The moisture requirements of moulds isolated from domestic dwellings

International Biodeterioration 25 (I 989) 259-284

The Moisture Requirements of Moulds Isolated from Domest i c Dwell ings

C. Grant"*. C. A. Hunter h:l:, B. Flannigan ~' & A. F. Bravery"~

"Department of the Environment. Building Research Establishment. Princes Risborough Laboratory, Princes Risborough, Aylcsbury. Buckinghamshire HPI7 9PX. UK

t'Dcpartment of Brewing and Biological Sciences. Heriot-Watt University. Edinburgh EHI IHX. UK

(Received 18 January 1988: accepted 24 June 1988)

ABSTRACT

Mouhl grawth on the damp internal su~wes of huihEngs is estimated to occur in some 2" 5 million dwellings in the UK. As moisture is regarded as the k~ T .fiwtor in promvting mouhl growth in buildings, a study was carried out to determine the ntoisture requirements fi)r growth of moukA" isolated from domestic d~tellinL:~, with the intention of providing buihling operators and practitioners with it!fi)rmation on crith'al internal humidity h've& Nineteen ,wecies of moukb which occurred in at h,ast 10% of ~hmtestic air samph,s were assessed fi)r their ability to grow at different h'vels of water activity (el ~, : A ,. = Rt l / I (X)). Certain of the~'e ~z,re seh,cted fi~r mort, ~h'taih'd studies of their A ,. requirements for growth on a range of buihling suhstrates ittchtding paint attd woodchip wallpaper.

The lowest A~, h,vd recorded for growth on malt e~'tract agar was O" 76 (76% Rlt) for Aspergillus repens, whih, fi)r A. vcrsicolor and several Penicil l ium ,wa'ies the minimum value was O. 79. On woodchip wallpw~er painted with an emulsion paint the A,, minimum for A. vcrsicolor attd P.

* Present address: Depa rtment of the Environment. Central Directorate of Environmental Protection, Romney House, 43 Marsham Street. London SWIP 3PY, UK. '~ Present address: Department of the Environment. Building Research Establishment. Princes Risborough Laboratory, Princes Risborough, Aylesbu .r.r.ry, Buckinghamshire HPI7 9PX, UK. ~To whom correspondence should be addressed.

259

~'.~_3 1989 Crown copyright.

260 C Grant. C A. Hunter. B. Flannigan. A. F. Brave~.

ch rysogenum was also found to be 0 ~ 79 at 25 °C. although at a temperature of 12 °C. which was closer to that found for wall surfaces in practice, the A ~. minima was elevated to O. 87for these two moulds. In general the study sh owed that increasing the temperature and the nutritional status of the substrate led to a reduction in the A,. requirements o f the mouhts. In building practice therefore the aim must be to maintain relative humMitv attd suoeace temperature conditions within dwellings at lewels such that mould susceptible substrates remain below A ~. 0.80 to avoid development of mouht growth.

INTRODUCTION

Damp internal surfaces of buildings are frequently affected by the growth ofmould (BRE, 198 !). Sanders and Cornish (1982) estimated that some 2 million dwellings in England (11-8% of the housing stock) had serious problems of dampness: extrapolated to the whole of the UK housing stock the indications were that 2.5 million dwellings were seriously affected, with condensation the cause in 60% of cases. A further 2 million dwellings had slight condensation problems. Confirmation of the extent of the problem in England was provided by the findings of the 1981 English House Conditions Survey (DOE, 1983) and, because of a number of factors including the greater Severity of the climate, it was considered likely that the problem was greater in Scotland (Scottish Affairs Committee, 1984). Mould problems are greatest in rented accommodation where they are a frequent cause of concern to the tenants (BRE, 1985).

The Building Research Establishment is investigating currently the efficacy of a number of measures to combat dampness caused by condensation, including improvements in thermal insulation, increasing background heating and reducing the moisture content of the air by means of extract fans and dchumidifiers (Cornish & Sanders, 1983). Fungicidal remedies are also available, including the use of toxic washes to kill mould growths on surfaces (BRE, 1982), and the use of fungicidal paints (Grant et al., 1986). As part of a major investigation undertaken to obtain a better understanding of the species of mould which grow in dwellings, an extensive survey of the air spora of occupied domestic dwellings was carried out and is reported elsewhere (Hunter et al., 1988). In conjunction with this, the range and frequency of occurrence of species present in mould growths on wails were examined.

Fundamental to the growth of such moulds on susceptible substrates is the presence of sufficient moisture. Scott (1957) introduced the concept of water activity (A,,) as the criterion ofwater availability to microorganisms. A,,. is numerically equivalent to RH/100 such that, for example,

Moisture requirements of moulds from domestic dwelfings 261

75% RH = A,,, 0-75 in a substrate in equilibrium with an atmosphere of that RH. Scott also reviewed earlier studies on the effect of relative humidity on the growth of moulds, yeasts and bacteria. In addition to Scott's work there have been numerous studies on the effect of Aw on the growth of microorganisms which cause deterioration of a diverse range of substrates: foodstuffs and other stored products (e.g. Scott. 1957: Ayerst. 1966. 1969: Pitt. 1975: Pitt & Hocking. 1977: Christian. 1980). grain (Fiannigan & Bana. 1980: Lacey et al.. 1980: Magan & Lacey. 1984). textiles (Galloway. 1934: Fermor & Eggins. 1980). paint (Eveleigh. 1961) and constructional materials (Coppock & Cookson. 1951). In the large majority of these studies.4 ,, requirements for germination and/or growth were determined on artificial media. Of those works cited above only Galloway (1934) and Coppock and Cookson (1951) employed substrates affected by mould growth in practice as substrates for laboratory studies. Materials used by Coppock and Cookson (1951) included distempered. painted and plastered wood. and brick with and without whitewash and cement render. Wood, in addition to such diverse materials as cheese and glass wool. was also used by Block (1953) in a study of humidity rctluircments for mould growth.

Mould growth in occupied dwcllings occurs on wall and ceiling surfaccs decorated prcdominantly with emulsion paint and wallpapcr although vinyl wall coverings arc sometimes affected: gloss paint on window joincry also supports the growth of moulds. It was considcrcd vital, therefore, that laboratory studies on thcA ,~. requirements of moulds found on damp surfaces in dwellings should bc carried out on substrates representative of those affected in practice and this tbrmcd an integral part of the work reported here. By reducing general moisture levels in dwellings, substrate moisture Icvcls will also be reduced, unless high levels result from direct water ingress, and once below a critical level will bc insufficient for mould growth to occur. One of the prime aims of the study therefore was to obtain data on theA,, levels which are limiting, so as to provide building practitioncrs with inlbrmation on critical humidity levels for the control of condensation-induced mould growth within dwellings.

MATERIALS A N D M E T H O D S

Test fungi

The moulds used in the study (Table 1) were selected from those which had been isolated from the air of more than 10% of the domestic

262 C Grant. C A. Hunter. B. Flannigan. A. F. Brave D'

TABLE 1 Moulds Isolated from Air in Dwellings" and Used in Water Activity Studies

Mould Percentage frequency of occurrence (%)

In all dweUings In air samples sampled where found

AIternaria alternata (Fries) Keissler 17.0

Aspergillus spp., including 74-5 A. repens (Corda) Saccardo A. versicolor (Vuillemin) Tiraboschi

Aureobasidium pullulans (de Bary) Arnaud 46.8

Cladosporium spp., including 88-7 C cladosporioides (Fresenius) de Vries C herbarum (Persoon) Link ¢r S. F. Gray C sphaerospermum Penzig

Fusarium spp., including 10.6 F. moniliforme Sheldon

Geom.vcespannorum (Link} Sigler and Carmichae[ 56.7

Mucor spp+. including 38.3 M. plumbeus Bonorden

Penicillium spp., including 95-7 P. br~wicompactum Dierckx P. chrysogenum Thorn P. nigricans Bainier ex Thorn P. spinulosum Thorn

Phoma herbarum Westendorp 36.2

Sistotrema brinkmannii (Brcsadola) J. Eriksson 51-1

Stachybotr2,'s atra Co rda 12.8

Ulocladium spp., including 61.7 U. chartarum (Preuss) Simmons U. consortiah, (Thumen) Simmons

2.6

52.0

13.8

73.7

I-0

2O-3

8.4

90.2

6.1

55.5

4.5

18-2

"Taken from Hunter et al. (1988). Nomenclature according to Catalogue of the Culture Collection of the Commonwealth Mycological Institute (Eighth Edition 1982 and 1983 Supplement}.

dwel l ings su r veyed ant i wh ich , wi th o n l y two excep t ions , were also i so la ted f r om m o u l d y wall su r faces wi th in dwel l ings i n c l u d e d in the air- spo r a su rvey ( H u n t e r et al., 1988). All the species were assessed for the i r A w r e q u i r e m e n t s for g e r m i n a t i o n a n d g rowth o n n u t r i en t agar , a n d

Moisture requirements of moulds from domestic dwelling~ 263

selected species were similarly assessed on a range of substrates representative of those affected by mould growth in dwellings.

Isolation and monitoring of moulds on walls

Areas of mould growth on walls were swabbed with culture swabs (Sterilin. $46) which were subsequently shaken in sterile Tween 80 (0.01%) and a series of dilutions prepared from the washings. Aliquots of each dilution were spread on to plates of 2% malt extract agar (MEA) containing antibiotics (Hunter et al.. 1988) and incubated. Washed swabs were streaked onto additional plates to check that no spores were missed. All plates were incubated at 25°C for recognition and eventual identification of mould cultures. Where areas of mould on walls were regularly monitored, swabs were taken at 150mm intervals along horizontal transects beginning and spaced at 250 mm above floor level: in particularly heavily colonised parts of the wall. samples were taken at 75 mm intervals along the horizontal transects. Spot samples were taken only from obviously mouldy areas.

Control of water activity

Water activity in the different substrates was controlled by the use of saturated salt solutions (Stokes & Robinson. 1949; Wexler & Hasegawa. 1954; Winston & Bates, 1960; Young. 1967) and solutions of potassium hydroxide (Solomon. 1951) to produce atmospheres of specific relative humidity (Rtl) within incubation chambers at the temperatures used in the different studies, in preparing and maintaining the humidity chambers the stipulations made by Wexler and Hasegawa (1954) and Winston and Bates (1960) regarding control and accuracy of RH were carefully observed. Values for the RH of the saturated salt solutions are given in Table 2. Where RH values were imprecisely defined in the literature or where solutions were used at temperatures different to those quoted against specific RH values, a check was made on the actual levels attained in the studies using a wet and dry bulb hygrometer-thermocouple arrangement (Anon. 1955).

Selection of temperatures for A,, studies

Previous studies of the A,~ requirements of mould fungi growing on nutrient media have typically been carried out at temperatures between 20-25°C and similar temperatures were used in the present work to provide comparison with published A,,, values.

264 C Grant. C A. Hunter. B. Flannigan. A. F. Braveo'

TABLE 2 Relative Humidity Values of Saturated Salt Solutions

Salt Temperature (° C)

5 12 18 20 25

BaCI2.2HzO 91.0' 90-7' KBr 83.0" KCI 88 .0" 86-5-88.0 '~ 85-0-86-5 h 85.0" KCIO~ K:CrO 4 88.0 h KH,PO4 98"0-99"0 h KNO3 96"5 h 95-5-96"0 h 94.0" 93.2 ~ KzSO.~ 98,5 h 98.0-99-0 h 97.2 ~ MgSO4.7 H,O 90.0 h NaCI 75,0 I' 76.0-76-5 ~' 76.0/' NH4CI 79.0-79-5 ~' 79.5 j' (NH4)2S04 82"5 h 80'5-81"0 h 80"5 h Pb(NO3), 97"0 l' ZnSO.~.71:I20 94-5/'

79.0 a 84.3 a 98.0 a

96-6'1 92.5 b

76.0 't

88.5 h

"Wcxlcr & tlascgawa (1954). ;'Winston & Bates (1960). "Yot, ng (1967). 'tStokcs & Robinson (1949).

To ob t a in i n f o r m a t i o n o n ac tua l t e m p e r a t u r e s in dwel l ings , tile t e m p e r a t u r e at the in te r io r ( r o o m side) s t t r facc o f the ex t e rna l walls o f an o c c u p i e d dwel l ing in West L o n d o n a f fec ted by m o u l d g rowth were m o n i t o r e d d u r i n g J a n u a r y - F c b r u a r y 1985. Wall su r face t e m p e r a t u r e s in the l iving r o o m (0-45 m a b o v e t l oo r level) a n d in the b a t h r o o m (1.0 m a b o v e f loor level) were m e a s u r e d at h a l f - h o u r l y in te rva l s us ing a su r face t e m p e r a t u r e p r o b e ( t he rmi s to r , type E U , G r a n t I n s t r u m e n t s Ltd) c o n n e c t e d to a m e m o r y r e c o r d e r ( type M R I - U , G r a n t I n s t r u m e n t s Ltd); m e a s u r e m e n t s o f a i r t e m p e r a t u r e a n d h u m i d i t y in the s a m e r o o m s were t aken s i m u l t a n e o u s l y us ing a m e m o r y r e c o r d e r fi t ted with a f ixed t h e r m i s t o r t e m p e r a t u r e s e n s o r a n d a c ap ac i t i v e h u m i d i t y s e n s o r ( type M F 2 UL, G r a n t I n s t r u m e n t s Ltd).

Da ta co l lec ted wcre a n a l y s e d by m i c r o c o m p u t e r . T h e w l p o u r pressures in each r o o m were c a l c u l a t e d f r o m the m e a s u r e d a i r t e m p e r a t u r e s a n d R H us ing an empi r i ca l f o r m u l a for the s a tu r a t ed v a p o u r p re s su re (BS 1339, 1965). T h e s e were t h e n c o m b i n e d with the s a tu r a t ed v a p o u r

Moisture requirements of moulds frorn domestic dwellings 265

pressures at the measured wall surface temperatures to give the RH values at the surface. It was assumed for the purposes ofcalculat ion that the air in each room was well mixed so that the vapour pressure at the wall surface would be close to that in the centre of the room. If condensation was occurring, the surface relative humidity, appeared to exceed 100%.

Based on the results of monitoring, temperatures of 5. 12 and 18°C were selected for studies of mould growth on building-related materials over a range of substrate A,,,s.

Substrates and incubation techniques

Nutrient agar For growth rate/temperature studies 1 ,ul ofspore suspension prepared in sterile Tween 80 (0.01%) and containing approximately l06 spores ml -~ was inoculated centrally on to triplicate MEA petri dishes and incubated at temperatures between 5 anti 37°C. Mean colony radii were calculated from three 120 ° opposed radial mcasurements on each dish.

For growth rate/A,, studies at 12 and 25°C, triplicate sterile MEA strips measuring approximatcly 1 0 x 3 0 x l m m were positioned on the underside of tile lids of steriliscd glass petri dishes (Sirockin & Cullimorc, 1969) containing appropriate saturated salt solutions. These wcre equilibratcd for 7-10 days and then inoculated as above. The mean ratc of radial extension was calculated from measurements of colony diameters made over periods of up to 21 (25°C) and 42 days (12°C).

Observations of spore gcrmination and growth at different Aw were made on hanging drop cultures of moulds. One drop of sterile molten MEA was placed central lyon a sterile coverslip (22 × 22 ram), allowcd to solidify, and the coverslip inverted onto the Vaseline-coated rim of a perspex cylinder (10 mm long × 13 mm ID) itself sealed with Vaseline onto a glass microscope slide and containing 0.5 ml of an appropriate saturated salt solution. After equilibration at 12°C and 25°C for 7-10 clays, triplicate drops were inoculated as above and examined micro- scopically daily for 10 days and subsequently at regular intervals up to 21 (25°C) and 42 days (12°C).

Woodchip paper Strips ofwoodchip wallpaper (40 x 10 mm), either plain or painted with a mould-susceptible white emulsion paint (Grant et al., 1986). were placed in minimal salts solution (Flannigan, 1977) and autoclaved at

266 C Grant. C A. Hunter. B. Flannigan. A. F. Brave~

109°C for 10 min and dried overnight. For assessment of the effects of an added carbon source, some strips were dipped in sterile 1% sodium carboxymethyl cellulose solution (NaCMC. low viscosity, BDH Ltd) and dried under aseptic conditions. All strips were suspended singly above 4 ml of appropriate saturated salt or potassium hydroxide solutions in sealed 150 × 16 mm rimless test-tubes and equilibrated for 14 days. For each species of mould, selected triplicate strips were inoculated using a dry, sterilised camel hair brush to transfer spores from M EA cultures. The tubes were incubated and observed regularly for 21 (25°C) and 42 days ( 12 °C). but in another series of experiments, emulsion painted woodchip paper strips (without added NaCMC) were also incubated at 18 and 5°C (for 29 and 70 days respectively).

Other wall coverings To compare the Aw requirements for germination of three species of Cladosporium on a range of substrates, squares of patterned wallpaper and a vinyl wall covering, in addition to plain and emulsion-painted woodchip paper, (40 × 40 mm) were sterilised by exposure to gamma radiation and positioncd over 50 ml of an appropriate saturated salt solution in crystallising dishes (90 mm diametcr × 55 mm dccp). To assist fixing onto the plastic mesh supports, the paper squares were cut with lugs (10 × 10 ram) protruding from two opposite sides. After equilibration for 10 days, triplicate squares were inoculated centrally with spores picked off sporulating cultures on the tip of a sterile, lightly- moistened needle and incubated at 20°C for 42 days inside sealed polythene bags to prevent changes in RH conditions within the dishes. A typical incubation chamber is illustrated in Fig. I.

Emulsion-l~ainted plaster Builders" gypsum finishing plaster (British Gypsum Ltd) was mixed (2 : 1) with sterile one-tenth strength minimal salts solution (Flannigan, 1977), poured into 30 mm diameter plastic petri dishes to a depth of 6 mm and allowed to cure at room temperature for 2-3 weeks. The exposed surface of the plaster "plug' was then painted with the same mould- susceptible emulsion paint used on the woodchip paper. Using a hot wire, four 90 ° opposed notches were cut in the rim of the base of each dish to facilitate gas exchange, and the painted surfaces sterilised by exposure to UV light for 15 min. The plugs were equilibrated for 14 days on a perforated platform over 500 ml of an appropriate saturated salt solution in groups of 10-12 in sealed, clear plastic boxes (220 × 115 × 80 mm). After central inoculation with selected moulds using a camel hair brush as before, the plugs were incubated and examined for growth over 21 (25°C)-70 days (5°C).

Moisture requirementc of mould~ from domestic dwellings 267

Fig. I. "l}'pical incubation chamber used for dclennination of .'l~, retluirenlcnls of mc~ulds.

I.in.~eed oil plant A mould-susceptible linseed oil paint (Braver)' et a/.. 19,~3) was painted 0il tO il central ;.lre,I (25 X ]5 i l l i l l ) o f glass nlicroscot~e slides ailcl stori l ised by t.'xpo,sure to gal i l i l la rad ia t ion after I l ion)ugh cto, ing in ihc laboratory. Fo l low ing equ i l i b ra l io i l for 1() days over appropr ia le saturated sail so lut ions in crysta l l is ing dishes, t r ip l icate painted slides wore inocul;-i icd wi th spores of Ceado.seJorittm sp|'), tiSillg a sterile n lo is lcnod needle, i i lcul)ated ;.it 20°C for42 d,ivs inside sealed pol.vtlierie t~ags and observed l])r spore gern l inat ion.

RESULTS AND I)ISC[JSSION

Range of nloulds isolaled from ~'alls

Apart [toni Mr,'or ldumheu.~ • and SLstotrema hrinkmannii, all moulds which had been isolated l'rom ihe air o f 2> 10% of sun cved domest ic

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Moisture requirements of moulds from domestic dwellings 269

dwellings (Hunter et al.. 1988). were also isolated by swabbing of mouldy wall surfaces within dwellings in the air spora survey (Table 1).

Wall surface temperatures

Wall surface temperature data are summarised in Table 3. In the intermittently-heated living room the mean wall temperature over the six weeks" monitoring period was 7.9°C, the lowest temperature recorded was 1.9°C and the highest 14.5°C: the largest temperature fluctuation during a single week was 10.5°C while over the whole sample the difference was 12-6°C. A similar pattern was observed in the bathroom although here the overall mean wall temperature was higher at 11.6°C, doubtless due to the fact that the bathroom was situated on the first floor of the dwelling and subject to thermal gain from heat input from various grotlnd floor sources.

The calculated overall mean surface RH values for the living room and the bathroom were similar (78% and 80% respectively) but there were differences between the amount of time that RH values at the wall surface exceeded pa rticula r levels. Not u nexpectedly the a mottnt of t i me that lhc bath room wall surfacc exceeded 70% R H was 50% highcr than in the living room. Results for RH values >80% wcrc broadly similar but at >90% and > 100% RH (free water at the wall surface) the living room wall surface was 'wetter" for more than twice as long as the bathroom. The explanation here is that in the bathroom, ambient RI! levels, and hence surface levels, rose sharply during bathing, although such inputs of moisture vapour wcrc of relatively short duration. In the living room on the other hand, moisture vapour input from the occupants, from an unvcnted liquefied petroleum gas convector heater and from the adjacent kitchen was sustained over much longer periods each day.

Temperature optima for growth

Maximum radial growth rates were determined tbr six selected moulds on 2% MEA (Fig. 2). These varied from 0-7 mm day -~ at 20°C for Cladosporium sphaerospermum (CS5) to 3 . 8 m m d a y -I at 25°C tbr Ulocladium consortiale. Optimum temperatures were 20°C for C. sphaerospermum (CS5) and Penicillium chrysogenum, 25 °C for Aspergillus versicolor, Penicillium brevicompactum and U. consortiale, and 30°C for Stachybotrys atra. Although S. atra spores germinated at 37°C (without further measurable growth), only P. chrysogenttm was able to grow at this temperature. However, all six moulds grew to some extent at 5°C

270 C Grant. C A. Hunter. B. Flannigan, A. F. Brave~."

• Aspergdlus vers~color AV1 x Cladosporlum sphaerospermurn CS5

Penlcllhum brevlcompactum ST2 Penlcilhum Chrysogenum ST4

• Stachybotrys atra SA 2 E} Ulocladlum consort la le UCO 5

40

35 :

30

I

2.5 •

~" 2 O

(...3

0.5

0 10 20 30 40

Tempera tu re - °C

Fig. 2. Effect of temperature on growth rate on 2% malt extract ag;lr.

indicating their ability to sustain growth over a wide temperature range once established on coh.l surfaces with faw)urable Aw conditions.

A,, optima for growth at 12 and 25°C

Growth rates for selected moulds on 2% MEA at different A ,~s at 12 and 25°C are plotted in Figs 3 and 4. The iowestA,,,optimum at 12°C was 0-87 for P. brevicompactum while the highest was 0.985 for tile other moulds with the exception o f P ch~sogemon (0.96). At 25 °C P. brevicompactum required a somewhat higherAw (0.885) tbr maximum growth than at the lower temperature, while that required by A. versicolor. S. atra and U consortiale was very slightly lower (0.98). The optimum forA. versicolor at 25°C was reduced to 0-965, the same value as for P. chrysogenum, which was virtually unchanged by the higher temperature.

For four of the six moulds the changes in Aw optima between 12 and 25°C were very small (+_0-005, equivalent to 0.5% RH), but for A. versicolor there was a reduction of 0.02 at 25 °C (its optimum temperature for growth). Unexpectedly, for P. brevicompactum there was an increase of

MoLvture requirements of moulds from domestic dwellings 27 i

2 0 • Aspergi l lus vers ico lo r AV1 Jt C ladospor ium sphaerospermum CS5 ~, PemcillJum b rev l compac tum ST2 o Penlcdhurn ch rysogenum ST4 • S tachvbo t r ys atra S A 2 o U loc lad ium consor t la le U C 0 5

1 5 ~

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0 ~ - - - - . - a . - - ~ _ ~ _ l _ . ~ o~--.--e-~"-"~___a._ I J 076 0 8 0 0 8 4 0 8 8 0 9 2 0 9 6 100

A w

Fig. 3. Effect of A,,, on growth rate on 2% malt extract agar at 12°C.

4 0 • Aspergdlus vers lco lo r AV 1 ~t C ladospor ium sphaerospermum CS5 ,'. Pemcdl ium b rev i compac tu rn ST2 o Penicdhum chrysogenum ST4 • S tachybo t r ys atra S A 2 c~ o

"~ 3 0 r~ U loc lad ium consor t i a le UCO 5 J ~

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g

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Fig. 4. Effect of Aw o n growth rate on 2% mal t extract aga r at 25°C.

,-) .~ _7_ C Grant. C A. Hunter. B. Flannigan. A. F. Braven.'

TABLE 4 A,, Minima for Growth of Moulds on 2% Malt

Extract Agar

Mould Temperature (° C)

12 25

A It. alternata (AAI) 0.91 0.89 A. repens (AR5) 0"79" 0-76 A. versicolor (AVI) 0.83 0-79 Aur. pullulans (AP3) 0.87 0.89 C. cladosporioides (CC I ) 0-83 0.84 C. sphaerosperrnum (CS5) 0.83 0-84 F moniliforme (FM4) 0.91 0-89 G. pannor~tm (GP2) 0"87 ~ 0.89 M. plumbeus (M P I ) 0.87 0.93 P. brevicompactum (ST2) 0.79 0.79 P. chr)'sogenum (ST4) 0.79 0.79 P nigricans (ST45) 0.83 0.79 P. spinulosum (ST8) 0-79 0,79 Ph, herbarum (Pill) 0.91 0,93 S. brinkmannii (SB I) 0.96 0,97 St. atra (SA2) 0.91 0.93 U. chartatTml (UCA4) 0.87 0.89 U. consortiah" (UCO5) 0-91 0.89

"Germination only.

0.015 in the A ,,. o p t i m u m at 25 °C (also its o p t i m u m growth temperatt , re). However. the effects o f such very small changes in ,,I,, opt ima at temperatt , res between 12 ° and 25°C seem t, nlikely to have any practical s ignif icance for mould growth in dwellings.

Minimum A . values for growth at 12 and 2 5 ° C

Table 4 summar i ses the data for m i n i m u m Aw values for growth at 12 ° and 25°C on 2% M E A for all o f tile moulds studied.

At 12°C tile lowest A,~ value at which spores germinated and growth cont inued was 0.79 for Penicillium spp. (P. brevicompactum. P. chr3"sogenum and P. sl~inulosunl); Aspergillus r~Twns germinated at this A ,~ but failed to sustain growth. The highest m i n i m u m A,~ was 0-96 for S. br inkmanni i .

At 25 °C m i n i m u m A ~,s were u n c h a n g e d for the three penicillia cited above, a l though Penicillium nigricans grew at 0.79 (at the lower temperatt , re its m i n i m u m was 0.83). The lowest recorded A,~ for

Moisture requirements of moulds frorn domestic dwellings 273

sustained growth was 0.76 for A. repens. Again. S. brinkmannii had the highest minimum A ,,, requirement (0-97). which is largely to be expected as this organism is normally associated with decaying window joinery where high moisture levels will prevail (Baker et al.. 1977: Savory. & Carey. 1979: Henningsson & Kiiarik. 1982).

Comparison of results obtained at 25°C in the present study with minimum Aw values tbr growth reported in the literature, generally on nutrient media at temperatures of 20-25°C (Table 5). shows good agreement in most cases. In the work reported here A. repens required a minimum A,v of 0.76 for growth whereas other workers have reported lower values of between 0.71-0.75. Conversely the strain of Cladosporium cladosporioi~h's (CC I ). isolated from a domestic air sample, grew down to an A,,, o f 0.84 while the strain isolated from wheat grain by Magan and Lacey (1984) required A~ of 0.88 for growth at 25°C. Similarly P. brevicompactum from the domestic air spora grew atA ,, 0.79 while strains reported on by Christian (1980) and Magan and Lacey (1984) required A w levels of 0.81 and 0.82 respectively.

TABI.E 5 A , Min ima Obta ined by Previous Workers

Mouhl Minimum A ~.

for spore germination fi~r growth

AIt. alternata 0.85 g i 0.88¢ A. rt7wns {).70;" 0.715:" 0.72: ~ 0.72" 1).7 I;"0.73-0.75:h0.75 x A. versicolor 0.75;z t 0.76:x 0.78;" 0-80: ~ 0.81 i 0-78: 'z~ 0.80 h C cladosporioides 0.8(¢ 0.88 x C. herbarum 0.85;" ~r 0-88" 0.90~: b: moniliforme {).87" M. plumbeus 0.93 '1 P. brevicompactum 0. 78; f 0- 80;~ 0- 81 ;h 0- 84 ~ 0. 81 ;,t 0. 82 x P chrysogenum 0-78; ~. f 0"8 l: h 0"84; t' 0-85 j 0"79 '1 P. spinulosum 0.80fJ 0.80 a Ptt. herbarum 0. 92" St. atra 0-85/0.93-0"9Y 0-94'" J

"Armol ik & Dickson (1956) bAvari & Allsopp (1983). "Ayerst (1969). dChris t ian (1980). "Eveleigh (1961). /Hock ing & Pitt (1979). XMagan & Lacy (1984).

hMislivec & Tuite (1970). iMislivec et al. (1975). JPanasenko (1944. 1967). kPitt & Chris t ian (1968). tSmith & Hill (1982). " S n o w (1949).

274 C Grant. C A. Hunter. B. Flannigan. ,4. F. Brave~'

From the publ ished data it can be seen that h igher A~, levels are required for sustained l inear growth o f moulds than for ge rmina t ion of their spores. In the major i ty of cases differences of a r o u n d +0-02 have been recorded (Table 5) a l though for Cladospor ium h e r b a n t m the difference was found to be +0-05 (Magan & Lacey. 1984). For S. atra. a

very, large difference o f +0.09 was found by Ayerst (1969), a l though this was after incubat ion for95 days. As the data reported by Magan & Lacey (1984) for a n u m b e r of field and storage fungi clearly show. asexual sporulat ion general ly requires higher A,,, levels still. Al though not reported in detail here, s imilar observat ions on differences in Aw levels required for sporula t ion were made in the present study.

Growth on building substrates

M i n i m u m A,~ values for growth of selected mouh.ls on woodchip wal lpaper are summar i sed in Table 6. On the paper treated with minimal salts solution alone, the min imunlA ws were without exception considerably higher than on M EA at both 12 and 25 °C. Thus while C sl~haerospermum (CS5), for example, grew at an Aw of 0.83 at 12°C on MEA, it failed to germinate on the woodchip paper even at an A,,, of 1.00 at this temperature, a l though it was capable o f growth at this A w level (1.00) at 25 °C. S. atra failed to grow at cither 12 or 25 °C on paper supp lemented with only minimal salts. An anoma lous result was obta ined tbr P. spinuloston which had an A ,~ m i n i m u m of 0.87 at 12°C, but rcquircd an A~, o f at least 0.97 for growth at 25°C on similarly unenr i ched paper.

TABLE 6 A,, Minima for Growth of Certain Moulds on Woodchip paper: Effect of Added Nutrients

Species of mouM 12 °C 25 °C

Untreated + NaCMC Untreated + NaCMC

A. versicolor (AV I ) 0-9 I C cladosporioid~:~" (CC 1 ) 0.98 C. ,~phaero.wennum (CS5) ng P. brevicompactum (ST2) 0.91 P chrysogenum (ST4) 0.87 P. spinulosum (STS) 0.87 St. atra (SA2) ng U chartamm (UCA4) 0-96 U consortiale (UCO5) 0.98

0.91 0.84 0.84 0-96 0-98 0.97 0-96 1.00 0.97 0.91 0.89 0.84 0.87 0-84 0-84 0-87 0-97 0.93 ng ng 0.98

0-96 0-98 0.93 0.96 0.97 0.97

ng = no growth.


TABLE 7 Aw Minima for Growth of Certain Moulds on Emulsion-Painted Woodchip Paper: Effect

of Added Nutrients

Species of mould 12 °C 25"C

Untreated + NaCMC Untreated + NaCMC

A. versicolor (AVI) 0-87 0.83 0.79 0.79 C. cladosporioides (CC1) 0-91 0.91 0-93 0-93 C. sphaerospermum (CS5) 0-96 0.87 0-93 0.89 P. brevicompactum (ST2) 0-87 0.83 0-84 0.79 P. chrysogenum (ST4) 0.87 0.83 0.84 0.79 P. spinulosum (STS) 0.83 0-83 0-89 0-89 St. atra (SA2) 0.96 0.96 0.98 0-97 U. chartarum (UCA4) 0.91 0.91 0.93 0.89 U consortiale (UCO5) 0-96 0.91 0.93 0.93

In general the effect of supplyir |g an addit ional carbon source (NaCMC) to simulate surface soiling with nutrient materials in tile domest ic situation, was to cause a reduction in the m i n i m u m A,, level at which growth would occur. Thus C. sphaero.v~ermttm (CS5)grew ;.it an A,~ of 0.96 at 12°C, for S. atra, al though NaCMC treatment was without effect at 12°C, growth did occur at an A,~ of 0.98 at 25°C.

Coat ing woodchip wallpaper with a mould-susceptible emulsion paint caused further reductions in m i n i m u m A ~ levels for ;.ill the moulds at both 12 and 25°C (Table 7). For C. sphaerospermttm (CS5) at 12 and 25°C respectively, m i n i m u m values of 0-96 and 0.93 were recorded, compared with no growth and 1.00 on unpain ted paper previously (Table 6). S. atra now grew a tA~ levels of 0-96 and 0.98 at 12 and 25°C (Table 7), where previously no growth had occurred (Table 6). P. spinulosum once again had a higher m i n i m u m A,,, requirement for growth at 25°C than at 12°C; a surprising result since its op t imum growth temperature has been reported as 26-28 °C (Domsch et al., 1980) and therefore one would expect growth at a lowerA w level at 25°C, than at 12 °C. ForA. versicolor the m i n i m u m A ,,, level on the emuls ion painted paper at 25°C (Table 7) was the same as that on MEA (Table 4), while for other moulds the values were considerably different; notably P. spinulosum, C. cladosporioides and C. sphaerospermum (CS5).

As on unpa in ted woodchip paper, the addi t ion of an extra source of carbon (NaCMC) generally caused a reduction in m i n i m u m A,,, levels and tended to have a more p ronounced effect at 12°C than at 25°C. Further, the magni tude of the effect was greater on the painted paper,

276 C. Grant. C. A. Hunter. B. Flannigan. A. F. Braveo'

where reductions in A , levels at 12 °C averaged 0.029 (equivalent to 2.9% RH) and ranged up to 0-09 (C. sphaerospermum CS5) (Table 7), as opposed to an average reduction of -0-01 ranging up to 0.04 (C. sphaerospermum CS5) on unpainted paper (Table 6). The effect of added nutrients in permitting growth at lower,4 ,,s than on plain substrates has been noted previously by Block (1953).

The A,, minima for growth of six selected moulds on emulsion painted woodchip paper and emulsion painted plaster at temperatures of 5, 12. 18 and 25 °C are plotted in Figs 5 and 6, respectively. The general trend of increasing temperature permitting growth at lower A w levels can be seen for both substrates although anomalous results were obtained for C. sphaerospennum (CS5) on painted paper, where an unexpected low A , value was recorded at 5°C (Fig. 5). and for S. atra on painted plaster, where a similar result was observed (Fig. 6). For C. sphaerospermum (CS5)

<

100

095

0.90

085

0 8 0

• Aspergil lus vers,color AV1 Cladosporlum sphaerospermum CS5 Penicillium brevicompactum ST 2

o Penicdlium chrysogenurn ST4 • Stachybotrys atra S A 2 u Ulocladlum consortiale UCO 5

"~" . A f t e r 70 dav~

I - - . 1 - L _ _ J 0.755 L ~0 15 20 25

T e m p e r a t u r e . ° C

Fig. 5. Effect of tempcraturc on A w m i n i m a l or growth on emulsion-paintedwoodchip paper.


• Asperglllus versicolor AV1 • Cladospor~um sphaerosperrnum CS 5 =, Peniclll~urn brewcompactum ST2 o Pemcdlium chrysogenum ST4 • Stachybotrys atra SA2

1 0 0 ~ U C O 5

0 8 5 ~ 5 Tecnperature - °C

Fig. 6. Effect of temperature on ,4,, minima tbr growth on emulsion-painted plaster.

on paper the difference in A,,, between 5 and 12 °C was +0.04, but for S. atra tile difference was only +0.01. Although after 21 days on painted paper at 25°C a minimum ,4, of 0.97 was recorded tbr S. alr~L when incubation was extended to 70 days. limited growth was noted at 0-93,4,,. For this mould, growth was evidently possible at a lower A , provided temperature (optimum 30°C - - see Fig. 2)and nutritional requirements were met; an A,, minimum of 0.93 was also recorded on MEA at 25°C (Table 4). Both Scott (1957) and Magan & Lacey (1984) have observed that when moulds are growing at sub-optimal temperatures, the A , minima are higher than at temperature optimal.

Comparison of the two sets of results at 12 and 25°C shows that. at the lower temperature, the nature of the base substrate (paper or plaster) had no effect on theA,~ minima obtained for four of the six moulds, although lbr C sphaerospermum (CS5) and U. consortiale lower minima (difference 0-04) were recorded on the painted plaster than on the painted paper. At the higher temperature all of the moulds were affected but there was no consistent overall pattern. For A. versicolor. P. hrevicompacmm and P. chrysogenum, iowerA ~, values were recorded on the painted paper than on the painted plaster (lower by 0.05-0.07) while lbr (7..v)haerospermum (CS5). S. atra and U. consortiale the reverse occurred although the differences in min imum A,,, levels were smaller (lower by 0.01-0.04).

Data on the comparison of min imum A,,, values for germination of spores of three species of Cladosporium on a range ofsubstrates at 20°C

278 C Grant. C A. Hunter. B. Flannigan. A. F. Braver3"

TABLE 8 A,~ Minima for Germination of Species of Cladosporium on Different Substrates at 20 °C

Substrate C. cladosporioides C. herbarum C. sphaerospermum (10HC8) (40HC8) (9KR 7)

2% Malt extract agar 0.85 0.85 0.81 Woodchip wallpaper 0-97 0-97 0-90 Patterned wallpaper 0-93 0.97 0-90 Emulsion-painted 0-97 1-00 0-97 woodchip paper Emulsion-painted 0-93 0.90 0.90 plaster Linseed oil paint on 1.00 0-97 0-93 glass Vinyl wall coveting 0.97 0.97 0.90

are summarised in Table 8. C. sphaerospermum (gKR7} had the lowest or equivalent A,, minima on all of the substrates tested. On MEA its A,, minimum was 0.81 which was a lower value than that recorded for growth ofa diffcrcnt strain of the mould (CS5) at 25 °C (Table 4), while on the same substratc both C chuh~Sl~Oriohh's (10HCS) altd C. herlmrum (40IIC8) had minima of 0.85, agreeing well with the value for growth of C,. cladosporiokh's (CCI) at 25°C (Table 4) and the published valucs tbr spore gcrmination of both species (Table 5).

The three species had different Aw rcquiremcnts on the different substratcs. This was sccn, for exzt topic, on the mould susceptible linseed oil paint where,,lw minima of 1.00, 0.97 and 0.93 were recorded for C. chuloslmriohh's (10HC8), C. herbarum (40HC8) and C. sphaeroslwrmum (gKR7), respectively. A somewhat unexpected result was the germination of spores of this last species at art A,~ level of 0-90 on the vinyl wall covering where the other two species required an A,,. of 0.97.

Sequential development of mould on walls

Regular monitoring of the papered surface of a kitchen wall revealed a succession of moulds as the paper gradually became wetter with condensation as winter progressed (Table 9). Colonisation of the papered surface progressed from those moulds which appeared while the paper was still comparatively dry - - Penicillium spp. and A. versicolor through to moulds which appeared in the later stages when conditions were very w e t - UIocladium spp., S. atra, etc. Where there was direct


TABLE 9 Succession of Moulds Developing on Wall Surfaces and Ranking of Species According toA,, Minima for Growth at 25 °C

Moulds Minimum A .. for growth on

MEA Woodchip wallpaper

Primary colonisers Penicillium spp. 0.79 0-84-0-89 A. versicolor 0.79 0.84

Secondary colonisers Cladosporium spp. 0.84 0.98-1-00

Tertiary colonisers UIocladium spp. 0.89 0.97-0-98 G. pannon~m 0.89 - - F. moniliforme 0-89 Ph. herbarium 0-93 - - St. atra 0.93 1.00"

"Inferred. as no growth occt, rred on woodchip paper without an added source of carbon.

ingress of water to tile substrate resulting from a structural fault in another dwelling. S. atnt was found nearest the point of ingress, with Penicillium spp. and A. versicolor nearer the drier ma rgi ns of the a fleeted area. Also shown in "litble 9 are the m i n i m u m /I,, requirements for growth ofa n u m b e r o f t h e moulds found on d a m p wall surlaces. It can be seen that in very broad terms they have A,,. minima for growth on MEA which tall into three classes: <0.80 (Penicilfium spp., A. versicolor), 0.80- 0.90 (Cladosporium spp.), and >0.90 (Ulochufium spp., S. atra, etc). Observations of successional colonisation token together with laboratory determinat ions ofAw min ima permit construction of 3 classes of mould fungi (Table 9) as tbllows:

I Primary colonisers ~A, , . < 0.80 II Secondary colonisers ~ A ~ 0.80-0.90

III Tertiary colonisers ~ A w > 0.90

Clearly, so-called tertiary, coionisers can exploit a surface early or immediately ifA~, condi t ions exceed 0.90 so, for example. S. atra and doubtless other "wet' surface colonisers could act as primary invaders given immediately favourable condit ions.

280 C Grant. C A. Hunter. B. Flannigan. A. F. Brave~."

TABLE 10 Suggested Generalised Sequence of Colonisation of Susceptible Substmtes

Based on Data Determined in Present Study (see Tables 4 and 9)

A ~. level in substrate

Species of moulds colonising substrates at:

12°C 25°C

<0-80 A. repens A. repens P. brewicompactum A. versicolor

(Primary P chrk'sogenum P brevicompactum colonisers) P spinulosum P. ch~sogenum

P. nigricans P. spinulosum

0.80--0.90 A. versicolor Air. alternata Aur. pullulans Aur. pulhdans

(Secondary C. cladosporioides C. cladosporiohh's colonisers) C. sphaerospermum C. sphaerospermum

G. pannonon E monil~rtne M. plumheus G. patltlOntttt P. nigricans U. chartan~m U. chartanon U. consortiale

>0.90 AIt. alternata M. plumheus F. moni l~rme Ph. herhanon

(Tertiary Ph. herharunt X hrinkmannii coloniscrs) X hrinkmannii St. atra

St. atra U. consortiale

On woodchip wallpaper without an addit ional carbon source, the species tested seemed to fit into only two groups ~ <0.90 and >0.90. On the whole, however, observations ofcolonisa t ion patterns in the field and the results obtained from the laboratory studies of Aw requirements correlated well and a generalised colonisat ion pattern can be proposed (Table 10). The results suggest a likely construction of a postulated colonisation sequence for the fungi listed in Table 4. Table 10 shows those moulds which could be expected to colonise susceptible substrates in the three broad A,, classes described above. Although evidence on colonisat ion of d a m p wall surfaces is l imited (Table 9), extrapolation to the other moulds studied provides a useful indicator of potential colonisers in practical situations.

C O N C L U S I O N S

The lowest A ~ level recorded for mould growth was 0.76 (at 25°C) forA. repens on MEA but growth was extremely slow. For A. versicolor and


several Penicillium spp routinely found in the air and on d a m p wall surfaces in dwellings the value was 0-79. At the opposite end o f the scale. the commonly-occur r ing wall moulds U. consortiale and S. atra had min ima of 0.89 and 0.93 respectively on MEA at 25°C. Under these condi t ions the highest recorded m i n i m u m A,~ value was 0-97 for S. brinkmatmii which, a l though a c o m m o n componen t of the air spora, was not found on wall surfaces.

Whilst the m i n i m u m A,, on bui lding substrates such as emuls ion painted woodchip wallpaper (with or without an added carbon source) was also found to be 0-79 forA. versicolor and P. cho'sogenum at 25 °C. the wall surface temperatures recorded within an occupied dwelling often lay below 12°C for much of the time during winter months. At 12 °C.A,, min ima for the most xerotolerant of the moulds tested increased to 0.83 for P spinulosum and 0.87 for A. versicolor and P. ch~'sogenttm, although with an added carbon source each of these moulds grew down toA w 0.83. Furthermore. calculations of relative humidity, and hence A,,, values at living room and ba throom wall surfaces in the same dwelling showed that levels in excess of 80% (0.80A~) occurred on average for only between roughly one-third and two-fifths of the time over a six wcck period during the coldest winter months. Mould fungi present either as ungcrmirmtcd sporcs or as myccl ium would thcrcforc be subject to t luctuating condi t ions of temperature and ,4 ,,. which would bc likely to adversely affect their ability to colonis t and survive on susceptible substrates.

Under experimental conditions, increasing the nutritional status had the effect of reducing the requirement of mould fungi for moistttrc. The inference from this is that surfaces which are soiled or covered with a susceptible paint or paper do not need to become as d a m p for mould to develop. Increasing the ambien t temperature also reduces the critical value of,'/w necessary for the growth of mould. However in normal housing conditions, a rise in temperature will cause A w to lall sharply unless it is accompanied by considerable generat ion of water vapour. The principle for building practice implied by these studies is that otherwise susceptible surfaces can be kept free of mould if relative humidit ies and tern pc ratu res at those su rfa ces a re main tai ned below,4 ,,; 0-80. Th is A ,~ will be achieved by various combina t ions o f air temperature and relative humidi ty in the room space according to the thermal properties of the wails themselves.

AC K N O W L E D G EM ENTS

The authors wish to acknowledge the help of Mr C. H. Sanders of the Building Research Establishment 's Scottish Laboratory for assistance in

282 C. Grant. C. A. Hunter. B. Flannigan. A. F. Brave~

the recording and computer analysis of the temperature and humidity. data, and also the co-operation of the local authority personnel and tenants in making properties available.

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