directly counting the colony forming units per Petri plate (CFU/pp). Species were identified by increasing the ITS region of the ribosomal DNA with BLAST, a computer tool. 3. Results 3.1. Frequency of Air Temperature FATs (%) The maximum and minimum temperatures were 27.6 ◦ C and 11.1 ◦ C, respectively, for the dwellings in Valdivia, and 27.8 ◦ C and 10.9 ◦ C, respectively, for the dwellings in Temuco (Table 3). All the temperature frequencies in the living rooms, bedrooms, and spaces under the floor during the weekly period were always between 5 ◦ C and 35 ◦ C, which is considered beneficial for fungus development according to the standard on the temperature frequencies defined for the appearance of the fungus (Table 4). The lowest outdoor temperature in Temuco was 5.4 ◦ C, and 5.8 ◦ C in Valdivia, greater than the lower range of 5 ◦ C. However, for the climate of these localities, lower temperatures could be occasionally found in winter. Table 3. Summary of the measurement of indoor and outdoor environmental variables between 4 November 2019 and 10 November 2019. City Dwelling Space Maximum Temperature (◦C) Minimum Temperature (◦C) Average Temperature (◦C) Maximum Relative Moisture (%) Minimum Relative Moisture (%) Average Relative Moisture (%) Maximum Ventilation (1/h) Minimum Ventilation (1/h) Average Ventilation (1/h) Valdivia 1 Under the floor 18.6 11.9 14.0 96.9 66.9 88.8 0.7 0.0 0.0 Living room 25.5 11.4 18.0 73.3 46.7 58.5 10.0 0.0 1.6 Bedroom 23.5 11.1 16.1 93.0 55.8 81.2 3.1 0.0 0.3 2 Under the floor 16.7 12.2 14.2 93.3 90.1 91.7 0.9 0.0 0.1 Living room 24.2 15.1 19.4 72.2 54.5 62.9 9.0 0.0 1.2 Bedroom 23.5 14.1 18.7 70.5 49.3 60.1 8.8 0.0 0.6 3 Under the floor 18.9 13.8 15.6 85.8 76.7 81.8 0.9 0.0 0.1 Living room 25.8 16.2 20.5 58.0 39.6 47.9 9.5 0.0 1.4 Bedroom 17.9 12.9 15.3 95.0 76.0 89.7 3.4 0.0 0.2 4 (witness) Under the floor 16.6 14.2 15.4 88.7 78.5 83.5 2.3 0.0 0.4 Living room 25.6 14.1 19.2 82.3 49.2 63.8 9.8 0.0 0.5 Bedroom 27.6 13.2 19.9 82.5 43.7 61.9 8.7 0.0 0.7 Outdoor 30.7 5.8 15.7 92.1 30.4 63.8 - - - Buildings 2024,14, 589 8 of 15 Table 3. Cont. City Dwelling Space Maximum Temperature (◦C) Minimum Temperature (◦C) Average Temperature (◦C) Maximum Relative Moisture (%) Minimum Relative Moisture (%) Average Relative Moisture (%) Maximum Ventilation (1/h) Minimum Ventilation (1/h) Average Ventilation (1/h) Temuco 1 Living room 26.1 10.9 17.0 75.9 45.5 62.5 12.1 0.0 0.8 Bedroom 27.2 12.7 18.2 66.2 43.2 57.7 8.0 0.0 0.4 2 Living room 21.3 14.0 17.3 69.3 54.9 63.0 1.3 0.0 0.1 Bedroom 27.8 14.7 20.0 73.2 58.3 64.4 2.1 0.0 0.1 3 (witness) Living room 26.7 14.3 19.4 76.5 49.9 59.5 1.1 0.0 0.1 Bedroom 27.6 13.5 19.3 75.1 45.3 59.1 1.5 0.0 0.1 4 Living room 25.5 14.1 19.1 67.0 44.1 54.7 0.6 0.0 0.0 Bedroom 25.0 14.9 19.2 63.5 46.2 55.5 8.0 0.0 1.1 Outdoor 28.8 5.4 15.8 86.9 35.9 63.6 - - - Table 4. Results of the frequency measurements in the wooden dwellings in Temuco and Valdivia for the appearance of the S. lacrymans rot fungus. Period between 4 November 2019 and 10 November 2019. Performance Dwellings in Valdivia Dwellings in Temuco Dw. 1 Dw. 2 Dw. 3 Dw. 4 (Witness) Dw. 1 Dw. 2 Dw. 3 (Witness) Dw. 4 Referential Standard Frequency of the weekly air relative humidity FRMs FRMs living room 70 (%) 4 46 0 8 6 1 0 0 ≥5% time greater than 70% FRMs bedroom 70 (%) 82 52 100 13 0 1 0 0 ≥5% time greater than 70% FRMs living room 90 (%) 0 0 0 0 0 0 0 0 ≥20% time greater than 90% FRMs bedroom 90 (%) 43 0 61 0 0 0 0 0 ≥20% time greater than 90% FRMs under the floor 90 (%) 55 100 0 0 - - - - ≥20% time greater than 90% Frequency of the weekly air temperature FATs FATs living room (%) 100 100 100 100 100 100 100 100 100% of time between 5 ◦ C and 35 ◦C FATs bedroom (%) 100 100 100 100 100 100 100 100 100% of time between 5 ◦ C and 35 ◦C FATs under the floor (%) 100 100 100 100 - - - - - Frequency of weekly ventilation rate FVRs FVRs living room (%) 39 49 41 69 75 100 100 100 ≤0% time lower than 1.8 (1/h) FVRs bedroom (%) 95 79 98 65 87 98 100 49 ≤0% time lower than 1.5 (1/h) FVRs under the floor (%) 100 100 100 89 - - - - ≤0% time lower than 1.5 (1/h) Spore concentration Concentration in living room (CFU/pp) 183 46 - 18 190 43 36 28 ≥52 (CFU/pp) Concentration in bedroom (CFU/pp) 90 43 22 - 67 32 - - ≥52 (CFU/pp) Concentration under the floor (CFU/pp) 124 85 102 52 - - - - ≥52 (CFU/pp) Buildings 2024,14, 589 9 of 15 3.2. Frequency of Relative Moisture FRMs As for the measurements under the floor in the city of Valdivia, Dwelling 2, which was the most affected by the active presence of the fungus, maintained moisture frequencies of 100% of the weekly period at about 90% of the threshold for the appearance of the fungus. This frequency was about 90% for 55% of the time in Dwelling 1, with a lower presence of fungi than Dwelling 2. In Dwelling 3, with the lowest presence of fungi, the relative moisture frequency under the floor never exceeded 90%, maintaining an average of 82%. In Dwelling 4 (witness), with no fungi, relative moisture frequency did not exceed 90%, and the average relative moisture frequency was 85% (Figure 3). Buildings2024,14,xFORPEERREVIEW9of16  FATsunderthefloor(%)100100100100----- FrequencyofweeklyventilationrateFVRs FVRslivingroom(%)3949416975100100100≤0%timelowerthan1.8(1/h) FVRsbedroom(%)95799865879810049≤0%timelowerthan1.5(1/h) FVRsunderthefloor(%)10010010089----≤0%timelowerthan1.5(1/h) Sporeconcentration Concentrationinliving room(CFU/pp)18346-18190433628≥52(CFU/pp) Concentrationinbedroom (CFU/pp)904322-6732--≥52(CFU/pp) Concentrationunderthe floor(CFU/pp)1248510252----≥52(CFU/pp) 3.2.FrequencyofRelativeMoistureFRMs AsforthemeasurementsunderthefloorinthecityofValdivia,Dwelling2,which wasthemostaffectedbytheactivepresenceofthefungus,maintainedmoisturefrequenciesof100%oftheweeklyperiodatabout90%ofthethresholdfortheappearanceofthe fungus.Thisfrequencywasabout90%for55%ofthetimeinDwelling1,withalower presenceoffungithanDwelling2.InDwelling3,withthelowestpresenceoffungi,the relativemoisturefrequencyunderthefloorneverexceeded90%,maintaininganaverage of82%.InDwelling4(witness),withnofungi,relativemoisturefrequencydidnotexceed 90%,andtheaveragerelativemoisturefrequencywas85%(Figure3).  Figure3.Behaviouroftheenvironmentalvariablesunderthefloor. Inthelivingroomsandbedrooms,nodwellingexceeded90%ofrelativemoisture during20%ofthetime,accordingtotherelativemoisturefrequenciesdefinedfortheappearanceofthefungus(Table4).Thisphenomenoncouldbeexplainedmainlybecauseof thespringmeasurementperiod,whenoutdoorrelativemoisturewasgreaterthan90%in particularcases,anddwellingsrecordedhighventilationrates.However,slightlygreater relativehumiditylevelsareexpectedforwinterbycomparingthelocalmeteorologywith thatduringtheweeksofthestudy(Table3). Thefrequencyof70%during5%ofthetimefortheappearanceofmoldwasexceeded inthelivingroomofDwelling2,andinthebedroomsofDwellings1,2,and3inValdivia (Table4).Therewasnomold,buttherewasfungiaftertheirvisualinspection.Thiscould berelatedtotheperiodiccleaningworksmadebyuserstomakethemdisappear,aswell astothesubstratecolonization,anantagonistbehaviourofrotfungi[49]. 0 5 10 15 20 25 30 35 30 40 50 60 70 80 90 100 04-11-2019 05-11-2019 06-11-2019 07-11-2019 08-11-2019 09-11-2019 Temperature(°C)andventilation(1/h) Relativehumidity(%) Time(day) Behaviouroftheenvironmentalvariablesunderthefloor HumidityV1 HumidityV2 HumidityV3 HumidityV4 TemperatureV1 VentilationV1 TemperatureV2 VentilationV2 TemperatureV3 VentilationV3 TemperatureV4 VentilationV4 LowerTem p erature,5°C Uppertemperature,35°C Ventilation ≤1.5(1/h) Relativehumidity≥ 90% Figure 3. Behaviour of the environmental variables under the floor. In the living rooms and bedrooms, no dwelling exceeded 90% of relative moisture during 20% of the time, according to the relative moisture frequencies defined for the appearance of the fungus (Table 4). This phenomenon could be explained mainly because of the spring measurement period, when outdoor relative moisture was greater than 90% in particular cases, and dwellings recorded high ventilation rates. However, slightly greater relative humidity levels are expected for winter by comparing the local meteorology with that during the weeks of the study (Table 3). The frequency of 70% during 5% of the time for the appearance of mold was exceeded in the living room of Dwelling 2, and in the bedrooms of Dwellings 1, 2, and 3 in Valdivia (Table 4). There was no mold, but there was fungi after their visual inspection. This could be related to the periodic cleaning works made by users to make them disappear, as well as to the substrate colonization, an antagonist behaviour of rot fungi [49]. 3.3. Frequency of Weekly Ventilation Rate FVRs (%) In all the living rooms, minimum ventilation frequencies were met 50% of the week as an average for no appearance of the fungus, and 48% of the time in bedrooms. However, Dwelling 4 (witness) in Valdivia was ventilated at lower rates (Table 4) because it has thermally improved one of the walls, which in turn improves the standards on air hermeticism. As for the ventilation frequency under the floor, Dwelling 4 (witness) met the minimum ventilation standard only 11% of the time, whereas Dwellings 1, 2, and 3 never exceeded the minimum ventilation threshold, concordant with the visual observations in which the fungus was in all these dwellings (Table 4). Figure 3represents ventilation behaviour under the floor in the city of Valdivia. 3.4. Spore Concentration CFU/pp As for the spore concentration under the floor, Dwellings 1, 2, and 3 presented fungus concentrations of 124, 85, and 102 CFU/pp, respectively. These values were compared