Controlled Formaldehyde Fumigation System

0099-2240/80/03-0480/08$02.00/0

Controlled Formaldehyde Fumigation System

N. R. ACKLAND,* M. R. HINTON, AND K. R. DENMEADE

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

Acomparative study of formaldehyde (HCHO) fumigation was carried out by controlledvaporization, using an electric vapor generator, and bythe Formalin-permanganate method. Determination of vapor levels as well as bactericidal actionshowed the generator to be more effective. Maximum achievablefumigant levels were temperature dependent andrelated to the equilibrium vapor concen-tration of HCHO. At a room temperature of21°C, vaporization ofmore than 2,000

ytg

ofHCHO per liter resulted in conversion of HCHO to paraformaldehyde, which condensed on surfaces and contributed to prolongedresidualvaporlevels. An electronic monitor is described which is capable of detecting HCHO levels as low as 10

Ag/liter

and can be used to monitorthe complete fumigation process. Formaldehyde fumigation has long been an

accepted methodof sterilization for areas where microbiological cleanliness is required. Fumiga-tion is usually carried out bymixing potassium permanganate and an excessof Formalin (40% formaldehyde) solution in a suitable container; sufficient heat isgenerated byoxidation ofpart of the formaldehyde (HCHO) with permanga-nate to vaporize the remaining formaldehyde

and water(1, 6, 7, 10). This method offumigation

isviolent, 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 either Formalin or paraformaldehyde, a polymer of HCHO which is converted toHCHOvapor upon heating (8).Formaldehydelevels werecompared infumigations of several workareasby 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 a depolymerizing agent (Imperial Chemical Industries, Melbourne, Australia) was used throughout. Para-formaldehyde powder (Koch-Light) was a white pol-ymerconsisting of 95% paraformaldehyde.

Areasfumigated.Alow-security area and a high-security area were monitored throughout the study. Thelow-security area was a large processing labora-tory;duringfumigation the air conditioning was turned off, but no attempt was made to seal air supply or exhaust ducts. The high-security area was a large sealedlaboratorywhose air supply was carefully con-trolled; exhaust air was passed through a gas-fired furnace to ensure microbiological security. The air conditioning for this area was turned off during

fumi-gation, and the airsupplyand exhaust outlet valves wereclosed.

Assay of HCHO. Initially, formaldehyde vapor concentrationswere measured by takingairsamples through rubber tubes protruding into the room at

varioussample points. Foursamplesweretaken per sampling time, using an evacuated liter flask. Forty milliliters of0.5M (NH4)2SO4wasaddedtoeachflask,

whichwasthen cooledto4'Ctodissolvethe formal-dehyde. One milliliter of a suitable dilution of the resulting solutionwasthen mixedwith1mlofafreshly prepared chromotropic acidreagent(1% chromotropic acid in 18 M H2SO4), and8 ml of18 M H2SO4 was added. After standing for 10 min, the mixture was diluted to afinal volume of 25 ml, and the optical density wasdetermined at540 nm. HCHOwas esti-matedfromastandardcurvein the range5 to40yg/ ml,and themeanconcentrationfrom thefoursamples wasdetermined.

Biological test systems. The spore suspension usedthroughoutwaspreparedfrom B.subtilisNCTC 8233 prepared by culture for 7 days on sporulation

agar (3) and suspension of the resultant growth in water.Thesuspension wassubjectedto twocyclesof heatingto65°Cfor30minfollowedbycentrifugation andresuspensionindistilledwater.Asamplewasthen stained and examined microscopically for spores(4). Glassrods 5mmin diameter and150 mmlong,witha

loop atoneend and a graduation mark50 mmfrom theother, were dipped into the spore suspension to

the mark. They were then suspended in a laminar-flow cabinettodry and stored inasterilejar. Before fumigation, rodswereplaced in different parts of the

areas tobe tested and transferred toa sterile

screw-capped test tube at the end of each procedure, imme-diatelyafter evacuationof thefumigant. Twenty mil-liliters of salinewasaddedtoeach tube to removethe spores from therods, andanestimate ofviable spores was obtained by plating dilutions of the suspension

ontonutrientagar. This was carriedoutwithin2hof removal of rods from thearea. Plateswereincubated

at37°C,and then colonieswerecounted after3days and checked after7days.

vaporizedon a500-Wheating plateinastainless-steel beaker. The apparatus usedsubsequently, shown in Fig. 1, consistsofastainless-steel5-litervessel with a 1,200-Wheating element.Sufficientcapacitywas pres-entto allow the addition ofwaterfor humidification andtocontaintheresultingfoam. A copperplatewas interposed between the element andthe bottom of the vessel to ensure auniform distribution of heatat a

thermostatically controlled temperature of approxi-mately170°C.

Electronicformaldehyde monitor. The appara-tus used (Fig. 2) was based on a Figaro 812 N-type semiconductor device, the conductivity of which changes in the presence of adsorbed molecules. No significant interference was observed from substances otherthan HCHO in the areas fumigated or by tem-perature changes normally encountered. Response timeof the sensor, obtained from Digitron Engineer-ing, Sydney, Australia, was approximately 20 s. The monitorwascalibrated in an enclosed system against HCHO released by paraformaldehyde vaporization. Good agreement was obtained between the weight of paraformaldehyde vaporized and the HCHO concen-tration as measured by chemical assay, which indi-cated that there was no loss of vapor in the test system.

RESULTS

HCHO levels by the

Formalin-perman-ganateand hotplate

vaporization

methods. For

Formalin-permanganate

fumigations, quan-tities of Formalin recommended have ranged

from 12 to 59 ml/m3 ofair space (6, 11), and

ratios forFormalin-permanganate have ranged from 3:1 to 5:3 (1, 10). In thefollowing experi-ments, fumigationswere carried out by using a

mixture of 17.8 ml of Formalin and 8.9 g of

potassiumpermanganate perm3 of airspace in alow-securityareaat room temperatures of 16, 24,and300C. In preliminaryexperiments, room airsamples weretaken from each of four

posi-tions, including the ceiling and the floor, and

HCHO levelswere determined by chemical as-say. Because no differences in concentration wereobserved betweenanyposition, allresults

from thesamplingpoints were averaged. HCHO

levelswerealso measured after vaporization of 12.5 ml of Formalin (70% of that used in the

Formalin-permanganate

fumigations

to allow for

losses duetopermanganate oxidation) on a hot

plateat a roomtemperature of27°C.

Results in Fig. 3 indicate that the highest levels ofHCHOwere obtained in the

Formalin-permanganate

method at thecommencement of

fumigation and then vapor levels dropped to base levels of 200 to 500 ug/liter by 4 h. The

initialconcentration of HCHO was temperature

dependent. Higherand morepersistent levels of HCHO were obtained aftervaporization by the hotplatemethod at aroomtemperature of 27°C,

FG. Electricvaporgenerator.

which could be

expected

to be a more efficient

sterilizing procedure.

With the

Formalin-per-manganate

method,

large

amountsofwater con-densate were observed on

objects

in the area after

vaporization.

Sur,vivalof B. subtilis after

ftumigation by

the

Formalin-permanganate

method. B.

subtilissporessurvivedonsomerodslocatedat various

places

ina

low-security

area

during

a 24-h

Formnalin-permanganate

fumigation

atan ini-tial temperature of 1800. Viable counts of 2 x

106 were obtained from control

rods,

but

only

two of nine rods

exposed

in the treated area

were

sterile;

viable counts of 20 to 600 were

obtained from the others. It is clear

that,

al-though

this

procedure

was

fairly

effective,

ster-ilizationwasnotachieved.

482 ACKLAND, HINTON, AND DENMEADE

SENSOR

DR IV

VOLTlAGE I SENSOR - MNPUT

SENSOR HEATER SUPPLY BlJFFE R A PA$ P .'.. CCOMPA--RATOFO --REFERENCE VOLTAGE

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

lC

24eC

27t 30t

FIG. 3. Comparison of Formalin-permanganatefumigationsat16, 24,and30°C andahot plate

vapori-zationofFormalinat27°C.

asealedjarat20°CincontrolledHCHOvapor

levels of 150and 300

Ag/liter

ofair,with relative

humidity maintained at approximately 100%.

The results inTable 1 show thatexposureto150 ,ug of HCHO per liter of air did not result in

sterilization even afterexposure for 6 h.

Expo-sure to 300 ,Ag/liter was much more effective,

andnoviablesporesweredetected at 3 h.

Use ofelectricvaporgenerator. In Table 2the resultsfromseveralfumigationsareshown

(procedures 1through 6), usingthe electric va-porgenerator (Fig. 1) and different amounts of Formalin inalow-securityareaatdifferent ini-tial temperatures. In procedure 1, persistent HCHO levels similar to those obtained bythe hot plate vaporization method (Fig. 3) were

achieved. No viableB. subtilisspores were de-tected after 24 h ofexposure on the 18 spore rods located inthe main laboratoryarea,buta

0.5% survivalwasdetectedon arodlocatedina

closedcupboard. Thefumigantlevel inthe

cup-boardwas160,ug/literafter 6 h.Procedure 2was

carried out with the same volume of Formalin

and an initial room temperature of 15°C. No

surviving spores could be detected after 7 h of exposure on 12 spore rods located around the room, but two located in cupboards showed a

survival of 1%.

Procedures 3 through 5, which were carried

out for different times and temperatures and withvaryingamountsofFormalin, are also de-scribed in Table 2. Inprocedure 6,a mixtureof

paraformaldehyde and water equivalent to8.9 ml of Formalinperm3of airwasused.Again,no

surviving spores could be detected from rods distributedthroughoutthe mainlaboratoryarea

inanyof theprocedures.

All subsequent fumigations were carried out with 3.7gofparaformaldehydeper m3ofroom

volume,which is converted toHCHO equivalent

tothatpresentin8.9mlof Formalin.Humidity

ineacharea wasincreasedto80%,alevel which

pg

fumigation by causing HCHO to dissolve in a

film ofmoisture aroundmicroorganisms, where it is more bactericidal than in vaporform (12). UseofFormalinat a room temperature of

200C

would have contributed approximately 30% to the relative humidity. More reliable humidity

control 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-causeofthetime involvedin chemical assays of

air samples. When the output of the monitor wasplottedagainstHCHOvaporlevelsobtained by chemical analysis, a power-law relationship was obtained (Fig. 4). Such a relationship is

particularly useful, since good sensitivity was

achievedatthelowervaporlevelsand the total

fumigationrange wascovered on the same scale.

Relationship between temperature and

equilibrium vapor concentration. The rela-tionship between temperature and the

equilib-riumvapor concentration is shown in Fig. 5. For

normal air-conditioned laboratoriesat

200C,

the

equilibrium vapor concentration is

approxi-mately2000

,ug

ofHCHOperliter.HCHOvapor

inexcessof theequilibriumvaporconcentration

will recondenseon thecoldestsurfaces,

produc-inga

paraformaldehyde film

oversurfaceswithin

theroom.This film slowlyevaporates,producing anunpleasant odor.

HCHO levels after fumigation. The

per-sistence of HCHOvaporaftercompletion of the

fumigation and restoration of ventilation is a

significant problem,

especially

in high-security laboratories. The extent of the problem can be seen from chartrecordings from the electronic monitorduring

fumigation

ofa high-anda

low-securityarea (Fig.6).

TABLE 1. Survivalof B. subtilis spores in controlled vapor levelsof 150 and 300pgof HCHO

perliterof air at 20°C yg of

Expo-HCHO/liter sure Total viable count %Survival of air time (h) Controls 0 2.5x 106 100 150 0.5 1.02x106 41 150 1.5 8.15x104 3.3 150 3.0 4.0x102 0.02 150 6.0 1.0X102 0.01 Controls 0 4.5x 106 100 300 0.5 4.0x105 8.9 300 1.0 6.5x 102 0.02 300 1.5 2.0x10 0.003 300 3.0 Nonedetected <0.0001 300 6.0 Nonedetected <0.0001

fumigations with the electric vapor generator and varying quantities of fumigant

Time Vapor after level(,ug Room comple- of dure Fumigant (ml/m3) temp tionof HCfO/

(0C) vapori- literof zation ar (h) ar 1 Formalin (17.8) 25 0 1,850 2 2,270 6 500 24 70 2 Formalin (17.8) 15 0 920 2 850 5 770 7 720 3 Formalin (17.8) 30 0 3,400 2 2,600 5 2,480 4 Formalin (8.9) 22 0 2,190 3 2,160 6 1,700 5 Formalin (4.4) 19 3 1,040 6 Paraformalde- 20 1 1,750 hyde (equiv-alent to 8.9 ml of For-malin)

Clearly,

therewas a

rapid

lossof

fumigant

in thelow-securityarea,throughvents,

leaks,

etc.,

butin thehigh-securityareaa

higher

levelwas

maintained. Although restoration of the venti-lationat 11h inthe

high-security

areaand12.5 hinthelow-securityarea

rapidly

removedmost

of the vapor, aresidual

HCHO

odor remained which was

paralleled by

a

high

monitor base level reading.

WVhen

ventilation was again stopped,at14h in the

low-security

areaand 17

h inthe

high-security

area, the level of

HCHO

rose,showing thatsome

revaporization

of form-aldehydewas

occurring.

The odorwas

particu-larly noticeablein the

high-security

area,where

a

vaporization

rateof40,ugof

HCHO/liter

per

hwasshownto occur

continuously.

Since 10air changesper houroccurred in the area,the

lab-oratory

atmosphere

would have contained an

average of 4 ,ug of HCHO per

liter,

a level in which it istoo

high

to work

(Table

3).

Vapori-zation at 3.7 g of

paraformaldehyde

perm3

re-sulted in

approximately

2

g/m3

(2,000

,ug of HCHO per

liter)

to condenseon walls and

fit-tings of the area, and 2 to 3

days

would be

required for the

dissipation

of the residual HCHO.This has beenconfirmed

by

observation. Attainment of

optimal

levels of HCHO

without

condensation. A controlled

484 AND DENMEADE 10-OUTPUT

mv1.0-mV1 0-1* 10 100

pgHCHO

Ilitre

AIR

1000 10000

FIG. 4. Response of monitortoformaldehydevaporlevels.

where the vaporlevel wasmaintainedbelow the

equilibrium concentrationtoavoidcondensation

ofHCHO. The comparator circuit on the

moni-tor (Fig. 2) enabled a simple ON-OFF control of theelectricalgenerators when the vapor

concen-trationrose to apredeterminedlevel.

During fumigation, the generator vaporized 1.1 g ofparaformaldehyde perm3 of room vol-ume(equivalentto1,100

,tg

ofHCHOperliter), and the vapor level was controlledto

approxi-mately 1,000 ,igofHCHO perliter over a 10-h

period. The lowestairtemperature recorded in

the room was 14°C, whereas the outside tem-perature dropped to 6°C. A slow rise in HCHO vaporlevel stilloccurred when the air condition-ing was turned off after 8 h ofventilation, and somecondensation ofHCHO appeared to have occurred on coldoutside surfaces.

Better results were obtained when the tem-perature was maintained above 20°C. The HCHO level wascontrolledat1,500,ug/liter, and vapor was quicklydistributed by a heating fan. Whenventilation was restored, the vapor level

dropped rapidly, and after 8 h there was no perceptibleHCHOodor.

DISCUSSION

The data presented above demonstrate sev-eraladvantagesoftheHCHOgenerator over the

Formalin-permanganate

method in HCHO fu-migation. Rapidly falling vapor levels in the permanganate method are due, in part, to the

formation ofwatercondensate on cold surfaces

which,

because of the

high partition

of

coeffi-cient ofHCHO in water

(13), rapidly

takes up

the HCHO vapor.

High

localized HCHO levels

generated

by rapid

vaporization

in thismethod alsoleadtoexcessivecondensation of

paraform-aldehyde.

From the standpoints ofsafety,

con-venience,

and

effectiveness,

theHCHO

genera-tor method was more

successful,

particularly

where

large

areasweretobe

fumigated.

It isnot

necessaryto

rapidly

leave theareaafteraddition of Formalin.

Instead,

it is

only

necessary to

adjust

atimer well in advance of the

fumigation,

and any necessary

sealing

of doors and other outletscanthenproceedatleisure and without thediscomfort of

leaking

vapor.The

importance

of

adequate sealing

is illustrated

by

the

rapid

loss of

fumigant

from a low-security area

(Fig.

6).

Slowdiffusion of vapor into closed

cupboards

was demonstrated in procedure 1.

Cupboards,

drawers,

and other such

fittings

in the room

must be

opened

to allow adequate

penetration

of

fumigant.

Results in

Fig.

3 show that effective HCHO

fumigation

ismarkedlytemperature

dependent

andthat slow release offumigantisessential to maintaineffective levels within thearea.

Vapor-ization of HCHOtolevels above the

equilibrium

vaporconcentrations,however,results in

exten-sive condensation of paraformaldehyde on all coldsurfaces.

Figure

6 illustratesparticular problems asso-ciated withHCHOfumigationofsecurityareas.

Although fumigant

was rapidly removed from

z

0

z

w

0

20000-C.)x

O 6000 8000 4000 / > I 7000-2 6000 S 5000 -J 4000-Xi 3000- 2000-1000

034

303 1/Tx103

32

3'1 20 30 °c 40 50

FIG. 5. Relationship between equilibrium vapor concentration of formaldehydeoverparaformaldehyde and absolute temperature.

the areawhen the ventilation was restored, the amount of paraformaldehyde present.

increase of temperature 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 fumigant condensedparaformaldehyde to vaporize, lead- concentration of 1,500 ,ug of HCHO per liter. At ing to a slow rise in HCHO concentration. In the this level, the restoration ofventilation has

rap-high-securityarea, the rise in HCHO level was idly removed all traces of thefumigant down to

ac-486

uo0

uo0

(q)

l;o

*

(l) ;jo*

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tk

to3

.eO x4) 5.. sta3 .2 2

b

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p_

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standards in various countries

Maximum allowa-bleformaldehyde Country concn(pg/liter of

air) U.S.FederalStandard 4,timeweighted

avg 6,ceiling 12,30-minceiling Great Britain 12 Hungary 1 Italy 5 Japan 6 U.S.S.R. 0.5

FederalRepublicofGermany 6 GermanDemocratic Republic 5

cepted as 1 ug/liter (11). This level complies

with most of the international standards

cur-rently inuse (Table 3).

LITERATURE CITED

1. Bartzokas, C.A., K. McCarthy, W. B. Shackleton, and B. F. Baker. 1978. Observations of theeffectsof formaldehydeon cockroaches and their flora. 1. Sur-vival of vaccinia virus infected cockroachesduring fu-migation with formaldehyde. J. Hyg.80:125-129. 2. BraswelI, J. R., D. R. Spiner, and R. K. Hoffman.

1970.Adsorption offormaldehydeby varioussurfaces duringgaseous decontamination.Appl. Microbiol. 20: 765-769.

Office, London.

4. Collins, C.H., andP. M.Lyne.1976. Microbiological methods,4thed., p. 110.Laboratorytechniques series. Butterworth & Co.(Publishers) Ltd., London. 5. Hoffman, R. K., and D. R. Spiner. 1970. Effect of

relativehumidity on the penetrability and sporicidal activity offormaldehyde. Appl. Microbiol. 20:616-619. 6. Lapen, R. F., and S. G. Kenzy. 1975. Effect of selective environmental treatments on the incidence of gross Marek's diseaselesions in chickens.Poult. Sci. 54:659-663.

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

8. Schifling,B., W. Weuffen, and H.Wigert. 1978. De-termination ofgaseous formaldehyde from

paraformal-dehyde tablets. 2. Studies on the use of

paraformalde-hyde tablets for bacterial count reduction, disinfection, coldsterilizationand sterile storage of medical instru-ments. Pharmazie33:103-104.

9. Stonehill, A. A., S. Krop, and P. M. Borich. 1963. Bufferedglutaraldehyde. Am. J. Hosp. Pharm.

20:458-465.

10. Tucker, J. F., E. G. Harry, and H. E.Wainman.1975. The effect offumigationwithmethyl bromide or form-aldehydeontheinfectivity of poultry house litter nat-urally contaminated withSalmonellavirchow. Br. Vet. J. 131:474-485.

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

12.Wade,A. 1977. Disinfectants andantiseptics,p. 520. In W.Martindale(ed.),Theextrapharmacopoeia,27th ed. ThePhannaceuticalPress,London.

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