Insulation Materials Susceptibility to Biological Degradation Agents: Molds and Subterranean Termites

Ci a ion: Nunes, L.; Dua e, S.; Pa acha, J.L.; Jones, D.; Paulmie , I.; Ku nik, M. Insula ion Ma e ials Suscep ibili y o Biological Deg ada ion Agen s: Molds and Sub e anean Te mi es. Appl. Sci. 2023,13, 11311. h ps://doi.o g/ 10.3390/app132011311 Academic Edi o : Ana Ma ins Ama o Recei ed: 7 Sep embe 2023 Re ised: 11 Oc obe 2023 Accep ed: 13 Oc obe 2023 Published: 14 Oc obe 2023 Copy igh : © 2023 by he au ho s. Licensee MDPI, Basel, Swi ze land. This a icle is an open access a icle dis ibu ed unde he e ms and condi ions o he C ea i e Commons A ibu ion (CC BY) license (h ps:// c ea i ecommons.o g/licenses/by/ 4.0/). applied sciences A icle Insula ion Ma e ials Suscep ibili y o Biological Deg ada ion Agen s: Molds and Sub e anean Te mi es Lina Nunes 1,2,* , Sónia Dua e 1, João L. Pa acha 1,3,4 , Dennis Jones 5, I an Paulmie 6 and Magdalena Ku nik 7 1S uc u es Depa men , Na ional Labo a o y o Ci il Enginee ing, A . do B asil, 101, 1700-066 Lisbon, Po ugal; [email p o ec ed] (S.D.); [email p o ec ed] (J.L.P.) 2CE3C, Cen e o Ecology, E olu ion and En i onmen al Changes & CHANGE, Global Change and Sus ainabili y Ins i u e, Uni e si y o he Azo es, 9700-042 Ang a do He oísmo, Po ugal 3Buildings Depa men , Na ional Labo a o y o Ci il Enginee ing, A . do B asil, 101, 1700-066 Lisbon, Po ugal 4CERIS—Ci il Enginee ing Resea ch and Inno a ion o Sus ainabili y, Ins i u o Supe io Técnico, Uni e si y o Lisbon, A . Ro isco Pais, 1049-001 Lisbon, Po ugal 5Wood Science and Enginee ing Di ision, Depa men o Enginee ing Sciences and Ma hema ics, Luleå Uni e si y o Technology, Fo ska ga an 1, 931 87 Skelle eå, Sweden; [email p o ec ed] 6Ins i u Technologique FCBA, Allée de Bou au BP227, 33000 Bo deaux, F ance; [email p o ec ed] 7Elici Plan , Le Châ aignie , 16220 Moulins-su -Ta doi e, F ance; [email p o ec ed] *Co espondence: [email p o ec ed] Abs ac : Insula ion ma e ials a e undamen al o dec easing ene gy losses and gua an eeing he - mal and acous ic com o in buildings, which may signi ican ly con ibu e o dec easing he ene gy consump ion ela ed wi h poo he mal building condi ions. These insula ion ma e ials should ha e a low suscep ibili y o biological deg ada ion agen s o dec ease he isks o deg ada ion o o he cons uc ion ma e ials, as well as dec ease possible heal h isks ela ed wi h he de elopmen o noxious biological deg ada ion agen s ega ding indoo ai quali y, o example, o dec ease possible s uc u al isks posed by hose agen s. The p esen s udy aimed a e alua ing he suscep ibili y o se e al insula ion ma e ials o mold g ow h and sub e anean e mi es’ a ack. Insula ion ma e ials, including expanded polys y ene (EPS), mine al wool (MW), and expanded co k agglome a e (ICB), we e es ed agains mold de elopmen , using ma i ime pine as a con ol. Th ee ypes o inocula- ions we e made: (1) na u al indoo inocula ion; (2) a i icial inocula ion using Aspe gillus nige and Penicillium uniculosum ; and (3) a i icial inocula ion using Au eobasidium pullulans. The suscep i- bili y o he insula ion ma e ials e e ed o, plus wood/glass ibe (WGF), was e alua ed o wo sub e anean e mi e species: Re iculi e mes g assei and Re iculi e mes la ipes. The expanded co k agglome a e showed a highe suscep ibili y o molds han he o he insula ion ma e ials es ed. The emaining ma e ials e ealed a good pe o mance, showing no g ow h o aces o g ow h o molds. All he ma e ials es ed showed suscep ibili y o sub e anean e mi es, wi h bo h species being able o c oss hem o ob ain access o he wood. Howe e , wood/glass ibe showed a nega i e e ec , which ansla ed in o lowe su i al a es and a ack deg ees o he wood. Some es ed ma e ials showed a good esis ance o he de elopmen o biological deg ada ion agen s, namely an o ganic ma e ial (coconu ibe ), a composi e o o ganic and ino ganic ma e ials (WGF), and an ino ganic ma e ial (EPS). These esul s indica e ha i is possible o pu sue he de elopmen o inno a i e and e ec i e insula ion ma e ials wi h a low suscep ibili y o biological deg ada ion agen s, ega dless o hei o ganic o ino ganic o igin. Keywo ds: insula ion ma e ials; biological deg ada ion; molds; ungal g ow h; sub e anean e mi es 1. In oduc ion In 2020, buildings con ibu ed o mo e han 30% o global ene gy- ela ed g eenhouse gas (GHG) emissions [ 1 ] and abou 40% o he wo ld’s ene gy consump ion, o which Appl. Sci. 2023,13, 11311. h ps://doi.o g/10.3390/app132011311 h ps://www.mdpi.com/jou nal/applsci Appl. Sci. 2023,13, 11311 2 o 12 app oxima ely 27% was a ibu ed o he esiden ial sec o [ 2 ]. To deal wi h his p ob- lem, se e al di ec i es we e deli e ed by he Eu opean Union (EU) aiming o minimize hea ing/cooling and elec ici y demands, and hus educing CO 2 emissions. Among he key policies o achie ing hese include he Ene gy Pe o mance o Building Di ec i e (EPBD), which was launched in 2010, and he Ene gy E iciency Di ec i e (EED), which was launched in 2012. Wi h he applica ion o hese documen s, he EU es ima ed a educ ion o 8%, 12%, and 17% on he ene gy equi ed o he hea ing/cooling o buildings in 2020, 2030, and 2050, espec i ely, compa ed o da a o 2005. Ne e heless, hese dec eases canno be achie ed i only new cons uc ion and new buildings a e conside ed, and a special look a he exis ing and e en olde buil he i age needs o be pe o med, as hese buildings ep esen mos o he Eu opean-buil pa imony. The use o p ope insula ion ma e ials and design has al eady been p o en as he bes s a egy o educing ene gy losses and gua an eeing he mal com o [ 3 , 4 ]. Addi ionally, he euse o cons uc ion building ma e ials was e has also been p omo ed as a sus ainable p ac ice [5]. The mal insula ion sys ems can be applied using h ee di e en applica ion echniques: o he ex e io —ex e io insula ion; o he in e io —in e io insula ion; and ia injec ion— ca i y insula ion. Ex e nal he mal insula ion composi e sys ems (ETICSs) a e o en applied on new cons uc ions. Howe e , when he e is a need o insula e buildings in an u ban con ex o e en buil he i age, he applica ion o an ETICS is equen ly no possible ( o example, due o excessi e na owing o pedes ian walkways in na ow s ee s), and he applica ion o in e io insula ion may be he only iable solu ion. The applica ion o his echnique comes wi h some disad an ages (such as educ ion in oom size) and p oblems, such as he de elopmen o he mal b idges [ 6 ], possible occu ence o in e s i ial condensa ion [7], os damage [8], and decay on imbe beam-ends [9], among o he s. I is known ha ai in il a ion in many o he exis ing buildings can esul in signi i- can hea losses, wi h losses as high as 33% being no ed in he UK [ 10 ]. As a esul o hese conce ns abou ene gy e iciency, along wi h socie y’s desi e o educe i s ca bon oo p in , buildings a e now being designed o be inc easingly ai igh . This lack o unwan ed en i- la ion can lead o dampness, mois u e damage in buildings, and inc eased le els o indoo ela i e humidi y. All hese condi ions s ongly con ibu e o mold g ow h, along wi h se e al physical and en i onmen al p ope ies (e.g., wa e con en , empe a u e, pH, and he ype (i.e., o ganic and non-o ganic) and hyg oscopic beha io o he ma e ials [11,12] ). P e ious esea ch has shown ha o ganic ma e ials, wi h ample nu ien s in hei com- posi ion, a e mo e p one o mold g ow h [ 13 ]. Fu he mo e, he de elopmen o new insula ion ma e ials, o en mul i-componen in na u e and comp ising na u al ma e ials and/o was es as pa o he composi ion o hei ma ix, se iously inc eases he isk o biode e io a ion [ 14 , 15 ]. The need o e alua e he biodeg ada ion isk o o ganic, ino ganic, and composi e insula ion ma e ials is he e o e o u mos impo ance o enable a co ec applica ion and use o hese ma e ials in cons uc ion sys ems. Acco ding o Ve die e al. [ 16 ], he mos common ungal gene a ound in indoo en i onmen s belong o he gene a Penicillium,Cladospo ium,Aspe gillus, and S achybo ys. The applica ion o an i ungal addi i es (e.g., sodium polybo a e o dichlo luanid) has been commonplace o a oid mold g ow h [ 17 ]. Ne e heless, such an applica ion always aises some conce ns ela ed o he eal e ec i eness o he an i ungal ea men o e ime, as well as wi h po en ial heal h conce ns [17,18]. In addi ion o ungal g ow h, he exposu e o he insula ion ma e ials o we condi ions esul ing om condensa ion o leakage may allow o he ac ion o o he bio ic deg ada- ion agen s, such as sub e anean e mi es. Wi h ega d o ungal g ow h, se e al s udies ha e been made wi h he aim o e alua ing he suscep ibili y o di e en cons uc ion and building ma e ials exposed o di e en condi ions [ 19 – 22 ]. Conce ning he esis ance o sub e anean e mi e a acks, e y ew s udies ha e been published [ 23 , 24 ]. Typically, e mi es do no use he insula ion ma e ial i sel as a ood sou ce, bu as a means o each- ing he wooden s uc u e o he building, o en p esen a high mois u e le els. As hey Appl. Sci. 2023,13, 11311 3 o 12 c oss he he mal insula ion laye , hey c ea e a se o unnels ha will na u ally a ec he he mal capaci y o he insula ion ma e ial and hus a ec he ene gy e iciency o he build- ing. Mo eo e , he physical p ope ies o he insula ion ma e ials p o ide a conside able amoun o in e nal empe a u e s abili y, inad e en ly p o iding a com o able habi a o e mi es [ 24 ]. A esis an he mal insula ion ma e ial would con ibu e o he p e en ion o sub e anean e mi e a acks and in es a ion in wooden elemen s o he buildings. The p esen s udy aimed o e alua e he suscep ibili y o se e al insula ion ma e ials o mold g ow h and sub e anean e mi e a acks (Bla odea: Isop e a: Rhino e mi idae) unde labo a o y condi ions, and o conside u he imp o emen s o insula ion ma e ial p ope ies ega ding bio ic deg ada ion. 2. Ma e ials and Me hods 2.1. Ma e ials Insula ion ma e ials, including expanded polys y ene om wo di e en manu ac u - e s (EPS-S and EPS-C), mine al wool (MW), and expanded co k agglome a e (Co), we e es ed (Figu e 1), using ma i ime pine (Pinus pinas e Ai on) as a con ol (C). In his s udy, he mine al wool (MW) used consis ed o ei he ock wool o glass wool. Glass wool is ob ained by mixing na u al sand and glass a a empe a u e be ween 1300 ◦ C and 1450 ◦ C, whe e he hea ed mass is o ced h ough o a ing nozzles, o en ia cen i ugal o ce, hus c ea ing ibe s. On he o he hand, ock wool is p oduced by mel ing di e en ypes o ocks (e.g., basal , dolos one, and diabase) a 1600 ◦ C, hus ob aining ibe s ha a e bound o- ge he using esins (o en e polyme ic mix u es) and oil ( o educe dus elease). Bo h glass wool and ock wool a e comme cialized as panels, el pipe sec ions, o olls. Expanded polys y ene (EPS) is composed o small sphe es o polys y ene, including an expansion agen (e.g., alipha ic alkanes, such as pen ane). This me hod allows o he p oduc ion o a igid closed-cell oam made o app oxima ely 98% ai and 2% plas ic. Insula ion co k boa d (Co) is a na u al, enewable, and ecyclable ma e ial composed o na u al co k ha can be applied on he acade o buildings, imp o ing hei ene gy pe o mance. No chemical adhesi es o addi i es a e used in he p oduc ion o insula ion co k boa ds. Appl. Sci. 2023, 13, x FOR PEER REVIEW 3 o 12 s udies ha e been made wi h he aim o e alua ing he suscep ibili y o di e en cons uc- ion and building ma e ials exposed o di e en condi ions [19–22]. Conce ning he e- sis ance o sub e anean e mi e a acks, e y ew s udies ha e been published [23,24]. Typically, e mi es do no use he insula ion ma e ial i sel as a ood sou ce, bu as a means o eaching he wooden s uc u e o he building, o en p esen a high mois u e le els. As hey c oss he he mal insula ion laye , hey c ea e a se o unnels ha will na u ally a ec he he mal capaci y o he insula ion ma e ial and hus a ec he ene gy e iciency o he building. Mo eo e , he physical p ope ies o he insula ion ma e ials p o ide a conside able amoun o in e nal empe a u e s abili y, inad e en ly p o iding a com o - able habi a o e mi es [24]. A esis an he mal insula ion ma e ial would con ibu e o he p e en ion o sub e anean e mi e a acks and in es a ion in wooden elemen s o he buildings. The p esen s udy aimed o e alua e he suscep ibili y o se e al insula ion ma e ials o mold g ow h and sub e anean e mi e a acks (Bla odea: Isop e a: Rhino e mi idae) unde labo a o y condi ions, and o conside u he imp o emen s o insula ion ma e ial p ope ies ega ding bio ic deg ada ion. 2. Ma e ials and Me hods 2.1. Ma e ials Insula ion ma e ials, including expanded polys y ene om wo di e en manu ac- u e s (EPS-S and EPS-C), mine al wool (MW), and expanded co k agglome a e (Co), we e es ed (Figu e 1), using ma i ime pine (Pinus pinas e Ai on) as a con ol (C). In his s udy, he mine al wool (MW) used consis ed o ei he ock wool o glass wool. Glass wool is ob ained by mixing na u al sand and glass a a empe a u e be ween 1300 °C and 1450 °C, whe e he hea ed mass is o ced h ough o a ing nozzles, o en ia cen i ugal o ce, hus c ea ing ibe s. On he o he hand, ock wool is p oduced by mel ing di e en ypes o ocks (e.g., basal , dolos one, and diabase) a 1600 °C, hus ob aining ibe s ha a e bound oge he using esins (o en e polyme ic mix u es) and oil ( o educe dus elease). Bo h glass wool and ock wool a e comme cialized as panels, el pipe sec ions, o olls. Ex- panded polys y ene (EPS) is composed o small sphe es o polys y ene, including an ex- pansion agen (e.g., alipha ic alkanes, such as pen ane). This me hod allows o he p o- duc ion o a igid closed-cell oam made o app oxima ely 98% ai and 2% plas ic. Insula- ion co k boa d (Co) is a na u al, enewable, and ecyclable ma e ial composed o na u al co k ha can be applied on he acade o buildings, imp o ing hei ene gy pe o mance. No chemical adhesi es o addi i es a e used in he p oduc ion o insula ion co k boa ds. (a) (b) (c) Figu e 1. De ailed images o he insula ion ma e ials EPS-S (a), MW (b), and Co (c). Fo e mi e esis ance es ing, wood plus glass ibe s (WGFs) and coconu ibe s we e also used. The WGF was o comme cial o igin, and he coconu ibe used was made o unp ocessed coconu ibe s, di ec ly om he p oduce . These ma e ials we e selec ed due o hei widesp ead use in Eu ope [25], whe e hey a e no only applied as a o m o in e- io insula ion, bu also as a o m o ex e io insula ion (i.e., ETICS). All samples we e Figu e 1. De ailed images o he insula ion ma e ials EPS-S (a), MW (b), and Co (c). Fo e mi e esis ance es ing, wood plus glass ibe s (WGFs) and coconu ibe s we e also used. The WGF was o comme cial o igin, and he coconu ibe used was made o unp ocessed coconu ibe s, di ec ly om he p oduce . These ma e ials we e selec ed due o hei widesp ead use in Eu ope [ 25 ], whe e hey a e no only applied as a o m o in e io insula ion, bu also as a o m o ex e io insula ion (i.e., ETICS). All samples we e placed in a clima ic oom a a empe a u e o 20 ◦ C ± 2 ◦ C and a ela i e humidi y (RH) o 65% ± 5% and we e main ained in hese condi ions un il hey we e equi ed o es ing. The samples ha we e inocula ed wi h molds we e p e iously s eam s e ilized in an au ocla e a 100 ◦ C o 20 min. Di e en samples we e cu om he insula ion panels: a se o nine samples o each s udied insula ion ma e ial, wi h dimensions o 40 × 40 × 30 mm 3 , was used o assess ungal g ow h; cylind ical pieces o each insula ion ma e ial ha we e la e compac ed in o Appl. Sci. 2023,13, 11311 4 o 12 glass ubes (see Sec ion 2.3) we e used o e alua e sub e anean e mi es’ abili y o c oss he ma e ials. 2.2. Fungal G ow h Tes s The samples we e inocula ed wi h h ee ungal species: Aspe gillus nige Tiegh., Penicillium uniculosum Thom, and Au eobasidium pullulans (De Ba y) G. A naud. These species we e selec ed conside ing hei p e alen p esence in in e io en i onmen s [ 13 , 16 ]. They a e also commonly e e enced o in he li e a u e and applicable s anda ds [ 13 , 16 , 26 ]. Th ee ypes o inocula ion we e applied: (1) na u al indoo inocula ion; (2) a i icial inocu- la ion using A. nige and P. uniculosum; and (3) a i icial inocula ion using A. pullulans, wi h h ee eplica es o each insula ion ma e ial being es ed. A spo e suspension o each ungus was used o a i icial inocula ions, acco ding o he me hod desc ibed in ISO 846 [ 27 ], and 2 mL o ungal suspensions was applied on each insula ion sample and con ol, using cul u e lasks ha we e p e iously s e ilized and p o ided wi h a s e ile cul u e medium (comp ising 40 g o mal , 20 g o aga , and 1 L o dis illed wa e ). Fo na u al indoo inocula ion, samples we e jus le in indoo condi ions a he labo a o y o se e al days wi hou a con olled empe a u e and RH. The cul u e lasks wi h inocula ed insula ion ma e ials we e closed and kep inside a cul u e chambe a T = 22 ◦ C ± 2 ◦ C and 70% ± 5% RH o ou weeks. The samples we e isually e alua ed o ungal g ow h weekly, conside ing he guidelines de ined in he s anda d ASTM D5590-00 [ 26 ] (Table 1). A e ou weeks o incuba ion, he ma e ials we e ca e ully emo ed om he es lasks, and he ungal g ow h and con amina ed a ea we e isually e alua ed wi h he help o an op ical mic oscope. Table 1. Guidelines o he isual assessmen o ungal g ow h adap ed om ASTM D 5590-00 [26]. In ensi y o G ow h E alua ion Co e ing o he Sample’s Su ace 0No g ow h appa en unde he mic oscope 0% 1 T aces o g ow h <10% 2 Ligh g ow h 10–30% 3 Mode a e g ow h 30–60% 4Hea y g ow h ( o comple e su ace co e age) 60–100% 2.3. Te mi e A ack Tes s The suscep ibili y o he insula ion ma e ials was e alua ed o wo sub e anean e mi e species: Re iculi e mes g assei Clémen and Re iculi e mes la ipes Kolla . The o me is one o he mos common species in he sou h o Eu ope, and he only one ha has been iden i ied in mainland Po ugal [ 28 , 29 ]. R. la ipes is an in oduced, hough well-es ablished, species in F ance. The e mi es we e collec ed in he ields om Po ugal (R. g assei) and F ance (R. la ipes) and we e main ained unde op imal condi ions un il es ing. The es s we e pe o med based on an adap a ion o he guidelines de ined in s anda d EN 118 [ 30 ]. Fi s , h ee cylind ical pieces o each insula ion ma e ial (MW, EPS-S, EPS-C, and ECA) we e cu and compac ed in o glass ubes. Then, he glass ubes we e s acked on pine wood pieces using a con ac glue, and a mix u e o sand and wa e in a 4:1 p opo ion was in oduced on he op o each ube. Two hund ed and i y wo ke s, wo soldie s, and wo nymphs we e in oduced in each glass ube. The ubes we e hen sealed wi h a piece o co on and an aluminum oil o a oid wa e e apo a ion (Figu e 2). The con ols we e also pe o med wi hou any insula ion ma e ial. Appl. Sci. 2023,13, 11311 5 o 12 Appl. Sci. 2023, 13, x FOR PEER REVIEW 5 o 12 s acked on pine wood pieces using a con ac glue, and a mix u e o sand and wa e in a 4:1 p opo ion was in oduced on he op o each ube. Two hund ed and i y wo ke s, wo soldie s, and wo nymphs we e in oduced in each glass ube. The ubes we e hen sealed wi h a piece o co on and an aluminum oil o a oid wa e e apo a ion (Figu e 2). The con ols we e also pe o med wi hou any insula ion ma e ial. The es specimens we e kep inside a condi ioned oom a 25 °C ± 2 °C and 80% ± 5% RH o ou weeks. A e his pe iod, he su i ing e mi es we e emo ed om he glass ube and coun ed, and he es specimens we e disassembled and cleaned. The su - i al a e (SR) o each es specimen was de e mined (Equa ion (1)): SR (%) = [(n . li e wo ke s/250) × 100]. (1) Figu e 2. Te mi e a ack es assembling, wi h, in his example, EPS-S as he insula ion ma e ial and he e mi e species Re iculi e mes g assei, a e 24 h o exposu e. E e y es specimen was isually examined, and he a ack deg ee was classi ied om 1 o 4, acco ding o he s anda d EN 118 [30]: 0—no a ack; 1—a emp ed a ack; 2— sligh a ack; 3—a e age a ack; and 4—s ong a ack. The analysis o a iance (ANOVA) s a is ical es was pe o med o assess he sus- cep ibili y o he di e en insula ion ma e ials o e mi es, along wi h a pos -hoc Tukey es , wi h a signi icance le el o p < 0.05. 3. Resul s 3.1. Resis ance o Fungal A acks Table 2 p esen s he a e age esul s o he isual assessmen deg ee o ungal g ow h. Table 2. Resul s o he isual assessmen a e o ungal g ow h, adap ed and complemen ed om he au ho s o [31]. Na u al Indoo Inocula ion A i icial Inocula ion wi h: A. nige and P. uniculosum A. pullulans Week 1 Week 2 Week 3 Week 4 Week 1 Week 2 Week 3 Week 4 Week 1 Week 2 Week 3 Week 4 Con ol 4.0 4.0 4.0 4.0 3.7 ± 0.6 3.7 ± 0.6 3.7 ± 0.6 4.0 3.0 4.0 4.0 4.0 EPS-C 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 EPS-S 0.0 0.7 ± 0.6 0.7 ± 0.6 0.7 ± 0.6 0.0 0.3 ± 0.6 0.3 ± 0.6 0.3 ± 0.6 0.0 0.3 ± 0.6 0.3 ± 0.6 0.3 ± 0.6 MW 0.0 0.0 0.0 0.3 ± 0.6 0.3 ± 0.6 0.3 ± 0.6 1.0 1.0 0.0 0.0 0.0 0.0 Co 2.0 2.3 ± 0.6 3.7 ± 0.6 3.7 ± 0.6 1.7 ± 0.6 2.3 ± 0.6 2.7 ± 0.6 2.7 ± 0.6 1.0 1.3 ± 0.6 2.0 2.0 Ra ing scale: 0—no g ow h; 1— aces o g ow h; 2—ligh g ow h; 3—mode a e g ow h; and 4— hea y g ow h. Figu e 2. Te mi e a ack es assembling, wi h, in his example, EPS-S as he insula ion ma e ial and he e mi e species Re iculi e mes g assei, a e 24 h o exposu e. The es specimens we e kep inside a condi ioned oom a 25 ◦ C ± 2 ◦ C and 80% ± 5% RH o ou weeks. A e his pe iod, he su i ing e mi es we e emo ed om he glass ube and coun ed, and he es specimens we e disassembled and cleaned. The su i al a e (SR) o each es specimen was de e mined (Equa ion (1)): SR (%) = [(n . li e wo ke s/250) ×100]. (1) E e y es specimen was isually examined, and he a ack deg ee was classi ied om 1 o 4, acco ding o he s anda d EN 118 [ 30 ]: 0—no a ack; 1—a emp ed a ack; 2—sligh a ack; 3—a e age a ack; and 4—s ong a ack. The analysis o a iance (ANOVA) s a is ical es was pe o med o assess he suscep- ibili y o he di e en insula ion ma e ials o e mi es, along wi h a pos -hoc Tukey es , wi h a signi icance le el o p< 0.05. 3. Resul s 3.1. Resis ance o Fungal A acks Table 2p esen s he a e age esul s o he isual assessmen deg ee o ungal g ow h. Table 2. Resul s o he isual assessmen a e o ungal g ow h, adap ed and complemen ed om he au ho s o [31]. Na u al Indoo Inocula ion A i icial Inocula ion wi h: A. nige and P. uniculosum A. pullulans Week 1 Week 2 Week 3 Week 4 Week 1 Week 2 Week 3 Week 4 Week 1 Week 2 Week 3 Week 4 Con ol 4.0 4.0 4.0 4.0 3.7 ± 0.6 3.7 ± 0.6 3.7 ± 0.6 4.0 3.0 4.0 4.0 4.0 EPS-C 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 EPS-S 0.0 0.7 ± 0.6 0.7 ± 0.6 0.7 ± 0.6 0.0 0.3 ± 0.6 0.3 ± 0.6 0.3 ± 0.6 0.0 0.3 ± 0.6 0.3 ± 0.6 0.3 ± 0.6 MW 0.0 0.0 0.0 0.3 ± 0.6 0.3 ± 0.6 0.3 ± 0.6 1.0 1.0 0.0 0.0 0.0 0.0 Co 2.0 2.3 ± 0.6 3.7 ± 0.6 3.7 ± 0.6 1.7 ± 0.6 2.3 ± 0.6 2.7 ± 0.6 2.7 ± 0.6 1.0 1.3 ± 0.6 2.0 2.0 Ra ing scale: 0—no g ow h; 1— aces o g ow h; 2—ligh g ow h; 3—mode a e g ow h; and 4—hea y g ow h. The esul s ob ained o he con ols (Cs) allowed o he alida ion o he es p oce- du e. Indeed, all con ols we e a ed as ou (hea y g ow h) a e ou weeks o incuba ion. Addi ionally, an app eciable le el o ungal g ow h was obse ed o co k samples (Co) a he end o he es , wi h all samples p esen ing mo e han 10% o hei su ace con amina ed and hus a ed as wo o mo e, ega dless o he ype o inocula ion (Figu e 3a). Fo he EPS-C samples, no g ow h was obse ed du ing he es (Figu e 3b). Howe e , his was no he case conside ing he EPS-S om a di e en manu ac u e , whe e aces o g ow h (i.e., <10% o he con amina ed su ace) we e de ec ed in some samples, especially when he Appl. Sci. 2023,13, 11311 6 o 12 na u al indoo inocula ion me hod was used (Figu e 3b). Fu he mo e, no ungal g ow h was obse ed in he MW samples o he a i icial inocula ion wi h A. pullulans, while he e we e only aces o ungal g ow h a e he i s week obse a ions o bo h A. nige and P. uniculosum inocula ion (Figu e 3c). When conside ing he na u al inocula ion, he esul s showed ha only one sample o MW showed aces o g ow h a he end o he es s o na u al inocula ion and was a ed as one. Appl. Sci. 2023, 13, x FOR PEER REVIEW 6 o 12 The esul s ob ained o he con ols (Cs) allowed o he alida ion o he es p oce- du e. Indeed, all con ols we e a ed as ou (hea y g ow h) a e ou weeks o incuba- ion. Addi ionally, an app eciable le el o ungal g ow h was obse ed o co k samples (Co) a he end o he es , wi h all samples p esen ing mo e han 10% o hei su ace con amina ed and hus a ed as wo o mo e, ega dless o he ype o inocula ion (Figu e 3a). Fo he EPS-C samples, no g ow h was obse ed du ing he es (Figu e 3b). Howe e , his was no he case conside ing he EPS-S om a di e en manu ac u e , whe e aces o g ow h (i.e., <10% o he con amina ed su ace) we e de ec ed in some samples, espe- cially when he na u al indoo inocula ion me hod was used (Figu e 3b). Fu he mo e, no ungal g ow h was obse ed in he MW samples o he a i icial inocula ion wi h A. pul- lulans, while he e we e only aces o ungal g ow h a e he i s week obse a ions o bo h A. nige and P. uniculosum inocula ion (Figu e 3c). When conside ing he na u al inocula ion, he esul s showed ha only one sample o MW showed aces o g ow h a he end o he es s o na u al inocula ion and was a ed as one. (a) (b) (c) Figu e 3. Aspec s o some samples a he end o he ungal es s. (a) Expanded co k agglome a e exposed o A. nige and P. uniculosum (le ) and o A. pullulans ( igh ). (b) Expanded polys y ene EPS-C (le ) and EPS-S ( igh ) submi ed o na u al indoo inocula ion. (c) Mine al wool exposed o A. nige and P. uniculosum (le ) and o A. pullulans ( igh ). The a ows and he do ed ci cles indi- ca e ungal g ow h. Figu e 3. Aspec s o some samples a he end o he ungal es s. ( a ) Expanded co k agglome a e exposed o A. nige and P. uniculosum ( le ) and o A. pullulans ( igh ). ( b ) Expanded polys y ene EPS-C ( le ) and EPS-S ( igh ) submi ed o na u al indoo inocula ion. ( c ) Mine al wool exposed o A. nige and P. uniculosum ( le ) and o A. pullulans ( igh ). The a ows and he do ed ci cles indica e ungal g ow h. 3.2. Te mi e Resis ance All con ols p esen ed a s ong a ack deg ee classi ied as a ou (s ong a ack) and su i al a es highe han 50%, which alida ed he e mi e a ack es [ 32 ]. Bo h e mi es we e able o c oss all ma e ials es ed, wi h he wood exhibi ing a high deg ee o a ack (classi ied as a ou ; Figu e 4), excep o MW, ha exhibi ed an a e age a ack when exposed o R. la ipes (Table 3). Appl. Sci. 2023,13, 11311 7 o 12 Appl. Sci. 2023, 13, x FOR PEER REVIEW 7 o 12 3.2. Te mi e Resis ance All con ols p esen ed a s ong a ack deg ee classi ied as a ou (s ong a ack) and su i al a es highe han 50%, which alida ed he e mi e a ack es [32]. Bo h e mi es we e able o c oss all ma e ials es ed, wi h he wood exhibi ing a high deg ee o a ack (classi ied as a ou ; Figu e 4), excep o MW, ha exhibi ed an a e age a ack when ex- posed o R. la ipes (Table 3). Figu e 4. Tes specimen o EPS-C (expanded polys y ene) wi h e mi es (Re iculi e mes g assei) clea ly c ossing he insula ion ma e ial. Bo h e mi e species’ su i al a es we e a ec ed by he ype o insula ion ma e ial used (F = 9.063; p < 0.001 o R. g assei, and F = 11.220; p < 0.001 o R. la ipes, espec i ely). Bo h species we e nega i ely a ec ed by he wood/glass ibe (WGF) ma e ial, as shown in Figu e 5, whe e he boxplo s o he su i al a e alues o e mi es belonging o his species show lowe alues in compa ison wi h e mi es in con ac wi h o he insula ion ma e ials. R. la ipes was no able o become es ablished on he wood/glass ibe ma e ial, which was e lec ed in he lowe a e age a ack deg ee e i ied (n = 3.0 ± 1.0; Table 3). Fo R. la ipes, signi ican ly lowe su i al a es, compa ed o he con ol (Figu e 5), we e ob- ained no only when in con ac wi h he WGF (p = 0.006), bu also wi h he coconu ibe (p = 0.027) and mine al wool (p = 0.006). This was he only ma e ial in which R. g assei showed a signi ican ly lowe pe o mance ega ding su i al a e in compa ison o he con ol (p < 0.001), also shown in Figu e 5. Table 3. Resul s o he insula ion ma e ials’ (con ol, coconu ibe , expanded co k (Co), EPS-C, EPS- S, WGF, and mine al wool (MW)) suscep ibili y o sub e anean e mi es: Re iculi e mes g assei and R. la ipes. Re iculi e mes g assei Re iculi e mes la ipes Su i al Ra e (%) A ack Deg ee Su i al Ra e (%) A ack Deg ee Con ol 74.0 ± 6.7 a 4.0 ± 0.0 56.9 ± 6.6 a 4.0 ± 0.0 Coconu ibe 73.3 ± 4.5 a 4.0 ± 0.0 24.1 ± 3.9 bc 4.0 ± 0.0 Co 68.5 ± 22.7 a 4.0 ± 0.0 58.7 ± 4.0 a 4.0 ± 0.0 EPS-C 75.1 ± 14.6 a 4.0 ± 0.0 51.3 ± 8.0 ac 4.0 ± 0.0 EPS-S 75.1 ± 10.5 a 4.0 ± 0.0 61.6 ± 7.2 a 4.0 ± 0.0 WGF 12.4 ± 17.2 b 4.0 ± 0.0 16.8 ± 12.1 bc 3.0 ± 1.0 MW 68.8 ± 4.5 a 4.0 ± 0.0 16.7 ± 21.4 b 4.0 ± 0.0 No e: di e en le e s in he same column ep esen signi ican di e ences among su i al a e al- ues o each sub e anean e mi e species. Figu e 4. Tes specimen o EPS-C (expanded polys y ene) wi h e mi es (Re iculi e mes g assei) clea ly c ossing he insula ion ma e ial. Table 3. Resul s o he insula ion ma e ials’ (con ol, coconu ibe , expanded co k (Co), EPS-C, EPS-S, WGF, and mine al wool (MW)) suscep ibili y o sub e anean e mi es: Re iculi e mes g assei and R. la ipes. Re iculi e mes g assei Re iculi e mes la ipes Su i al Ra e (%) A ack Deg ee Su i al Ra e (%) A ack Deg ee Con ol 74.0 ±6.7 a4.0 ±0.0 56.9 ±6.6 a4.0 ±0.0 Coconu ibe 73.3 ±4.5 a4.0 ±0.0 24.1 ±3.9 bc 4.0 ±0.0 Co 68.5 ±22.7 a4.0 ±0.0 58.7 ±4.0 a4.0 ±0.0 EPS-C 75.1 ±14.6 a4.0 ±0.0 51.3 ±8.0 ac 4.0 ±0.0 EPS-S 75.1 ±10.5 a4.0 ±0.0 61.6 ±7.2 a4.0 ±0.0 WGF 12.4 ±17.2 b4.0 ±0.0 16.8 ±12.1 bc 3.0 ±1.0 MW 68.8 ±4.5 a4.0 ±0.0 16.7 ±21.4 b4.0 ±0.0 No e: di e en le e s in he same column ep esen signi ican di e ences among su i al a e alues o each sub e anean e mi e species. Bo h e mi e species’ su i al a es we e a ec ed by he ype o insula ion ma e ial used (F = 9.063; p< 0.001 o R. g assei, and F = 11.220; p< 0.001 o R. la ipes, espec i ely). Bo h species we e nega i ely a ec ed by he wood/glass ibe (WGF) ma e ial, as shown in Figu e 5, whe e he boxplo s o he su i al a e alues o e mi es belonging o his species show lowe alues in compa ison wi h e mi es in con ac wi h o he insula ion ma e ials. R. la ipes was no able o become es ablished on he wood/glass ibe ma e ial, which was e lec ed in he lowe a e age a ack deg ee e i ied (n = 3.0 ± 1.0; Table 3). Fo R. la ipes, signi ican ly lowe su i al a es, compa ed o he con ol (Figu e 5), we e ob ained no only when in con ac wi h he WGF (p= 0.006), bu also wi h he coconu ibe (p= 0.027) and mine al wool (p= 0.006). This was he only ma e ial in which R. g assei showed a signi ican ly lowe pe o mance ega ding su i al a e in compa ison o he con ol (p< 0.001), also shown in Figu e 5. The esul s ob ained o EPS-S and EPS-C p esen ed no signi ican di e ences be ween each o he o bo h species, leading o he conclusion ha hese comme cially a ailable insula ion ma e ials p esen ed simila c ossing suscep ibili ies o he e mi es in each espec i e es . Appl. Sci. 2023,13, 11311 8 o 12 Appl. Sci. 2023, 13, x FOR PEER REVIEW 8 o 12 Figu e 5. Boxplo o he su i al a e (%) o he sub e anean e mi es Re iculi e mes la ipes ( ed boxplo s) and Re iculi e mes g assei (blue boxplo s) ela ed o he di e en insula ion ma e ials s ud- ied: coconu ibe (coconu ), ma i ime pine con ol (con ol), expanded co k (ECA), expanded poly- s y ene: EPS-C and EPS-S, wood/glass ibe s (WGF), and mine al wool (Wool). The esul s ob ained o EPS-S and EPS-C p esen ed no signi ican di e ences be- ween each o he o bo h species, leading o he conclusion ha hese comme cially a ail- able insula ion ma e ials p esen ed simila c ossing suscep ibili ies o he e mi es in each espec i e es . 4. Discussion The de elopmen o new insula ion ma e ials is usually made wi h he objec i e o enhancing he mal, acous ic, o e en s uc u al ea u es o he cons uc ion, as well as on a ci cula economy pe spec i e, p io i izing he use o ecycled and biobased ma e ials in encou aging he g ea e use o mo e sus ainable cons uc ion ma e ials ( o example, hose sugges ed by he au ho s o [5,33]); howe e , he suscep ibili y o biological deg a- da ion agen s is some imes o e looked. Gi en ha hese insula ion ma e ials will be ap- plied in close p oximi y o o he cons uc ion ma e ials, i is impo an ha hei pe o - mance, unde condi ions ha may igge he de elopmen o biological deg ada ion agen s ( o example, leakages [19]) is app op ia e; o he wise, hese agen s may ins all hemsel es and e en ac as a sou ce o con amina ion o he o he cons uc ion ma e ials, he eby lowe ing he o e all pe o mance o he cons uc ion sys em. In e ms o mold de elopmen , he co k agglome a e showed a highe le el o sus- cep ibili y han he o he insula ion ma e ials es ed, wi h mo e han 10% o he con ami- na ed a ea no ed a he end o he ials, ei he o he na u al o a i icial inocula ion o molds (Figu e 3a). The emaining ma e ials demons a ed good pe o mances, showing no g ow h o aces o g ow h o ungi (Figu e 3b,c). All he ma e ials showed suscep ibil- i y o sub e anean e mi es, wi h bo h species being able o c oss all ma e ials o gain access o he wood. In ac , co k is a na u al ma e ial wi h an o ganic ma ix composed o lignin, sube in, and polysaccha ides (cellulose and hemicellulose) [31,34]. Indeed, o ganic-based ma e i- als like co k [13,20,35] a e mo e ulne able o ungal deg ada ion, as hey p o ide ample nu ien s ia hei cons i uen componen s capable o sus aining ungal g ow h. Fu he - mo e, he ungal g ow h o co k exposed o a i icial inocula ion wi h A. nige and P. uniculosum was highe han when co k was exposed o a i icial inocula ion using Au. pullulans. In a p e ious s udy, Hy ä inen e al. [19] epo ed ha Penicillium spp. was he Figu e 5. Boxplo o he su i al a e (%) o he sub e anean e mi es Re iculi e mes la ipes ( ed boxplo s) and Re iculi e mes g assei (blue boxplo s) ela ed o he di e en insula ion ma e ials s udied: coconu ibe (coconu ), ma i ime pine con ol (con ol), expanded co k (ECA), expanded polys y ene: EPS-C and EPS-S, wood/glass ibe s (WGF), and mine al wool (Wool). 4. Discussion The de elopmen o new insula ion ma e ials is usually made wi h he objec i e o enhancing he mal, acous ic, o e en s uc u al ea u es o he cons uc ion, as well as on a ci cula economy pe spec i e, p io i izing he use o ecycled and biobased ma e ials in encou aging he g ea e use o mo e sus ainable cons uc ion ma e ials ( o example, hose sugges ed by he au ho s o [ 5 , 33 ]); howe e , he suscep ibili y o biological deg ada ion agen s is some imes o e looked. Gi en ha hese insula ion ma e ials will be applied in close p oximi y o o he cons uc ion ma e ials, i is impo an ha hei pe o mance, unde condi ions ha may igge he de elopmen o biological deg ada ion agen s ( o example, leakages [ 19 ]) is app op ia e; o he wise, hese agen s may ins all hemsel es and e en ac as a sou ce o con amina ion o he o he cons uc ion ma e ials, he eby lowe ing he o e all pe o mance o he cons uc ion sys em. In e ms o mold de elopmen , he co k agglome a e showed a highe le el o suscep i- bili y han he o he insula ion ma e ials es ed, wi h mo e han 10% o he con amina ed a ea no ed a he end o he ials, ei he o he na u al o a i icial inocula ion o molds (Figu e 3a). The emaining ma e ials demons a ed good pe o mances, showing no g ow h o aces o g ow h o ungi (Figu e 3b,c). All he ma e ials showed suscep ibili y o sub e anean e mi es, wi h bo h species being able o c oss all ma e ials o gain access o he wood. In ac , co k is a na u al ma e ial wi h an o ganic ma ix composed o lignin, sube in, and polysaccha ides (cellulose and hemicellulose) [ 31 , 34 ]. Indeed, o ganic-based ma e ials like co k [ 13 , 20 , 35 ] a e mo e ulne able o ungal deg ada ion, as hey p o ide ample nu i- en s ia hei cons i uen componen s capable o sus aining ungal g ow h. Fu he mo e, he ungal g ow h o co k exposed o a i icial inocula ion wi h A. nige and P. uniculosum was highe han when co k was exposed o a i icial inocula ion using A. pullulans. In a p e ious s udy, Hy ä inen e al. [19] epo ed ha Penicillium spp. was he mos common ungal gene a ound in samples belonging o he wood g oup, which included na u al co k (one o he building ma e ials conside ed in his s udy) being p esen on 61.2% o all samples; he o al p e alence o Penicillium sp. plus Aspe gillus sp. was 78.1% agains he 25.1% o Au eobasidium sp., which o e s a possible jus i ica ion o he esul s ob ained in he p esen s udy. Co k was shown o be suscep ible no only o molds bu also o bo h e mi e species, which was expec ed due o he na u al composi ion o co k ega ding he Appl. Sci. 2023,13, 11311 9 o 12 nu ien s a ailable, which include componen s ha e mi es h i e o in wooden ood sou ces, such as glucose o xylose [36,37]. Coconu ibe is also a na u al-based ma e ial. This ma e ial was only es ed agains bo h sub e anean e mi e species, and hei beha io s we e di e en , as he su i al a e o R. la ipes was signi ican ly lowe han he con ols, while R. g assei did no show his e ec (Figu e 5). Coconu ibe s a e cu en ly being s udied o composi es and o ano he bio-based ma e ial de elopmen , and i s mois u e esis ance has been no ed as one o hei ad an ageous cha ac e is ics [ 38 ]. Sub e anean e mi es a e dependen on a high mois u e con en o he ma e ials hey h i e in, and since he beha io o coconu ibe s owa ds mois u e is con a y o he needs o he e mi es, i s p esence may nega i ely impac e mi e su i al a es. Howe e , as R. g assei we e no a ec ed, u he in es iga ions need o be pu sued. Di e en e mi e species may exhibi a di e en beha io owa ds eeding p e e ences; o example, coconu wood demons a ed highe a ack deg ees when exposed o Mas o e mes da winiensis F ogga han o Cop o e mes acinaci o mis (F ogga ) [ 39 ]. P e ious s udies ha e s a ed he na u al du abili y o coconu ibe agains e mi es as du able (Re iculi e mes san onensis Fey aud; [40]). Wood/glass ibe is an example o a hyb id p oduc , using o ganic (wood ibe s) and ino ganic (glass) ma e ials. Simila es s ega ding ungal g ow h assessmen (wi h Aspe gillus sp. and Penicillium sp.) we e p e iously pe o med in-house, and hei esul s showed a low esis ance o he ma e ial o ungal g ow h (Nunes, unpublished), indica ing ha an o ganic ma e ial which is a o able o ungal de elopmen will enable ungal coloniza ion o he composi e ma e ials. Molds appea o h i e on wood and glass ibe s composi es, on he con a y o decay ungi and e mi es [ 41 ]. Indeed, sub e anean e mi es eac ed nega i ely agains he WGFs. Te mi es need o cons uc galle ies o c oss he insula ion ma e ials, bu doub s emain as o whe he hey eed on hose ma e ials o i hey use i o cons uc ing he mud ubes wi hou inges ing hem. The nega i e e ec e i ied in he e mi e’s su i al a e (Figu e 5), and a lowe a ack deg ee o he wood ega ding R. la ipes, could be explained by ei he con ac o ia inges ion. The inges ion o glass may in e e e wi h he diges i e ac ’s in eg i y, which may explain he esul s ha we e ob ained. Mine al wool showed a good esis ance o bo h molds and R. la ipes, al hough i showed aces o g ow h bo h in he na u ally inocula ed samples and he samples inocu- la ed wi h A. nige and P. uniculosum (Figu e 3c). In ac , he suscep ibili y o he ungal g ow h o an ino ganic ma e ial, like MW, is expec ed o be na u ally lowe when com- pa ed wi h an o ganic ma e ial [ 20 , 31 , 42 ]. The only sample showing aces o g ow h o na u al indoo inocula ion could ha e been p e iously con amina ed wi h some o ganic dus pa icles, hus con ibu ing owa ds he inc ease in ungal g ow h suscep ibili y [ 43 ]. Mine al wools usually exhibi a lowe wa e holding capaci y and mois u e con en , and mic oo ganisms may use dus s o wa e accumula ed on hese ma e ials o su i e, no deg ading he ino ganic ma e ial, as i is a poo nu ien sou ce [ 20 , 42 ]. R. la ipes showed a lowe su i al a e when exposed o MW, compa ed o he con ols, which was no e i ied on he o he e mi e species (Figu e 5), and his ac may be linked o eeding p e e ences o o he di e en ial suscep ibili y o he e mi es when in con ac wi h his ma e ial, and u he in es iga ions on his need o be pu sued. When conside ing EPS, i s bio-suscep ibili y o ungal g ow h is e y low, in acco - dance wi h he esul s published by he au ho s o [ 43 ]. This ino ganic ma e ial was easily c ossed by e mi es, al hough i has been hough ha as e mi es a e no able o eed on plas ics, hei con ac wi h his ma e ial may igge hei in ica e symbio ic communi y in o p ocessing plas ic pa icles i inges ed du ing he cons uc ion o hei galle ies. The abili y o e mi es and i s symbion s o p ocess di e en kinds o plas ics and wood plas ics composi es has been ecen ly hypo hesized, al hough u he in es iga ions on his a e needed [ 44 , 45 ]. Rega dless o he pu a i ely low nu i ional alue o plas ics, he e mi e holobion ( he e mi e oge he wi h i s symbion s [ 46 , 47 ]) may bene i om aking some ene gy ou o i and p ocessing his ma e ial, e en i acciden ally inges ed. The in e es in