APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Dec. 1978, p. 889-8970099-2240/78/0036-0889$02.00/0Copyright © 1978 American Society for Microbiology

Vol. 36, No. 6

Printed in U.S.A.

Identification of Detergents as Components of WastewaterSludge That Modify the Thermal Stability of Reovirus and

EnterovirusesRICHARD L. WARD`8 AND CAROL S. ASHLEY2

Sandia Laboratories, Division 4535, Albuquerque, New Mexico 87185,' and University ofNew Mexico,

Albuquerque, New Mexico 871062Received for publication 18 September 1978

The agent in wastewater sludge previously shown to reduce the heat requiredto inactivate reovirus (R. L. Ward and C. S. Ashley, Appl. Environ. Microbiol.34:681-688, 1977) was "separated" from other sludge components and analyzedby infrared spectroscopy. The infrared spectrum of this material was quite similarto the spectra of commercial anionic detergents, and subsequent analyses of thefractionated sludge samples revealed that anionic detergents in sludge were

copurified with the virucidal activity. Further measurements on the virucidalactivities of specific detergents revealed that ionic detergents reduce the heatrequired to inactivate reovirus, that cationic detergents are more active thananionic, and that nonionic detergents are inactive. Several detergents were alsoshown to protect poliovirus and other enteroviruses against inactivation by heat.These results indicate that ionic detergents are the major component in waste-water sludge that reduce the thermal stability of reovirus and, in addition, thatdetergents are able to protect enteroviruses against heat.

Viruses belonging to the family Reoviridae arecommon pathogens of enteric origin which areroutinely isolated from wastewater sludge. Reo-virus is the prototype of viruses in this familyand has been chosen to represent the group forstudies involving enteric virus inactivation insludge.During an investigation on heat inactivation

of enteric viruses in wastewater sludge, it wasshown that sludge contains a component thatgreatly reduces the heat required to inactivatereovirus (15). It was also shown that this agentdoes not accelerate heat inactivation of polio-virus, an enteric virus of the family Picornaviri-dae. Instead, a partially purified sludge fractioncontaining this agent was quite protective ofpoliovirus against heat inactivation. This resultindicated that the agent may be the componentof sludge previously found to protect poliovirusagainst inactivation by heat (18).

It has now been shown that wastewater sludgedoes, in fact, contain a component that bothaccelerates heat inactivation of reovirus and re-tards that of poliovirus and other enteroviruses.This component has been identified as surfac-tants or detergents.

MATERIALS AND METHODSCells and virus. Reovirus (type 3, strain Dearing)

was grown and plaqued on L-cells in the manner given

in a previous publication (16). The undiluted prepa-ration of this virus contained about 4 x 10' plaque-forming units (PFU)/ml. The growth and plaquing ofall three strains of enteroviruses (poliovirus type 1,strain CHAT; poliovirus type 2, strain 712-Ch-2ab; andcoxsackievirus B1) were carried out on HeLa cells asalso described previously (14). The starting titers ofthese enterovirus preparations were about 4 x 109, 5x 109, and 5 x 109 PFU/ml, respectively.

Purification of the sludge agent. The procedureused to purify the agent in sludge that causes a reduc-tion in the heat required to inactivate reovirus is basedprimarily on results reported in our previous publica-tion (15). There it was shown that the agent is origi-nally associated with sludge solids but can be washedfrom these solids by blending with water. The agentobtained in the sludge washes was found to be resistantto heat (250 to 300°C for 30 min) and soluble at highpH, but it precipitated at low pH. It was subsequentlyshown that, after hexane extraction of the agent, itsactivity is found either in an interphase between theaqueous and hexane phases or dispersed between theinterphase and aqueous phase. These combined resultswere used to design a procedure to separate the agentfrom other sludge components.The general scheme for the separation procedure is

presented in the flow diagram of Fig. 1. More specifi-cally, 4 liters of anaerobically digested sludge (sample1) at pH 6.2 containing 9% solids was centrifuged at18,000 x g for 20 min. The supernatant (sample 2) wasdecanted, and the pellet was resuspended (sample 3)by blending with 2 liters of distilled water at pH 11.6.The solids were again pelleted by centrifugation and

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890 WARD AND ASHLEY

rewashed with another 2 liters of distilled water at pH12.0. The combined washes (no. 2 and no. 3 superna-tants, sample 4) were evaporated to dryness on a hotplate and then heated at 250°C for 30 min. The badlycharred material that resulted was resuspended in 4liters of distilled water by blending at pH 12.0. Aftercentrifugation at 18,000 x g for 20 min, the volume ofthe supernatant (sample 5) was reduced to 250 ml byevaporation, and its pH was adjusted to 2.0. Afteranother centrifugation (24,000 x g, 20 min), the pelletwas resuspended in 450 ml of distilled water at pH 12.2and again centrifuged (8,000 x g, 20 min). The volumeof the supernatant was increased to 4 liters with dis-tilled water (sample 6), the pH was adjusted to 10.6,and the material was blended with an equal volume ofhexane. After separation of the phases by centrifuga-tion (4,000 x g, 12 min), about one-third of the volumewas a clear, colorless upper phase, about one-third wasa clear, amber lower phase (sample 7), and the re-mainder was a viscous, white interphase (sample 8).The three phases were separated, the interphase wasevaporated to dryness, and this material was analyzedby infrared spectroscopy. Samples obtained during thevarious extraction steps were all adjusted to volumesequivalent to the initial 4 liters and assayed for viru-cidal activity against reovirus.

Heat treatment and infectivity assay of vi-

ruses. Sludge or detergent samples to be assayed fortheir effects on the rate of heat inactivation of reovirusor enteroviruses were adjusted to the designated con-centrations in tris(hydroxymethyl)aminomethane(Tris) buffer (0.1 or 0.01 M) at pH 8.5. After making a10-fold dilution of virus directly into these samples,the vials were flamed, incubated for the appropriatetimes and temperatures, and placed in an ice bath. Allsamples were sonically treated and assayed for infec-tious virus by the plaque assay as previously described(14, 16). Sodium dodecyl sulfate (SDS; 0.1%, wt/vol)was added just before sonic treatment to those samplesnot already containing known concentrations of deter-gents. The recovery of PFU was always determinedrelative to an unheated control sample and calculatedas percentage of recovery or surviving fraction of PFU.Measurement of detergent concentration. The

concentration of anionic detergents in the varioussludge samples was determined by the methylene bluemethod (1) as modified by Wang (13). To extract thesedetergents from sludge solids, 0.5-mnl samples of sludgecontaining less than 10% solids were blended with 9.5ml of absolute ethanol and filtered, a procedure de-scribed by Maurer et al. (6). One-milliliter samples ofthe extracted detergents were then shaken for 30 s ina separatory funnel with 15 ml of distilled water, 0.3ml of methylene blue solution (125 mg of methylene

SLUDGE (sample 1)pH 6.2 centrifuge

SUPERNATANT (sample 2)

SUPERNATANT (sample 4)dry, heat,resuspend,centrifuge pH 12.0

PELLET

RESUSPENDED PELLET (sample 3)pH 11.6 centrifuge

' IRESUSPENDED PELLET

pH 12.0|centrifuge

PELLET

SUPERNATANT (sample 5)pH 2 0centrifuge

SUPERNATAN T

PELLET

AQUEOUS (sample 7)

RESUSPENDED PELLETpH 12.2centri fuge

SUPERNATANT (sample 6)pH 10.6 hexone extraction

INTERPHASE (sample 8) HEXANE

pry

I R AnalysisFIG. 1. Flow diagram for the separation of the virucidal agent from sludge. IR, Infrared.

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DETERGENTS AND THERMAL STABILITY OF VIRUSES 891

blue and 2 ml of concentrated sulfuric acid adjusted to100 ml with distilled water), 1 ml of buffer (7.5 g ofNaH2PO4H20, 12.4 g of Na2HPO4 7H20, 26.3 g ofcitric acid, and 10 ml of concentrated sulfuric acidadjusted to 250 ml with distilled water), and 10 ml ofchloroform. After allowing the phases to separate, thechloroform phase was removed and its optical densitywas measured at 650 nm. To determine the concentra-tion of anionic detergents in a sample, the opticaldensities of known amounts of SDS were determinedby this method, and the results were used to generatea standard curve of optical density versus concentra-tion. Concentrations of anionic detergents in the un-known samples were determined from this curve andexpressed as equivalents of SDS.

RESULTSAnalysis of fractionated sludge for viru-

cidal activity. To identify the agent in waste-water sludge that causes a reduction in the heatrequired to inactivate reovirus, this agent wasseparated from other sludge components accord-ing to the general scheme presented in Fig. 1.Virucidal activity was monitored after each stepby measuring the effect of a sample of fraction-ated sludge on the rate of reovirus inactivationat 45°C. For this assay, the pH values of thesamples were carefully adjusted and maintainedat 8.5, because pH was shown to have a largeeffect on the virucidal activity of the sludgeagent (15).

Figure 2 shows the results obtained after eachof the major purification steps. The unfraction-ated, anaerobically digested sludge used in thispreparation (sample 1) had very little activitybefore removal of its liquid fraction (sample 2)by centrifugation. The finding that the virucidalactivity associated with the resuspended solids(sample 3) was much greater than the originalsludge indicates that a large portion of someprotective material was removed with the liquidfraction but the virucidal agent stayed associ-ated with the solids. The virucidal material wasthen washed from the sludge solids with distilledwater (sample 4), dried, and heated to cause thedecomposition of much of the remaining solidmaterial. This resulted in no detectable loss ofvirucidal activity (sample 5). Precipitation atlow pH followed by the removal of undissolvedmaterial by centrifugation at high pH (sample6) also resulted in no loss of activity. Hexaneextraction of this material caused the virucidalactivity to be distributed between the lower,aqueous phase (sample 7) and the interphase(sample 8), but no activity was found in theupper, hexane phase. Because all of the virucidalactivity was, at times, found in the interphasewhen lower concentrations of sludge were usedin this experiment, it appeared that the materialin the interphase might be of greater purity.

CL

2-2c 10

* 10 0 1-1 2

A .

10 -

I 0 5 10 15 20Time (min)

FIG. 2. Rate of heat inactivation of reovirus insamples offractionated sludge. Reovirus was diluted10-fold into samples of fractionated sludge (see Fig.1) adjusted to pH 8.5 in 0.01 M Tris and heated at45°C for the times specified. After sonic treatment in0.1% SDS, the samples were assayed for recoverablePFU. Samples are numbered as shown in Fig. 1.Symbols: *, Tris buffer; A, sample 1; O, sample 2;U, sample 3; A, sample 4; 0, sample 5; *, sample 6;*, sample 7; 0, sample 8.

Therefore, it was analyzed by infrared spectros-copy for the purpose of identifying the virucidalagent.Infrared spectral analysis of the "puri-

fied" sludge agent. The infrared absorbancypattern of the material obtained in the inter-phase after hexane extraction of the sludge agentwas determined as a solid (KBr wafer) afterdrying. This pattern (Fig. 3A) could not be di-rectly related to the published patterns of anyhighly purified compound. However, there aremany similarities between this pattern and thoseof several commercial anionic detergents. Forexample, the pattern for an alkyl aryl sulfonatecalled Super Pearl (Fig. 3B), manufactured byColgate-Palmolive Co., has a number of peaksof relatively similar size in nearly identical po-sitions. This result led to the suggestion that theunknown material might be a detergent. Thephysical properties of the agent (e.g., texture,foaming ability, color) and the chemical char-acteristics which allowed its purification in themanner described are in agreement with thisconclusion.Detergent measurement of fractionated

sludge samples. The initial suggestion that thesludge agent might be a detergent prompted ananalysis of the detergent concentration in thevarious sludge fractions obtained during the pu-

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892 WARD AND ASHLEY

CM-1

_t I

z

tYz

44

V-

I

z49

WAVELENGTH (MICRONS)

WAVELENGTH (MICRONS)

FIG. 3. Infrared absorbancy patterns of fractionated sludge containing the virucidal agent (A) and ofSuper Pearl, an alkyl aryl sulfonate (B). Sample 8 shown in Fig. 1 was dried and made into a potassiumbromide wafer of 0.45-mm thickness and then analyzed by infrared spectroscopy. A portion of the originalspectrum ( ) was magnified fivefold (--- -) for better definition of the peaks. The infrared pattern ofSuperPearl (Colgate-Palmolive Co.) was reproduced from a publication of Sadtler Research Laboratories, Inc.(1964). Permission for the publication herein of Sadtler Standard Spectra has been granted, and all rightsare reserved, by Sadtler Research Laboratories, Inc.

rification of this material (see Fig. 1). There area number of methods used to measure detergentconcentration in wastewater and its sludge, butthe most common method is the methylene bluetechnique (1). Although this method will notdetect cationic or nonionic detergents, it shouldstill be a fairly reliable indicator of detergentconcentration because about 70% of the surfac-tants manufactured for use in commercial deter-gents are anionic (3).The quantity of anionic detergents in 4 liters

of digested sludge before the purification of thevirucidal agent was found to be 3.1 g, calculatedon the basis of SDS as the standard (Table 1).About 80% of this material was recovered in thetwo washes (sample 4), and nearly 50% was stillpresent after drying and heat treatment (sample5). Furthermore, the low pH precipitation stepwas found to cause no additional loss of anionicdetergents (sample 6). During hexane extractionthe detergents became distributed between theaqueous phase (sample 7) and the interphase

TABLE 1. Recovery of anionic detergents in varioussludge fractions during purification of the virucidal

agent

Sample no.' Equivalents of % RecoverySDS (g)b1 3.1 1002 0.3 103 2.9 934 2.5 815 1.5 496 1.5 497 0.9 298 0.5 16

a Fractionated samples are numbered as presentedin Fig. 1.

bQuantity in 4 liters of sludge.

(sample 8) in a manner comparable to the dis-tribution of virucidal activity (see Fig. 2). Theseresults show that detergents are present insludge in quite high concentrations and werecopurified with the virucidal agent during this

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DETERGENTS AND THERMAL STABILITY OF VIRUSES 893

study. Therefore, all that remained to positivelyidentify detergents as the antiviral agents insludge was to demonstrate that these com-pounds are virucidal at concentrations compa-rable to that found in sludge.Virucidal activity of SDS. A standard an-

ionic detergent present in many commercial sur-factants is the compound SDS. This compoundis available in a highly purified form and was,therefore, tested for its virucidal effect on reo-virus.SDS at concentrations below and above that

found for anionic detergents in sludge rapidlyinactivated reovirus at 450C, whereas no detect-able inactivation occurred under these condi-tions in the absence of the detergent (Fig. 4).Furthermore, this compound is virucidal at tem-peratures well below 45°C (Fig. 5), a propertyalso found for the sludge agent (15).These results show that if the anionic deter-

gents in sludge have virucidal activities compa-rable to that of SDS, the concentration of thesedetergents should be sufficient to cause the ob-served effects on reovirus. Therefore, the viru-cidal activities of a number of standard anionicdetergents in commercial products were mea-sured. To determine the structural features of adetergent required for activity, the virucidal ef-fects of several cationic detergents, nonionic de-tergents, and compounds smaller than deter-gents but with similar structures were measuredas well.

-1

U 1030.

.0 -4c

%" 10

.;i

Time (min)

FIG. 4. Effect ofSDS on the rate of heat inactiva-tion of reovirus. Reovirus was diluted 10-fold into0.1% SDS buffered at pH 8.5 with 0.1 M Tris (A),0.01% SDS buffered in the same manner (U), or Trisbuffer alone (0), heated at 45°C for the times speci-fied, and analyzed for recoverable PFU.

-110' -2.2 10

10-4510\

0 10 20

Time (hr)FIG. 5. Effect of temperature on the rate of heat

inactivation ofreovirus by SDS. Reovirus was diluted10-fold into 0.1% SDS buffered atpH 8.5 with 0.1 MTris, heated at 35°C (0), 30°C (A), or 21°C (O), andanalyzed for recoverable PFU. The control sample inbuffer alone (0) was heated at 40°C.

Relationship between detergent struc-ture and virucidal activity. The structuralfeatures of detergents and detergent-like com-pounds required for virucidal activity againstreovirus were determined by measuring theamount of virus inactivation in 0.1% (wt/vol)solutions of each compound at pH 8.5 during 20min at 450C. The results of this experiment showthat there is a clear distinction between activeand inactive compounds (Table 2). Accordingly,several conclusions can be drawn from the data.The first conclusion is that the hydrophobic

regions of these compounds must have a crucialminimal size to affect the rate of heat inactiva-tion of reovirus (see compounds 1 through 6, 8,9). For example, sodium decyl sulfate (com-pound 6) differs from SDS (compound 7) byonly two methylene groups but has very littleactivity under conditions where SDS is ex-tremely active. A similar comparison can bemade between nonyltrimethylammonium bro-mide (compound 9) and dodecyltrimethylam-monium chloride (compound 20).A second conclusion is that detergents must

be ionized in order to have virucidal activity.Several nonionic detergents were tested (com-pounds 10 through 14), but none contained de-tectable activity even though the hydrophobicregions of these molecules closely resembledthose of active ionic compounds.A third conclusion that can be made from the

results of Table 2 is that active detergents canbe either positively or negatively charged (seecompounds 7, 15 through 21). However, asshown more explicitly in Fig. 6, the positively

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TABLE 2. Relationship between virucidal activities and structures of detergents and detergent-likecompounds

Virucidal activity"Compound Structural formula (% recovery of PFU

after 20 min, 45°C)

Control (Tris buffer) 1001. p-Toluenesulfonic acid CH3C6H4SO3 1002. Octanoic acid CH3(CH2)6COO 713. Triethylamine (CH3CH2)3NH+ 884 Hexyl sodium sulfate CH3(CH2)50SO;-Na+ 1005. Octyl sodium sulfate CH3(CH2)70SO3-Na+ 1006. Decyl sodium sulfate CH3(CH2)90SO:-Na+ 597. SDS CH3(CH2)11OSO3-Na+ 0.0038. 2-Naphthalenesulfonic acid CjoH7SO3- 469. Nonyltrimethylammonium bro- CH3(CH2)8N+(CH3)3Br- 97

mide10. Ethosperse LA-4b CH3(CH2),,0(CH2CH20)5H 10011. Igepal Co-630b CH3(CH2)8C6H40(CH2CH20)9H 10012. Clindrol 200-Lb CH3(CH2),0CON(CH2CH20H)2 10013. Brij 58 CH3(CH2)150(CH2CH20)2oH 10014. Remex 690 CH3(CH2)8C6H4O(CH2CH2O)ioH 10015. Lauroyl sarcosine CH3(CH2),0CON(CH3)CH2COO- 0.00316. Sarkosyl 0* CH3(CH2)5CH=CH(CH2)5CON(CH3)CH2COO- 0.00217. Standapol ES-40b CH3(CH2)i30CH2CH20SO:3- 0.418. Alipal Co-436b CH3(CH2)8C6H40 (CH2CH2O)4SO3 0.0119. Sodium dodecyl benzene sulfonateb CH3(CH2)iiC6H4SO3-Na+ <0.00120. Dodecyltrimethylammonium chlo- CH3(CH2),,N+(CH3)3CP- <0.001

ride21. BTC-824 P_100b CH3(CH2)13N+(CH3)2CH2C6H5CP- <0.001

a Reovirus was diluted 10-fold into a 0.1% solution of each compound in 0.1 M Tris (pH 8.5), heated for 20min at 45°C, and assayed for recoverable PFU. The percentage of recovery of infectious virus was determinedrelative to an unheated control in Tris buffer.

b These compounds were gifts of: (10) Glyco Chemicals, Inc.; (11 and 18) GAF Corp.; (12) Clintwood ChemicalCo.; (16) Geigy Chemical Corp.; (17) Standard Chemical Products; (19) Alcolac, Inc.; and (21) Onyx ChemicalCo.

charged compounds tested were much more vi-rucidal than those with negative charges whenpresent at the same molar concentration. Fur-thermore, the positively charged detergentBTC-824 P-100 was extremely virucidal, asshown by the finding that it caused the inacti-vation of more than 99.99% of the viruses whenthe sample was held at 4°C in this experiment.

In summary, these results show that ionicdetergents have virucidal activities against reo-virus comparable to or greater than SDS. It isconcluded, therefore, that they are the agents inwastewater sludge responsible for reducing theheat required to inactivate reovirus.Protective effect of detergents against

heat inactivation of enteroviruses. In a pre-vious publication (15) it was suggested that theagent in sludge responsible for accelerated heatinactivation of reovirus may be the same agentthat was found to protect poliovirus against in-activation by heat (18). Because it has now beenestablished that the former is ionic detergents,it was of interest to measure the effect of deter-gents on heat inactivation of poliovirus andother enteroviruses.

The initial experiment was to determine theeffect of SDS on the rate of heat inactivation ofpoliovirus type 1, strain CHAT. SDS is protec-tive of poliovirus (Fig. 7), and the maximumeffect appears to be at a concentration of about0.1% or 1 g/liter, a value nearly equivalent tothat found for anionic detergents in digestedsludge (see Table 1). This concentration of SDSwas shown to be even more protective of twoother enteroviruses, poliovirus type 2, strain 712,and coxsackievirus Bi (Fig. 8). Therefore, it isclear that at least this detergent has oppositeeffects on the rates of heat inactivation of rep-resentative members of two different enteric vi-rus groups, reoviruses and enteroviruses.The effects of other detergents and detergent-

like compounds on heat inactivation of polio-virus (CHAT) were then measured. Most com-pounds that were inactive against reovirus (seeTable 2) also had little or no effect on heatinactivation of poliovirus (Table 3). Similarly,most compounds that accelerated heat inacti-vation of reovirus were protective of poliovirus.However, there are several notable exceptions(see compounds 2, 10, 12, 18, 19). Although sev-

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DETERGENTS AND THERMAL STABILITY OF VIRUSES 895

', AeX ,105

0 1 2 3

Tim. (hr)FIG. 6. Comparative effects of several ionic deter-

gents on the rate of heat inactivation of reovirus.Reovirus was diluted 10-fold into 0.003 M solutionsof each detergent in 0.1 M Tris at pH 8.5, heated at350C for the times indicated, and assayed for recover-able PFU. Samples were kept at 4°C at all timesbefore titrating, except while being incubated at 35°C.Detergent symbols: A, Alipal Co-436; *, Sarkosyl 0;U, lauroyl sarcosine; O1, Standapol ES-40; 0, SDS;A, sodium dodecyl benzene sulfonate; O, dodecyltri-methylammonium chloride; *, BTC-824 P-100.

eral explanations for these discrepancies can beproposed, the reasons are not immediately ap-parent from structural considerations. It can beconcluded, however, that detergents are at leastone of the compounds of wastewater sludge thatprotect enteroviruses against inactivation byheat.

DISCUSSIONThe agent in wastewater sludge that reduces

the heat required to inactivate reovirus wasshown to have several unique properties thatallow its separation from other sludge compo-nents (15). In retrospect, these properties cor-relate well with the eventual identification ofthe agent as ionic detergents, particularly an-ionic detergents. Although cationic detergentsare produced in significant amounts (3) and usedin commercial products which eventually be-come a part of sewage, they should have beenlost in the purification procedure used in thisstudy. This conclusion is based on the fact thatanionic detergents become uncharged at low pHvalues, which causes them to become insolublein aqueous solutions, but cationic detergents re-tain their charge at low pH and remain watersoluble. Therefore, the low-pH precipitation stepused in the purification procedure should have

removed cationic detergents not removed in pre-vious steps. The correlations between the in-frared spectroscopy pattern of the purified agentand the patterns of several commercial anionicdetergents support this conclusion.Although detergents are apparently the main

components in sludge that accelerate heat inac-tivation of reovirus, this does not eliminate thepossibility that other sludge components havethis activity as well. Furthermore, sludge alsocontains compounds that have the opposite ef-fect on heat inactivation of reovirus and counterthe effect of detergents. A significant amount ofthis protective material was removed with thesupernatant after the initial centrifugation ofsludge because more virucidal activity was ex-pressed by the resuspended pellet than by theoriginal sludge. A more detailed examination ofthe effects of the protective material will bemade in the accompanying paper (17).Very few studies have been published con-

cerning the effects of detergents on nonenvel-oped viruses, but even from these few it is evi-dent that one detergent, SDS, has distinctlydifferent effects on different strains of virus.Adenovirus is extremely sensitive to SDS, asshown by the finding that incubation at roomtemperature with 0.012% SDS causes rapid dis-ruption of adenovirus particles (10). Adeno-as-sociated virus, a parvovirus, is much less readilydissociated by this compound, a conclusion

100Oia.~ ~

0

0C

10

S

3

'n10

Time (min)FIG. 7. Effect of SDS on the rate of heat inactiva-

tion ofpoliovirus. A lysate ofpoliovirus type 1, strainCHAT, was diluted 10-fold with 0.1 M Tris buffer,pH8.5 (0), or solutions buffered in the same mannercontaining 0.001% SDS (*), 0.01% SDS (U), 0.1%SDS (-), or 1.0%o SDS (A). After heat treatment at450 C for the times specified, the samples were assayedfor recoverable PFU.

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Q10 \

. 0

10

10- * '*'~-2

10

0 10 20 30 40 50

Time (min)FIG. 8. Protective effect of SDS on heat inactiva-

tion ofpoliovirus type 2 strain 712 (A) and coxsack-ievirus Bl (B). Virus preparations were diluted 10-fold into either 0.1% SDS buffered atpH 8.5 with 0.1M Tris (0) or Tris buffer alone (a), heated at 45°C,and assayed for recoverable PFU.

reached through electron microscopic examina-tion of virion disruption (9). Finally, humanrhinovirus type 2 is stabilized by SDS becauseits inactivation by both heat and acid is retardedby this compound (5). Rhinoviruses belong tothe same virus family as poliovirus and otherenteroviruses, the Picornaviridae. For this rea-son it is not surprising that enteroviruses werealso found, in the present study, to be protectedby SDS against inactivation by heat. However,the reasons why SDS and other detergents havesuch contrasting effects on different strains ofnonenveloped viruses remain to be determined.

It has been reported that a variety of com-pounds with very different structures protectpoliovirus against inactivation by heat (cf. 4, 8,11, 12). Detergents now represent another classof compounds with this activity. Because themechanism by which protection occurs has notbeen determined, it is not possible to predictwhether these compounds operate through com-mon or different pathways.The practical significance of the data reported

here is dependent on several factors. One factoris that the results found for the strain of reovirusexamined in this study be applicable to othermembers of the family Reoviridae, especiallyrotaviruses, which are thought to be the humanviruses of greatest import in this group (2). An-

other important factor is that the concentrationof ionic detergents in sludge be sufficient tocause reovirus inactivation, even in the presenceof protective substances found in sludge. Bothraw and anaerobically digested sludges from theSewage Treatment Plant in Albuquerque con-sistently contained more than 500 mg-equiva-lents of SDS per liter. It is impossible to predictwhether this quantity will be found in all waste-water sludges, but detergents should be one ofthe more uniform components of wastewater.For example, a study made in Japan indicatedthat the methylene blue-active substances inraw sewage were consistently between 5.7 and14 mg/liter (7).A fmal factor to be mentioned concerning the

applicability of these results is that the temper-atures and pH values of the sludge during treat-ment must be high enough and maintained forsufficient periods of time. It has already beenshown (15) that adequate rates of reovirus in-activation may require temperatures in excess of20°C and pH values greater than 8 in sludgescontaining the expected concentrations of deter-gents. However, the general applicability of

TABLE 3. Protective effects of detergents anddetergent-like compounds against heat inactivation

ofpoliovirusProtectiveactivitya (%

Compound recovery ofPFU after 20min, 45°C)

Control (Tris buffer) ............... 0.0011. p-Toluenesulfonic acid ...... 0.0052. Octanoic acid 553. Triethylamine.0.0024. Hexyl sodium sulfate 0.025. Octyl sodium sulfate 0.056. Decyl sodium sulfate 0.0037. SDS 148. 2-Napthalenesulfonic acid 0.039. Nonyltrimethylammonium bromide 0.008

10. Ethosperse LA-4 0.2111. Igepal Co-630 0.0112. Clindrol 200-L 3013. Brij 58 .....0.00814. Remex 690 ....0.00115. Lauroyl sarcosine 1116. Sarkosyl 0 2.517. Standapol ES-40 1618. Alipal Co-436 0.0119. Sodium dodecyl benzene sulfonate ... 0.0120. Dodecyltrimethylammonium chloride 2.221. BTC-824 P-100 ....... .. 0.95

a Poliovirus strain CHAT was diluted 10-fold into0.1% solutions of each compound in 0.1 M Tris (pH8.5), heated at 45°C for 20 min, and assayed for re-coverable PFU. The percentage of recovery was de-termined relative to an unheated control in Tris buffer.

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DETERGENTS AND THERMAL STABILITY OF VIRUSES 897

these results in controlling the spread of virusesin the environment can be fully assessed onlyafter more extensive experimentation.

ACKNOWLEDGMENTS

We thank T. M. Myers for performing the infrared spectralanalysis of the unknown sludge components. We also express

our sincere gratitude to the several companies listed in Table2 that donated samples of their detergents.

This work was supported by Division of Advanced Systemsand Materials Production Division, U.S. Department of En-ergy, Washington, D.C., and the Municipal EnvironmentalResearch Laboratory, U.S. Enviromnental Protection Agency,Cincinnati, Ohio, Interagency Agreement E(29-2)-3536/EPA-IAG-D6-0675.

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