APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 1980, p. 480-487 Vol. 39, No. 30099-2240/80/03-0480/08$02.00/0

Controlled Formaldehyde Fumigation SystemN. R. ACKLAND,* M. R. HINTON, AND K. R. DENMEADE

Bioengineering Department, Research and Development Division, Commonwealth Serum Laboratories,Melbourne, Australia

A comparative study of formaldehyde (HCHO) fumigation was carried out bycontrolled vaporization, using an electric vapor generator, and by the Formalin-permanganate method. Determination of vapor levels as well as bactericidalaction showed the generator to be more effective. Maximum achievable fumigantlevels were temperature dependent and related to the equilibrium vapor concen-tration of HCHO. At a room temperature of 21°C, vaporization of more than2,000 ytg ofHCHO per liter resulted in conversion of HCHO to paraformaldehyde,which condensed on surfaces and contributed to prolonged residual vapor levels.An electronic monitor is described which is capable of detecting HCHO levels aslow as 10 Ag/liter and can be used to monitor the complete fumigation process.

Formaldehyde fumigation has long been anaccepted method of sterilization for areas wheremicrobiological cleanliness is required. Fumiga-tion is usually carried out by mixing potassiumpermanganate and an excess of Formalin (40%formaldehyde) solution in a suitable container;sufficient heat is generated by oxidation of partof the formaldehyde (HCHO) with permanga-nate to vaporize the remaining formaldehydeand water (1, 6, 7, 10). This method of fumigationis violent, messy, and potentially explosive (7).In this paper, an alternative procedure is de-scribed for fumigation by controlled vaporiza-tion, in an electrical vapor generator, of eitherFormalin or paraformaldehyde, a polymer ofHCHO which is converted to HCHO vapor uponheating (8). Formaldehyde levels were comparedin fumigations of several work areas by the con-trolled vaporization and Formalin-permanga-nate methods, and the effectiveness of each pro-cedure was determined from the survival of re-sistant Bacillus subtilis spores (2, 5, 9, 12).

MATERIALS AND METHODS

Reagents. A commercial Formalin solution con-sisting of 40% HCHO containing 10% methanol as adepolymerizing agent (Imperial Chemical Industries,Melbourne, Australia) was used throughout. Para-formaldehyde powder (Koch-Light) was a white pol-ymer consisting of 95% paraformaldehyde.Areas fumigated. A low-security area and a high-

security area were monitored throughout the study.The low-security area was a large processing labora-tory; during fumigation the air conditioning was turnedoff, but no attempt was made to seal air supply orexhaust ducts. The high-security area was a largesealed laboratory whose air supply was carefully con-trolled; exhaust air was passed through a gas-firedfurnace to ensure microbiological security. The airconditioning for this area was turned off during fumi-

gation, and the air supply and exhaust outlet valveswere closed.Assay of HCHO. Initially, formaldehyde vapor

concentrations were measured by taking air samplesthrough rubber tubes protruding into the room atvarious sample points. Four samples were taken persampling time, using an evacuated liter flask. Fortymilliliters of 0.5 M (NH4)2SO4 was added to each flask,which was then cooled to 4'C to dissolve the formal-dehyde. One milliliter of a suitable dilution of theresulting solution was then mixed with 1 ml of a freshlyprepared chromotropic acid reagent (1% chromotropicacid in 18 M H2SO4), and 8 ml of 18 M H2SO4 wasadded. After standing for 10 min, the mixture wasdiluted to a final volume of 25 ml, and the opticaldensity was determined at 540 nm. HCHO was esti-mated from a standard curve in the range 5 to 40 yg/ml, and the mean concentration from the four sampleswas determined.

Biological test systems. The spore suspensionused throughout was prepared from B. subtilis NCTC8233 prepared by culture for 7 days on sporulationagar (3) and suspension of the resultant growth inwater. The suspension was subjected to two cycles ofheating to 65°C for 30 min followed by centrifugationand resuspension in distilled water. A sample was thenstained and examined microscopically for spores (4).Glass rods 5 mm in diameter and 150 mm long, with aloop at one end and a graduation mark 50 mm fromthe other, were dipped into the spore suspension tothe mark. They were then suspended in a laminar-flow cabinet to dry and stored in a sterile jar. Beforefumigation, rods were placed in different parts of theareas to be tested and transferred to a sterile screw-capped test tube at the end of each procedure, imme-diately after evacuation of the fumigant. Twenty mil-liliters of saline was added to each tube to remove thespores from the rods, and an estimate of viable sporeswas obtained by plating dilutions of the suspensiononto nutrient agar. This was carried out within 2 h ofremoval of rods from the area. Plates were incubatedat 37°C, and then colonies were counted after 3 daysand checked after 7 days.

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FORMALDEHYDE FUMIGATION SYSTEM 481

Electric vapor generator. Initially, HCHO wasvaporized on a 500-W heating plate in a stainless-steelbeaker. The apparatus used subsequently, shown inFig. 1, consists of a stainless-steel 5-liter vessel with a1,200-W heating element. Sufficient capacity was pres-ent to allow the addition of water for humidificationand to contain the resulting foam. A copper plate wasinterposed between the element and the bottom of thevessel to ensure a uniform distribution of heat at athermostatically controlled temperature of approxi-mately 170°C.

Electronic formaldehyde monitor. The appara-tus used (Fig. 2) was based on a Figaro 812 N-typesemiconductor device, the conductivity of whichchanges in the presence of adsorbed molecules. Nosignificant interference was observed from substancesother than HCHO in the areas fumigated or by tem-perature changes normally encountered. Responsetime of the sensor, obtained from Digitron Engineer-ing, Sydney, Australia, was approximately 20 s. Themonitor was calibrated in an enclosed system againstHCHO released by paraformaldehyde vaporization.Good agreement was obtained between the weight ofparaformaldehyde vaporized and the HCHO concen-tration as measured by chemical assay, which indi-cated that there was no loss of vapor in the testsystem.

RESULTSHCHO levels by the Formalin-perman-

ganate and hot plate vaporization methods.For Formalin-permanganate fumigations, quan-tities of Formalin recommended have rangedfrom 12 to 59 ml/m3 of air space (6, 11), andratios for Formalin-permanganate have rangedfrom 3:1 to 5:3 (1, 10). In the following experi-ments, fumigations were carried out by using amixture of 17.8 ml of Formalin and 8.9 g ofpotassium permanganate per m3 of air space ina low-security area at room temperatures of 16,24, and 300C. In preliminary experiments, roomair samples were taken from each of four posi-tions, including the ceiling and the floor, andHCHO levels were determined by chemical as-say. Because no differences in concentrationwere observed between any position, all resultsfrom the sampling points were averaged. HCHOlevels were also measured after vaporization of12.5 ml of Formalin (70% of that used in theFormalin-permanganate fumigations to allow forlosses due to permanganate oxidation) on a hotplate at a room temperature of 27°C.

Results in Fig. 3 indicate that the highestlevels of HCHO were obtained in the Formalin-permanganate method at the commencement offumigation and then vapor levels dropped tobase levels of 200 to 500 ug/liter by 4 h. Theinitial concentration ofHCHO was temperaturedependent. Higher and more persistent levels ofHCHO were obtained after vaporization by thehot plate method at a room temperature of 27°C,

FG. Electric vapor generator.

which could be expected to be a more efficientsterilizing procedure. With the Formalin-per-manganate method, large amounts of water con-densate were observed on objects in the areaafter vaporization.

Sur,vival ofB. subtilis after ftumigation bythe Formalin-permanganate method. B.subtilis spores survived on some rods located atvarious places in a low-security area during a 24-h Formnalin-permanganate fumigation at an ini-tial temperature of 1800. Viable counts of 2 x106 were obtained from control rods, but onlytwo of nine rods exposed in the treated areawere sterile; viable counts of 20 to 600 wereobtained from the others. It is clear that, al-though this procedure was fairly effective, ster-ilization was not achieved.Twofcurther experiments were carried out in

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482 ACKLAND, HINTON, AND DENMEADE

SENSORDR IV

VOLTlAGEI SENSOR - MNPUT

SENSORHEAT ER

SUPPLY

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REFERENCEVOLTAGE

FIG. 2. System block diagram of the electronic monitor with controller.

lC

24eC27t

30t

FIG. 3. Comparison of Formalin-permanganate fumigations at 16, 24, and 30°C and a hot plate vapori-zation ofFormalin at 27°C.

a sealed jar at 20°C in controlled HCHO vaporlevels of 150 and 300 Ag/liter of air, with relativehumidity maintained at approximately 100%.The results in Table 1 show that exposure to 150,ug of HCHO per liter of air did not result insterilization even after exposure for 6 h. Expo-sure to 300 ,Ag/liter was much more effective,and no viable spores were detected at 3 h.Use of electric vapor generator. In Table

2 the results from several fumigations are shown(procedures 1 through 6), using the electric va-

por generator (Fig. 1) and different amounts ofFormalin in a low-security area at different ini-tial temperatures. In procedure 1, persistentHCHO levels similar to those obtained by thehot plate vaporization method (Fig. 3) were

achieved. No viable B. subtilis spores were de-tected after 24 h of exposure on the 18 sporerods located in the main laboratory area, but a0.5% survival was detected on a rod located in a

closed cupboard. The fumigant level in the cup-

board was 160 ,ug/liter after 6 h. Procedure 2 wascarried out with the same volume of Formalinand an initial room temperature of 15°C. Nosurviving spores could be detected after 7 h ofexposure on 12 spore rods located around theroom, but two located in cupboards showed a

survival of 1%.Procedures 3 through 5, which were carried

out for different times and temperatures andwith varying amounts of Formalin, are also de-scribed in Table 2. In procedure 6, a mixture ofparaformaldehyde and water equivalent to 8.9ml of Formalin per m3 of air was used. Again, no

surviving spores could be detected from rodsdistributed throughout the main laboratory areain any of the procedures.

All subsequent fumigations were carried outwith 3.7 g of paraformaldehyde per m3 of roomvolume, which is converted to HCHO equivalentto that present in 8.9 ml of Formalin. Humidityin each area was increased to 80%, a level which

pg

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FORMALDEHYDE FUMIGATION SYSTEM 483

has been shown to increase the effectiveness offumigation by causing HCHO to dissolve in afilm of moisture around microorganisms, whereit is more bactericidal than in vapor form (12).Use of Formalin at a room temperature of 200Cwould have contributed approximately 30% tothe relative humidity. More reliable humiditycontrol could be achieved with paraformalde-hyde, which was also more convenient to use.Use of electronic HCHO monitor. The

electronic monitor (Fig. 2) was developed be-cause of the time involved in chemical assays ofair samples. When the output of the monitorwas plotted againstHCHO vapor levels obtainedby chemical analysis, a power-law relationshipwas obtained (Fig. 4). Such a relationship isparticularly useful, since good sensitivity wasachieved at the lower vapor levels and the totalfumigation range was covered on the same scale.Relationship between temperature and

equilibrium vapor concentration. The rela-tionship between temperature and the equilib-rium vapor concentration is shown in Fig. 5. Fornormal air-conditioned laboratories at 200C, theequilibrium vapor concentration is approxi-mately 2000 ,ug ofHCHO per liter. HCHO vaporin excess of the equilibrium vapor concentrationwill recondense on the coldest surfaces, produc-ing a paraformaldehyde film over surfaces withinthe room. This film slowly evaporates, producingan unpleasant odor.HCHO levels after fumigation. The per-

sistence ofHCHO vapor after completion of thefumigation and restoration of ventilation is asignificant problem, especially in high-securitylaboratories. The extent of the problem can beseen from chart recordings from the electronicmonitor during fumigation of a high- and a low-security area (Fig. 6).

TABLE 1. Survival of B. subtilis spores incontrolled vapor levels of 150 and 300 pg ofHCHO

per liter of air at 20°Cyg of Expo-

HCHO/liter sure Total viable count % Survivalof air time (h)

Controls 0 2.5 x 106 100150 0.5 1.02 x 106 41150 1.5 8.15 x 104 3.3150 3.0 4.0 x 102 0.02150 6.0 1.0 X 102 0.01

Controls 0 4.5 x 106 100300 0.5 4.0 x 105 8.9300 1.0 6.5 x 102 0.02300 1.5 2.0 x 10 0.003300 3.0 None detected <0.0001300 6.0 None detected <0.0001

TABLE 2. HCHO vapor levels during a series offumigations with the electric vapor generator and

varying quantities offumigantTime Vaporafter level (,ug

Room comple- ofdure Fumigant (ml/m3) temp tion of HCfO/

(0C) vapori- liter ofzation

ar(h) ar

1 Formalin (17.8) 25 0 1,8502 2,2706 500

24 702 Formalin (17.8) 15 0 920

2 8505 7707 720

3 Formalin (17.8) 30 0 3,4002 2,6005 2,480

4 Formalin (8.9) 22 0 2,1903 2,1606 1,700

5 Formalin (4.4) 19 3 1,0406 Paraformalde- 20 1 1,750

hyde (equiv-alent to 8.9ml of For-malin)

Clearly, there was a rapid loss of fumigant inthe low-security area, through vents, leaks, etc.,but in the high-security area a higher level wasmaintained. Although restoration of the venti-lation at 11 h in the high-security area and 12.5h in the low-security area rapidly removed mostof the vapor, a residual HCHO odor remainedwhich was paralleled by a high monitor baselevel reading. WVhen ventilation was againstopped, at 14 h in the low-security area and 17h in the high-security area, the level of HCHOrose, showing that some revaporization of form-aldehyde was occurring. The odor was particu-larly noticeable in the high-security area, wherea vaporization rate of 40 ,ug of HCHO/liter perh was shown to occur continuously. Since 10 airchanges per hour occurred in the area, the lab-oratory atmosphere would have contained anaverage of 4 ,ug of HCHO per liter, a level inwhich it is too high to work (Table 3). Vapori-zation at 3.7 g of paraformaldehyde per m3 re-sulted in approximately 2 g/m3 (2,000 ,ug ofHCHO per liter) to condense on walls and fit-tings of the area, and 2 to 3 days would berequired for the dissipation of the residualHCHO. This has been confirmed by observation.Attainment of optimal levels of HCHO

without condensation. A controlled fumiga-tion was carried out in a high-security area

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484 ACKLAND, HINTON, AND DENMEADE

10-

OUTPUT

mv1.0-mV1

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10 100pg HCHOIlitre AIR

1000 10000

FIG. 4. Response of monitor to formaldehyde vapor levels.

where the vapor level was maintained below theequilibrium concentration to avoid condensationof HCHO. The comparator circuit on the moni-tor (Fig. 2) enabled a simple ON-OFF control ofthe electrical generators when the vapor concen-tration rose to a predetermined level.During fumigation, the generator vaporized

1.1 g of paraformaldehyde per m3 of room vol-ume (equivalent to 1,100 ,tg of HCHO per liter),and the vapor level was controlled to approxi-mately 1,000 ,ig of HCHO per liter over a 10-hperiod. The lowest air temperature recorded inthe room was 14°C, whereas the outside tem-perature dropped to 6°C. A slow rise in HCHOvapor level still occurred when the air condition-ing was turned off after 8 h of ventilation, andsome condensation of HCHO appeared to haveoccurred on cold outside surfaces.

Better results were obtained when the tem-perature was maintained above 20°C. TheHCHO level was controlled at 1,500 ,ug/liter, andvapor was quickly distributed by a heating fan.When ventilation was restored, the vapor leveldropped rapidly, and after 8 h there was noperceptible HCHO odor.

DISCUSSIONThe data presented above demonstrate sev-

eral advantages of the HCHO generator over theFormalin-permanganate method in HCHO fu-migation. Rapidly falling vapor levels in thepermanganate method are due, in part, to theformation of water condensate on cold surfaces

which, because of the high partition of coeffi-cient of HCHO in water (13), rapidly takes upthe HCHO vapor. High localized HCHO levelsgenerated by rapid vaporization in this methodalso lead to excessive condensation of paraform-aldehyde. From the standpoints of safety, con-venience, and effectiveness, the HCHO genera-tor method was more successful, particularlywhere large areas were to be fumigated. It is notnecessary to rapidly leave the area after additionof Formalin. Instead, it is only necessary toadjust a timer well in advance of the fumigation,and any necessary sealing of doors and otheroutlets can then proceed at leisure and withoutthe discomfort of leaking vapor. The importanceof adequate sealing is illustrated by the rapidloss of fumigant from a low-security area (Fig.6). Slow diffusion of vapor into closed cupboardswas demonstrated in procedure 1. Cupboards,drawers, and other such fittings in the roommust be opened to allow adequate penetrationof fumigant.

Results in Fig. 3 show that effective HCHOfumigation is markedly temperature dependentand that slow release of fumigant is essential tomaintain effective levels within the area. Vapor-ization ofHCHO to levels above the equilibriumvapor concentrations, however, results in exten-sive condensation of paraformaldehyde on allcold surfaces.

Figure 6 illustrates particular problems asso-ciated with HCHO fumigation of security areas.Although fumigant was rapidly removed from

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VOL. 39,1980 FORMALDEHYDE FUMIGATION SYSTEM 485

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FIG. 5. Relationship between equilibrium vapor concentration of formaldehyde over paraformaldehydeand absolute temperature.

the area when the ventilation was restored, the amount of paraformaldehyde present.increase oftemperature which occurred after the The value of maintenance of a uniform tem-entry of warmer conditioned air allowed the perature was particularly apparent at a fumigantcondensed paraformaldehyde to vaporize, lead- concentration of 1,500 ,ug of HCHO per liter. Ating to a slow rise in HCHO concentration. In the this level, the restoration of ventilation has rap-high-security area, the rise in HCHO level was idly removed all traces of the fumigant down toparticularly rapid, due probably to the large the level of human detection, commonly ac-

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486 ACKLAND, HINTON, AND DENMEADE APPL. ENVIRON. MICROBIOL.

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FORMALDEHYDE FUMIGATION SYSTEM 487

TABLE 3. Formaldehyde vapor: working-levelstandards in various countries

Maximum allowa-ble formaldehydeCountry concn (pg/liter of

air)

U.S. Federal Standard 4, time weightedavg

6, ceiling12, 30-min ceiling

Great Britain 12Hungary 1Italy 5Japan 6U.S.S.R. 0.5Federal Republic of Germany 6German Democratic Republic 5

cepted as 1 ug/liter (11). This level complieswith most of the international standards cur-

rently in use (Table 3).

LITERATURE CITED

1. Bartzokas, C. A., K. McCarthy, W. B. Shackleton,and B. F. Baker. 1978. Observations of the effects offormaldehyde on cockroaches and their flora. 1. Sur-vival of vaccinia virus infected cockroaches during fu-migation with formaldehyde. J. Hyg. 80:125-129.

2. BraswelI, J. R., D. R. Spiner, and R. K. Hoffman.1970. Adsorption of formaldehyde by various surfacesduring gaseous decontamination. Appl. Microbiol. 20:765-769.

3. British Pharmacopoeia. 1973. Her Majesty's StationeryOffice, London.

4. Collins, C. H., and P. M. Lyne. 1976. Microbiologicalmethods, 4th ed., p. 110. Laboratory techniques series.Butterworth & Co. (Publishers) Ltd., London.

5. Hoffman, R. K., and D. R. Spiner. 1970. Effect ofrelative humidity on the penetrability and sporicidalactivity of formaldehyde. Appl. Microbiol. 20:616-619.

6. Lapen, R. F., and S. G. Kenzy. 1975. Effect of selectiveenvironmental treatments on the incidence of grossMarek's disease lesions in chickens. Poult. Sci. 54:659-663.

7. Robinson, P. J. 1978. Fumigation incident. Chem. Ind.(London) 18:723-724.

8. Schifling, B., W. Weuffen, and H. Wigert. 1978. De-termination of gaseous formaldehyde from paraformal-dehyde tablets. 2. Studies on the use of paraformalde-hyde tablets for bacterial count reduction, disinfection,cold sterilization and sterile storage of medical instru-ments. Pharmazie 33:103-104.

9. Stonehill, A. A., S. Krop, and P. M. Borich. 1963.Buffered glutaraldehyde. Am. J. Hosp. Pharm. 20:458-465.

10. Tucker, J. F., E. G. Harry, and H. E. Wainman. 1975.The effect of fumigation with methyl bromide or form-aldehyde on the infectivity of poultry house litter nat-urally contaminated with Salmonella virchow. Br. Vet.J. 131:474-485.

11. U.S. Department of Health, Education and Welfare.1976. Occupational exposure to formaldehyde. NationalInstitute of Occupational Safety and Health, publ. no.77-126. U.S. Department of Health, Education and Wel-fare, Washington, D.C.

12. Wade, A. 1977. Disinfectants and antiseptics, p. 520. InW. Martindale (ed.), The extra pharmacopoeia, 27th ed.The Phannaceutical Press, London.

13. Walker, J. F. 1944. Formaldehyde, p. 52. Reinhold Pub-lishing Corp., New York.

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