25386 LO

r-^r-N ,-, •"-••«w/i_ wt.|\l | Ite,

FOR COMMUNITY WATER SUPPLY ANDSANIiATION (IRC)

. 20, No. I, pp. 37-43, 1936:at Britain. All rights reserved

0043-1354/86 $3.00 + 0.00Copyright © 1986 Pergamon Press Ltd

LOW TECHNOLOGY WATER PURIFICATION BYBENTONITE CLAY FLOCCULATION AS PERFORMED

IN SUDANESE VILLAGESVIROLOGICAL EXAMINATIONS*

EBBA LUND and BIRTH NISSENThe Department of Vet. Virology and Immunology, The Royal Veterinary and Agricultural University

of Copenhagen, 13 Bulowsvej, DK-1870 Copenhagen, Denmark

(Received October 1984)

Abstract—Viruses were removed from various types of water by flocculntion with natural bentonite claysfrom the banks of the Nile as well as with pure bentonite. The flocculation technique employedcorresponds to that used to clarify Nile water in Sudanese villages. If proper flocculation occurred 3 to4 log,0 units of virus could be removed. Some of the conditions for floe formation were examined.

Key words—water purification, bentonite clay, bentonite, flocculation, Sudanese villages

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INTRODUCTION

Jahn (1976) and Jahn and El Fadil (1984} havedescribed liow the river Nile during the flooa seasonin Sudan, beginning in June and lasting throughSeptember, is highly turbid. Suspended solids may beas high as 8000 m g l ~ ' . In the villages, waterpurification by flocculation has been employed forover 100 hundred years (Jahn, 1977). One of themethods employed in the villages is the addition of asuspension of "rauwaq", clarifier, which is a clayfound in varying quality at the river banks. The mainflocculating component is montmorillonite (ben-tonite) (Jahn, 1977). Jahn (1981) describes howpounded rauwaq is prepared as a suspension in adeep plate or calabash and stirred for 10-20 min witha stick or spoon. The suspension is then added to theturbid water in an earthenware jar or other watercontainer. If the woman, who carries out thepurification, has chosen the proper concentration forthe particular water and rauwaq, floes will form andsettle. Concentrations above and below the properone will leave the water turbid. Satisfactoryclarification will take at least 1 h. Jahn has made apreliminary investigation in 1974 on the removal ofcoliforms by the rauwaq (clarifier) clay and reportsthat it is general knowledge in the villages thatfamilies who treat their water have lower incidencerates of gastroentestinal disturbances than otherfamilies.

Jahn inspired us to look into the hygienic aspectsof the purification and provided the clay materials. Astudy was set up where field work was done in Sudan,and more complicated studies including the viro-logical studies were carried out in Denmark. Bacte-

•Supported by grants from DAN1DA (the Danish Inter-national Development Agency).

riological examinations [preliminary reported byMadsen and Schlundt (1983)], parasitological exam-inations and virological examinations in continuationof a preliminary work by Lund and Jahn have beencarried out. The present report deals with the viro-logical examinations. Preliminary results on the viro-logical work were presented by Lund and Jahn at the2nd International Conference on the Impact of ViralDiseases on the Development of African and MiddleEast countries, Nairobi, 1980.

MATERIAL AND METHODSThe rauwaq's (bentonite clays) and benlonite employed

Samples of rauwaq of good quality collected in NorthSudan and brought to Copenhagen by special messenger in1979 were: rauwaq Uqda and rauwaq Gir al. During a visitin 1980 the senior author picked up rauwaq samples asindicated by villagers. These samples were from Wad ElSaid, Alti and Eseba. The rauwaq samples were estimatedfor turbidity by appearance in 500 ml bottles after additionto Copenhagen tap water to which had been added I / I 0 ofcell culture maintenance medium. The pH was around 1.3and room temperature 20-23°C. The samples were left tostand for 3 h at room temperature and observed for tur-bidity. In Table 1 the observations are indicated. An optimalconcentration is seen for the Uqda type, but for the othersonly minimal doses were found for flocculation.

Pure bentonite (Sigma B-3378) was a good flocculant,when added to waters of different composition under similarconditions as employed for the rauwaq. The water bottleswere inspected with the results indicated in Table 2. As maybe seen from Table 2, bentonite could also flocculate inwater, if the ionic strength was sufficient.The water types employed

In all 7 different types of water were employed in theexperiments. The purpose was, and also in ihe experimentscarried out in Copenhagen, to simulate as closely as possiblethe water conditions in Sudan during river flooding periods.Therefore, the artificial Nile water was probably close to theoptimal for the flocculation experiments. This water wasprepared by Madsen and Schlundt in the following way: to1.01. of Copenhagen tap water was added 6.64 g of mudfrom the Nile bank and 0.1 g of Bacto Pepton.

37

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3& EBBA LUND and BIRTE NISSEN

Table 1. Eslimalion of fl peculation ability of ruuwaq added lo Copenhagen tup water with 10% cell culture maintenance medium, pH 7.4

Type of rauwaq

Turbidity*

lOgl ' 20g|-

*Turbidity was observed by visual inspection for the various concentrations added.

30gl 'UqdaGir alWad El SaidAlti

Eseba

Turbid

Turbid at

Slightly turbid

l - 3 0 g l - '

ClearClear

Turbid

Turbid

Clear

Turbid

Somewhat turbid

Slightly turbid

Somewhat turbid

Clear

Turbid

Clear

Clear

Table 2. Flocculation when bentonite was added to water of various compositions

Turbidity*In tap water In demineralized water With 10% culture medium

Bentonite(gl ')

0.51.02.03.05.0

With nofurther

addition

TurbidTurbidClearClearClear

With 10%culturemedium

TurbidSlightly turbid

ClearClearClear

With nofurther

addition

TurbidTurbid

Very turbidVery turbid

—

With266 mg 1 'of CaCI,

TurbidTurbid

Slightly turbidClear

—

And nofurther

addition

TurbidTurbidTurbidTurbid

—

With 133 mg|- 'of CaCI;Turbid

Slightly turbidClearClear

—

With 266 mg|-.'of CaCljTurbid

Slightly turbidClearClear

—*For characteristics of tap water and demineralized water see Table 3.fTurbidily was observed by visual inspection.{The same results were obtained with no addition of CaClj or employing 133 or 266 mg I"

The results obtained with the Nile waters, the artificialNile water and the Copenhagen tap water were not essen-tially different in the present experiments. Flocculation wasobtained in all cases. The characteristics of the watersemployed are given in Table 3, where the pond water is froma highly turbid duck pond of the University park.

Viruses and cell cultures employedThe human enterovirus Coxsackie B3 employed was

originally isolated from biologically treated Copenhagenwaste water. It is a virus type which has repeatedly beenfound in waste water all over the world and must beconsidered a good indicator for faecal virus pollution. Thetilers obtained were around lO'^TCID^ml" ' when titratedin tube cultures of Ilela cells, a human cell line whichoriginated from a cervix carcinoma. The cells were grown at37°C in a medium consisting of Eagle's solution with 10%of new born calf serum with antibiotics. The maintenancemedium consisted of the same medium with only 2% calfserum. The tttra'.ions were carried out by CPE (cytopathiceffect) determinations employing 3 tube cultures for each10-fold dilution step with 0.1 ml inocula and the tilercalculated in TCID^ per 0.1 ml.

A strain of human adenovirus type 1 was also isolatedfrom waste water and included in the present work, because

it has a surface and size quite different from the entero-viruses. It is commonly found in the intestinal tract, but issomewhat less resistant to environmental factors than theenteroviruses. It was grown and employed in the same wayas the coxsackievirus. The liters varied somewhat, but wasbetween 1060-10'5TCID iomt-' during the exptrienls.

The bovine parvovirus (HADEN virus) employed wasreceived from the Danish Slale Veterinary Virus ResearchInslilule, Lindholm. It was titrated in secondary calf kidneycells received as primary cultures from Lindholm. Themedia employed were like the ones for the Hela cells exceptfor the use of horse serum instead of calf serum. The literobtained was around lO'^TCID^ ml" '. This virus type wasincluded, although specifically bovine, because parvovirusesare extremely resistant toward environmental factors (Sri-vastava and Lund, 1980) and a good human type is notavailable for routine work.

Experimental procedureThe slurries as they were made up from bentonite clays

in Sudan involved stirring or shaking by hand. Preliminaryexperiments showed that floccualtions could be obtainedemploying a Griffin flask shaker with the same resulls aswilh manual procedures, mechanically shaken slurries wereemployed in the experiments. The desired amount of rau-

Table 3. Characterization of the various water types employed in the experiments*

Tap waterPond waterArtificial Nile waterBlue Nile Sept.

82 (flooding season)Blue Nile Apnl

83 (dry season)White Nile April 83Irrigation

Canal. Soba.April 83

Demineralized water

Turbidity(FTU)

231400MOO

7.8

48170

COD(permanganate)

( m g l - ' j

7.0166.0167.0167.0

12.6

20.051

Totalsolids

(mg l - 1 )425660

53852397

340

5251570

Total

°dHf

13.520.213.58.6

5.7

3.59.5

hardness

CaCOj270404270172

114

70190

Conductivity(mS m ')

56.2—

57.2203

16.5

19.537.0

25-10

PH827.57.98.0

7.8

8.07.9

"The analyses were k ind ly performed by the Copenhagen Water Works Laboratory. We gratefully acknowledge this assistance.tOne degree of hardness (1 dH) corresponds lo l O m g l " 1 of CaO

Virus removal by clay flocculation

waq or bentonite svas suspended in 100ml of the water tobe tested and mechanically shaken for 30 min. The virus wasadded after removing a sample for titration to make a finaldilut ion of 1:100 or up to 1:10,000, and the total watersample was made up to 1000ml. Apparently no differencewas found in the removal capacity, whether a high tiler ofvirus or a more diluted material was employed, but at leastlO^TCIDjo/O.l ml was desired in the flocculation experi-ments in order to get a good estimation of the removalcapacity. The sample stayed overnight at room temperatureafter careful mixing by hand. Flocculations were in pre-liminary experiments carried out at 20 and at 37=C. Nodifference in flocculation efficiency was found between thetwo temperatures. For each test two flasks were set up. Inone the floes were allowed to sediment spontaneously, andthe content of the other one was centrifuged at 5000-7000£for 20-30 min. Both supernatants were titrated for virus. Nosignificant differences in liters were observed between thetwo supernatants, and the removal efficiency of theflocculation is in the tables indicated as the average of thetwo values, thus improving the accuracy of the titrations.The floes from the centrifugation were eluted employing10% beef extract at pH 9.0 for the elution with a dilutionfactor of 1:5. After centrifugation at 1000^ for 30 min andreadjustment to pH 7.4 the eluate was titrated, and the virusliters expressed as amount of virus per g wet wt of centri-fuged floes. Throughout the paper the log units employedare loglo units.

Persistence of infectious virus in the sedimentsIn connection with corresponding bacteriological exam-

inations suspensions of coxsackievirus were diluted 1:10with Nile mud to which rauwaq had been added to simulatea floe. The mixture was then distributed in Petri dishes witha layer of around 10mm in thickness. They were left opento dry exposed to Sudan sunlight. Samples were exposed inthe open in such number that three could be taken out eachday for 7 days and then less frequently for 30 days in al l .The Petri dishes were then closed with tape and kept in thecold until examined in Copenhagen (air transportation bypersonal messenger). In the laboratory a I g sample fromeach dish was eluated wi th beef extract, pH 9, to which wasadded 1000 1U of penicillin and 1000 g of streptomycin, andthe eluates were centrifuged at 7000 # for 30min. The pHwas adjusted to 7.0 and then titrated in Hel.a cell tubecultures employing O.I ml inocula. The eluates from theunexposed mud samples had tilers between 10SO andl060TCIDso/0.1 ml, the undiluted original virus suspensioncontained lO^TCID^/O. I ml. The samples which had beenexposed 24 h (13-14 April) contained in one sample10"?TCIDW/0.1 ml, and in no other samples virus could bedemonstrated. The April temperature was around 32°C, andthere was sunshine most of the days. To support this resulta laboratory experiment was carried out, where 10 g of soilwas mixed with 3 ml of virus suspension and kept in 1 gamounts open at 32aC in an incubator. On each day asample was removed and eluated like the Sudan weatherexposed samples. The zero sample eluate containedlO^TCIDjo/O.! ml. After 24 h, the liter was 1009 and laterno virus was demonstrated. Thus the drying alone at 32"Cwas able to inactivate the virus in <48h.

EXPERIMENTS AND RESULTS

The experiments were carried out during a periodof 5 years, and in this period the sensitivity of the cellcultures and the tilers of the viruses employed variedsomewhat. The actual differences in liter of the virusemployed were however li t t le more than experimentalerror. Therefore, it seems justified to compile theindiv idual results obtained from the various series of

experiments. For the tilers obtained for virus ad-sorbed to the floes the amount adsorbed per g wet wtis indicated. The weight of the floes varied somewhatbetween the experiments but was within the interval15-22g. The concentration of virus per g of floes didnot give information that could tell much about theadsorbing capacity of the floes but serve as a controlof the experimental procedure, because the differencein liter between the added virus and the one of thetreated water could have been the result of virusinactivation rather than removal in the flocculation.In addition the fact that the liter of the eluates wasseveral logs higher than the one of the correspondingsupernatant serve to stress the fact that virus didadsorb to the floes and could be eluted. Therefore thesediments could give potential hygienic problems.

Experiments employing various bentonite clays (rau-waqs)from Sudan on Copenhagen tap water to removecoxsackievirus

Five different rauwaqs were tested as flocculants.The concentrations employed.were gauged by the factthat the two of them (from Uqda and Gir al in NorthSudan) were known locally to be of good quality, andthe indications from the experiments shown in Tables1-3 were employed. The results from the experimentsto remove coxsackievirus are shown in Table 4. Itmay be seen that rauwaq efficiently removes virusfrom the water. Reliable removal was obtained whenthe good flocculants were employed. With too high aconcentration of the clay no proper flocculation andlitlle removal of virus was obtained. Even the poorerrauwaq qualities could remove 2 log,0 units of virus.A strong adsorption to the fides was indicated withno virus inactivalion, but reversible binding.

Experiments employing a high turbidity duck .pondwater in an attempt to flocculate coxsackievirus .

The pond water employed was turbid and with ahigh load of organic material. In Table 5 the resultsare compiled. It may be seen that compared with theresults of Table 4 on tap water the results show lessefficient or erratic removal of virus. Thus if the wateris too heavily loaded the removal efficiency can beApparently very much reduced.

Experiments employing pure hentonite on variouswaters to remove co.tsackievirus

The flocculaling, removing factor in a claydefinitely depends on the bentonite present, but evenwith chemical and mineralogical analysis of the ben-tonite clays employed it would be impossible topredict the removal capacity for viruses. It wastherefore considered of interest to work with purebentonite also. One single batch of bentonite wasused throughout the experiments, because even forpure bentonite Ihe aclivity is not given by the mineralalone. As may be seen in Table 6, excellent removalwas obtained with I g I" ' of bentonite e.xcept for the

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Table 4. Experiments employing various bentonite clays (rauwaqs) from Sudan on Copenhagen tap waterto remove coxsackievirus B3

Log virus liter

Bentonite clay

Rauwaq. UqdaI g l - '

30gl- 'Rauwaq, Gir al

Rauwaq. AJti30g|- '

Rauwaq, Eseba20 g I " '

Rauwaq. Wad El Saul

In untreatedwater

675.12.72.46.12.85.03.84.44.4

4.03.9

2.6

2.6

56

In treatedwater

Virus adsorbed to floesVirus removal (log10 TCIL^Mj g" l wet

(%) wt of floes)

6.73.40.10.74.1O.I2.20.80.83.4

0.70.2

0.0

0.3

2.4

09899.7989999.899.899.999.9790

99.9599.98

99.8

99.5

99.4

694.74.84.85.35.15.44.84.04.8

4.03.8

3.3

4.0

5.8

Table 5. Experiments employing bentonite clays on duck pond water with coxsackievirus added

FlocculamRauwaq, Uqda

I g l - "

l O g l - '30g|-"

Rauwaq, Gir al

Log virus tilerper nil untreated

water

Log virus tilerper ml treaied

walerVirus removal

Virus adsorbed to floes(log.oTCIDy, g - ' wel

wt of Hoes)

6.72.46.12.83.84.44.4

4.03.9

6.72.45.10.52.92.34.2

2.32.6

00

9099.587.499.236.9

98.095.0

6.85.3504.84.83.84.0

3.84.5

•Waler sample lurbid after ireatment.

White Nile and the Irrigation Canal water, but evenin these cases around 99% removal was obtained.

Experiments with low conductivity water and CaCl2 toremove coxsackievirus

To examine the importance of the ionic strengthfor flocculation and removal capacity of virus in

water some experiments were carried out at pH 7.4using demineralized water. As may be seen in Table7 bentonite could not efficiently flocculate under suchconditions, and no virus removal was demonstrated.In accordance with the preliminary observations ofTable 2 turbidity could be removed if CaCl2 wasadded. Even when flocculation was obtained the virusremoval was less than optimal.

Concentrationof bentonite

Table 6. Experiments employing pure benlonile to remove coxsackievirus from various waters; Virus adsorbed

to ftocs (log loVirus removal TCID J 0g~ 1 wet

(%) wt of floes)

UP

Water type

Tap water

ArtificialNile waterBlue Nile

water*White Nile

w.alertIrrigation

Canal. Soba'J

Log virus tiler Log virus tilerper ml untreated per ml treated

water water

3.83.83.9

3.8

3.8

3.8

0.70.80.2

0.5

2.1

1.7

99.9299.9099.98

99.95

98.0

99.2

5.04.75.7

5.0

4.4

3.4

•Brought to Copenhagen after collection in May 198?.tSome tu rb id i t y left in treated water.^Visible par t i cu la r mat te r still present in treated water.

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Virus removal by

Table 7. Experiments employing bentonite on demineralized water (2Turbidity Log virus titer

Bentonite after per ml untreatedconcentration CaCl; added treatment water

1 None Turbid 443 None Very turbid 4.41 133mg|-' Clear 4.4

Clear 4.43 266 mg|-' Clear 4.4

Experiments to remove adenovirus by flocculationSome of the waters and flocculants employed for

the removal of coxsackievirus were examined for theremoval of adenovirus. The results seen in Table 8indicate that the removal of adenovirus functionsclosely in the same way with around the sameefficiency as for coxsackievirus.

Experiments to remove bovine parvovirusCorresponding to the experiments on adenovirus

the ability of rauwaq or pure bentonite to removeparvovirus by flocculation was examined. The resultsare shown in Table 9. It seems possible that theremoval of parvovirus is somewhat less efficient thanthe removal of coxsackievirus, but the differencedemonstrated is small and probably not significant.

DISCUSSION

Flocculation by bentoniteThe purpose of the present work was not to look

into kinetic or other studies related to flocculationtheories, but to study virus removal under conditionssimulating what could take place in Sudanese vil-lages. It seems that during winter (Jan. -March) theair temperature in Northern Sudan is around

Table 8. Experiments employing bentonite clay and bentoniteLog virus titer

per ml untreatedFlocculam Water waterRauwaq. Gir al Copenhagen 6.0

I g l " ' t ap water10 g l ~ ' Tap water 63

Rauwaq, Cir al Artificial 5.510 g T ' Nile water

Bentonite0.2 g l ~ ' Tap water 5.81 g l ~ ' Tap water 5.0

Table 9. Experiments toLog virus liter

per ml untreatedFlocculant Water waterRauwaq. Uqda Copenhagen

tap waterI g l - 1 3 .7I 0 g | - ' 2.8

Bcn'.oniteI g l - ' 3 .42 g | - ' 2 . 9

Ftcmoniie ArtificialNile water

1 K 1 " ' ' 2-8

•fr^-f^~j^7^-l^/ fttJ'T"*Tii'- 1 TI • r r - iir<" T" t_r— Vfr-mi - i._i.-V""- •- —r" " ~ """ "-^ m-"- ' ---•••• — • — ——— • — - — -• - -s

clay flocculation 41

5 mS m" ' ) to which virus suspensions were added in dilution 1/1000Log virus titer Virus cone, in sedimentper ml treated Virus removal (log,0 units of TCIDy, !

water (%) g ~ ' we-t wt) f4.7 No removal 4.2 • - 14.4 0 4.22.7 • 98.0 6.43.4 90.0 6.223 99.2 6.3

20-29DC and the water temperature 20-25°C. Insummer the air can by .40-44°C and the watertemperature 29-3 1°C. It was found that theflocculation apparently did not change in the region20-37°C and that the pH around or slightly above 8.0after addition of bentonite clay was optimal forflocculation. It is important for the removal capacitythat the floes be formed slowly, i.e. during one toseveral hours and be allowed to settle spontaneously.It seems that it was possible to simulate theflocculation in the laboratory and study some of thefactors of importance.

Flocculation in water treatmentFlocculation is routinely a part of the conventional

waterworks treatment to remove turbidity. The floesare often obtained by the addition of alum, and it isrecognized that the efficiency of flocculation dependsvery much on the water quality. Langelier et al.(1952) found that a preadjustment of buffer capacityand pH may improve the flocculation. Optimumflocculation requires that an equilibrium be obtained,in which many parameters are involved, such asturbidity, particle size distribution, exchange capac-ity, pH and alkalinity. The exchange capacity of awater can be increased by the addition of negativelycharged colloids, such as activated silica, bentonite

to remove adenovirus from tap water and artificial Nile waterLog virus titer Virus cone, in floesper ml treated Virus removal (log,0 units of TCIDM

water (%) g ~ ' wet wt) ' .5.2 84.2 6.7

3.1 99.94 7.02.7 99.8 6.1 '

3.5 99.5 6.71.0 9999 4.7

remove bovine parvovirusLog virus titer Virus adsorbed to floesper ml treated Virus removal (log,u units of TC1DM

water (%) g ~ ' wet wt)

3.0 80.1 3.20.0 99.8 4.4

1.0 99.6 5.20.0 99.9 4.8

08 99.0 3.7

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42 EBBA LUND and BIRTE NISSES

and various other materials. Such substances maygreatly improve flocculation and clarification. See,for example, Libor et ul. (1973), Kirch (1974) andWorthington (1978).

Fuller''s earthBentonite has been used to full woollen cloth and

was then called fuller's earth. Fuller's earth is anatural mineral containing bentonite. It consists ofvolcanic ash and can therefore be found in manygeological strata. The quality of fuller's earth notonly depends on the mineral contents but also on theexposure of the earth to the open air. Minerals fromtop layers are more active than from deeper layers.The ion exchange capacity can be for instance 27 mequiv. for a fuller's earth, and pure bentonite canhave a capacity of maybe 90 m equiv. The quality ofbentonites for fulling has to be empirically deter-mined. The same is the case for their flocculationcapacity for turbid wates. In all experiments carriedout by the group we used either specified rauwaqs orone single batch of bentonite, and we had to test theirflocculation activity and then use a specific batch forcomparative studies. This corresponds to the experi-ence in the Sudan villages.

Removal of viruses with clayIn the study by Carlson et al. (1968) natural waters,

clay suspensions (kaolinite, montmorillonite or illite)were added to water and then phage T2 or poliovirus1. The virus content of the supernatant after centri-fugation at 1900/j was determined in samples withand without clay addition. There was thus no possi-bil i ty to distinguish between removal by clay andvirus inactivation, but it was found that "naturalriver turbidity" could do the same as the defined clayminerals, i.e. removal of up to I-21og|0 units of virus.It was also found that resuspension of virus couldtake place if the ionic strength was lowered; that infact the process was completely reversible. It wasindicated that there would be competition for ad-sorption sites on the clay between viruses and otherproteinaceous materials.

As has been pointed out repeatedly (e.g. Lund etal., 1969; Lund, 1971; Schaub and Sagik, 1975;Wellings et al., 1976) and now has been generallyaccepted by all who work with environmentalsamples, the enteric viruses, as far as they have beenexamined, have affinity for solids, be it clays orparticulate matter from faeces. The adsorption is aphysical-chemical reversible reaction, and the solidassociated viruses are probably more resistant tospontaneous inac t iva t ion than free viruses. In a sew-age treatment plant about 50% of the virus loadfollows the primary sludge (Lund, 1971). Clay miner-als play an important role in soil microbiology (e.g.Filip, 1979). Among the normally applied chemicalflocculants in \s-ater and waste water treatments area lum, lime and ferrous and ferric chloride. These are

all good virus removing compounds but wouldprobably in many cases require for instance pro-longed sedimentation or sand filtration for efficientremoval.

The results of the present report indicate that evenunder primitive conditions such as with bentoniteflocculation in a water jar, very good removal ofenteric virus may be obtained. The removal is notnecessarily followed by a clarification as this woulddepend on the nature of the turbidity, but a clarifiedwater would have improved hygienic quality from avirological point of view.

Flocculation and sedimentation alone in water-works cannot be expected to give more than about99% removal. In the report by van Olphen et al.(1984) raw surface water contained from 0 PFU in1001. to 5 PFU1-' raw surface water, but 11 of 55samples of partially purified water were virus positivewith up to 3 PFU 1"' . The treatment by coagulationand sedimentation followed by rapid sand filtrationwas found insufficient for complete virus removal.

Chemical disinfections can give a treatment thatwill remove or inactivate up to 6 log,0 units of virus(WHO, 1979). According to South African experi-ence (e.g. Grabow et al., 1980) even 12 log,,, units ofvirus can be expected to be removed by advancedwaste water treatment for water reuse. Nonetheless,a 3-4 log,0 unit reduction as found in the presentreport under optimal conditions is a very consid-erable hygienic improvement.

The choice of viruses for the studyCompared with bacteriological examinations viro-

logical ones are complicated and time consuming.There is however a great advantage from the experi-mental point of view which of course also is reflectedin the real life situation that viruses do not multiplyin the environment, i.e. outside the proper living cells.In addition the enteric viruses employed are verystable at a wide pH range also at temperatures likehere between 20-37°C, so that the time factor is of noimportance in the flocculation experiments.

The coxsackievirus employed in the study canalmost be considered a viral indicator for faecalpollution, as it is very frequently reported from allover the world. Addy and Otatume (1976) have oneof the few African reports. It is a virus which isrelatively stable in the environment, but typical for anenterovirus it does not withstand drying very well, asmay be seen in the present report. The adenoviruswas included, because it is chemically and structurallya very different enteric virus, and so is the parvovirus.All three types were removed with about equalefficiency. It would have been interesting to include arotavirus in the study, both because it is probably themost important agent for infantile diarrhea, andbecause it has been reported (Smith and Gerba,1982), that rotavirus is less efficiently removed inwaste water treatments than cnteroviruscs.

"•?&}'• '

Virus removal by clay flocculation 43

Persistence of virus in the floesAs can be seen from the results of the present work

high liters of infectious virus are contained in thefloes. The native habit of disposing with the sedi-ments from the water by throwing it over the wall ofthe yard and out in the village street could then be ahazardous procedure. To get an impression of thepotential public health problem of the floe disposalthe experiments of the present report where viruscontaining mud was exposed to the Sudan weatherwere carried out. The virus contents were within 24 hreduced to a not demonstrable level from the added1050-10lSOTCIDs0/0.1 ml. Laboratory experiments in-dicated that the drying alone would destroy the viruswithin 48 h. Consequently, the risk of spreading viralinfections with unsanitary disposal of rlocs is consid-erably reduced under dry and sunny conditions as inSudan. The removal from the water is not connectedwith any virus inactivation, but with a concentrationin the (Iocs. Therefore the improved water qualitydepends entirely on a proper separation of the floesfrom the water. It could even be imagined, that anamount of water with residual floes would be ofpoorer hygienic quality than the untreated water.

What would be a sufficient removal of virus to make apotable water?

The question of acceptable virus standards fordrinking water has not been solved but much dis-cussed (e.g. WHO, 1979; IAWPRC Study Group,1983; Feachem et a/., I9S1). It is not easy to solve theproblem. For piped supplies it might be reasonable todemand a "virus-free" water, but we do not have themethods to guarantee that. In fact we have no gooddefinition for "virus-free". Among other things be-cause we do not even have proper cultivation meth-ods for a number of the relevant human pathogenicviruses. A number of piped supplies have containeddemonstrable amounts of virus.

Would a 3-4 log,0 unit removal of virus be ade-quate? As quoted by Feachem et ul. (1981) pollutedsurface water has been reported to contain up to 300infectious units 1~'. If this number is realistic then a3~4log|0 unit removal would probably be an im-portant improvement of water. There is strong epi-demiological evidence that the most important spreadof enteric virus is from person to person. Con-sequently a further improvement of drinking watermay from a virological point of view not be soimportant to prevent spread of infections and diseaseas improvement of sanitary conditions in general.This question is going to be further discussed in alater paper by the whole group.

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