8) Pergamon

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War. ScL Tech. Vol. 31, No. 5-6, pp. 55-62, 1995." Copyright ~ 1995 IAWQ

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REMOVAL AND INACTIVATION OFVIRUSES BY DRINKING WATERTREATMENT PROCESSES UNDER FULLSCALE CONDITIONS

A. H. Havelaar*, M. van Olphen*'** and J. F. Schijven*

• Laboratory ofWater and Food Microbiology, National Institute ofPublic Healthand Environmental Protection. P.O. Box 1,3720 BA Bilthoven, The Netherlands•• KIWA Research and Testing. P.O. Box lO72, 3430 BB Nieuwegein.The Netherlands

ABSTRACT

Risk-based evaluations of the hygienic quality of drinking water require accurate data on removal andinactivation of pathogens by different steps of the treatment chain. The continuing trend to reduce chemicaldisinfection leads to an increased interest in the effect of other processes. based on physical removal orbiological inactivation. This study reports data on the removal and inactivation of entero- and reoviruses bythree such processes. For comparison. data on a variety of model organisms are also reported. All studieswere carried out in the winter period because the concentration of viruses is then at its maximum, and thereducing capacities of the processes are at their minima. Storage in three reservoirs in series (averagedetention time 7 months) reduced the concentration of enteroviruses by a factor of 400-1,000, river bankfiltration was highly effective, reducing enteroviruses by a factor of at least 10,000. The effect ofcoagulationlflocculationlsedimentationlfiltration processes was highly variable, and was better when rapidsand filtration was included. The removal of F-specific RNA bacteriophages most closely followed that ofviruses in these three processes.

KEYWORDS

Drinking water; viruses; storage reservoirs; river bank filtration; coagulation.

INTRODUCTION

Recent developments in quantitative risk assessment have shown that drinking water treatment processesmust meet high standards with regard to removal of pathogenic micro-organisms. Based on an acceptablelevel of risk of 1 infection per 10,000 consumers per year, in the USA target values for pathogenic viruses(such as rota- or poliovirus) are recommended to be less than 2-3 per 10-7 per litre of drinking water (Regliet 01., 199\). It is not feasible to demonstrate compliance with such low concentrations by direct monitoringof the treated water. The virological safety of drinking water must be assured by measurements of theconcentration of pathogens in the source water and the removal and/or inactivation by treatment (Havelaar.1993). Ideally, the information must also describe the variability in time and in place of the pathogenconcentrations in drinking water. Such information can be derived from different sources. The most relevant

.5.5

56 A. H. HAVELAAR el al.

data can be obtained under full scale operating conditions, but such studies are limited to the first stages ofthe treatment process because of the detection limit of presently available methods. Additional informationcan be generated under pilot scale conditions (eventually seeded with pathogens to circumvent the detectionlimit problem). It is also possible to use data from model organisms (process indicators), which normallyoccur in significantly higher concentrations, provided that the removal of these indicators is related to that ofrelevant pathogens.

This study will describe the removal of entero- and reoviruses from surface waters by three types of drinkingwater treatment processes under field scale conditions: storage in surface reservoirs, river bank filtration andcoagulation/flocculation/sedimentationlfiltration. This paper is a summary of a series of papers where moredetails can be found (Van Olphen et al., 1992; 1993; Havelaar et al., 1993 and one manuscript inpreparation). The removal of various groups of model organisms will be related to that of the viruses. Wehave shown in an earlier paper that in the source waters, the concentration of F-specific RNA•bacteriophages was the best predictor of the concentration of viruses (Havelaar et al., 1993). All studieswere done in the winter season (November - March) because the virus concentrations in the river water areat their maximum, and the effectivity of the treatment processes is least.

MATERIALS AND METHODS

Treatment plants

fA). Stora~e reservoirs Waterwinnin~bedrjif "De Brabantse BjesboSch" (WEB). Three interconnectedreservoirs (38, 33 and 13 x 106 m3) are used in series, the average retention time during these studies (1983•5) was resp. 12-13, 12-13 and 5 weeks; hence the total retention time was approx. 7 months. By naturalcirculation and air injection at the bottom, each of the reservoirs can be considered as a well mixed system.In the last reservoir, the pH is raised by addition of sodium hydroxide for partial softening to 1.55 mmoVl.Before transport to several treatment plants, the pH is fixed at 9 by addition of sulfuric acid or sodiumhydroxide. The total production in the study period was 120 x 106 m] per year. Samples were taken at theinlet (River Maas) and at the outlet of each of the three reservoirs "De Gijster", "Honderd en Dertig" and"Petrusplaat".

(B) Riyer bank filtration plants Remmerden and Zwijndrecht The effect of river bank filtration was studiedat two locations selected for the following criteria: full operating conditions, water from the first aquiferwells that produce almost exclusively river bank filtrate and only a low percentage of ground water, sho";distance and residence time from river to well and availability of hydrological data. The characteristics ofthe wells are as follows: Remmerden well #1 draws suboxic water, 3 80% originating from the river Rijn at adistance of 30 m and with a modal residence time of 2 weeks. Zwijndrecht wells #7 and #19 draw anoxicwater, 3 90% originating from the river "Oude Maas" (a side-arm of the Rijn) at 25 and 30 m resp., with amodal residence time of 10 weeks.

(C). Coa~lation plants Watertransportmaatschappii Rjin-KennemerJand (WRKl. GemeentewaterJeidin~en

Amsterdam (GWA) and GemeenteJjjk Waterbedrijf Oronjn2en (OWGl. The WRK treatment plant Useswater from the Lekkanaal (a side-arm of the Rijn) which is coagulated with FeCI] (3 mgll as Fe) at a pH of7.3. After flocculation and sedimentation in a basin, the pH is raised to 8.0 with sodium hydroxide and thewater is filtered over rapid sand filters (2 m1b) and transported a.o. to the treatment works at Leiduin.Samples were taken of the raw water and one day later from the filtered water after transport to Leiduin. Theretention time of the water in the transport pipelines varied between 15 and 30 hours. The OWA treatmentplant uses water from the Amsterdam-Rijnkanaal (also a side-arm of the Rijn), which was mixed I: I withwater seepage water from the Bethunepolder. The water was coagulated in two steps with FeCI] (7 and 5mgJI, resp.) at a pH of 7.2-7.6 and after flocculation is allowed to settle in a sedimentation basin. Sampleswere taken of the raw water and the water after sedimentation. The OWG treatment plant uses water fromthe Drentsche Aa (a regional river), which is stored in a small reservoir (residence time 0.5=1 day) andcoagulated with polyhydroxy-aluminiumchloride (Sachtoklar, 3.5 - 8.5 mgll as AI) at a pH of 6.5 -7.0. After

Removal and inactivation of viruses S7

flocculation, the water was passed over lamella separators for sedimentation and filtered ground water wasadded if the temperature was < 4 DC; followed by aeration, activated carbon filtration, rapid and slow sandfiltration. Samples were taken of the raw water, the water after sedimentation and after rapid sand filtration.

MicrobiQl02ical methods

Enteroviruses (ENTVIR), reoviruses (REOVIR), F-specific RNA bacteriophages (FRNAPH), somaticcoliphages (SOMCPH), tQtal (TOTCOl) and thennotolerant (THCOl) coliform bacteria, faecal streptococci(FSTREP) and spores Qf sulphite-reducing c1Qstridia (SSRC) were determined as previously described(Havelaar et al., 1993; Havelaar and Nieuwstad, 1985). Colony counts at 37 (COLCOUNT 37) and 22 DC(COLCOUNT 22) were determined using pour plates and Plate Count Agar, incubated for resp. 2 and 3days. Aeromonas (AEROM) was determined by membrane filtration on ampicillin-dextrin agar according toHavelaar et al. (1987).

Statistical methods

All data were IOlog-transfonned before calculations and analysis. For all processes, Decimal Reduction(DR) values were calculated as the difference of the (mean) log-concentrations of the inflowing andoutflowing water. For coagulation processes, the relation between the DR values of (entero- or reo)virusesand model organisms or turbidity was calculated by correlation analysis using the MlNlTAB statisticalsoftware (Minitab, 1991).

RESULTS AND DISCUSSION

Stora~e reservoirs

Table 1 gives a summary of the concentrations of micro-organisms in the river and reservoir water in twoconsecutive winter seasons (Van Olphen et al., 1992; Havelaar et aI., 1992).

Table 1. Reduction of the concentration of viruses and indicator organisms by storage of river water in theBiesbosch reservoirs

Sampling Years Geometric mean concentration per litrepoint (winter

season)ENTVIR REOVIR FRNAPH SOMCPH THeOL FSTREP

River water '83-'84 1.6 3.4 not done not done 2600 690'84-'85 \.6 \.9 1200 6200 2500 450

De Gijster '83-'84 0.13 0.76 not done not done 620 300'84-'85 0.25 0.77 80 980 590 170

Honderd en '83-'84 <0.003 0.055 not done not done 330 170Dertig

'84-'85 0.015 0.078 5 130 100 50Petrusplaat '83-'84 <0.001 0.005 not done not done <50 <60

'84-'85 0003 0.006 2 50 20 20Average DR '83-'84 >3.20 2.83 notdene not done >1.72 >\.08value

'84-'85 2.73 2.51 2.78 2.08 2.10 1.34Number ofsamples was 7-9 in '83·'84; 4 for viruses and 14·]7 for model organisms in '84-'85

The geometric mean concentration of enteroviruses in the river Meuse was 1.6 per litre in both winters, themajority of isolates being typed as Coxsackie B (88 and 94%, respectively). The DR value of theenteroviruses was > 3.20 in the '83-'84 winter and 2.73 in the '84-'85 winter; all reservoirs contributed to the

58 A. H. HAVELAAR et al.

overall reduction. Coxsackie B viruses remained dominant in all reservoirs, although the proportion ofuntypable strains increased from 0 to 29%. The concentration of reoviruses in the raw water was somewhathigher than the enteroviruses, and the overall reduction was smaller. The reduction of FRNA-phages wasvery similar to the enteroviruses, whereas somatic coliphages, thermotolerant coliforms and faecalstreptococci were all reduced to a lesser extent.

A more detailed interpretation of the difference in behaviour of the various groups of micro-organisms in thereservoirs can be made on the basis of the variation of their concentrations in time and place (see Figure I).The concentration of enteroviruses in the river water showed a distinct peak in January-February, and thesame pattern was visible in the reservoirs. The FRNA-phages followed basically the same pattern, whereasthe behaviour for the other organisms was quite irregular. The reduction of the faecal bacteria and SOMCPHfrom one reservoir to the next was less, and occasionally the concentration in a later reservoir exceeded thatin the previous one. This indicates that recontamination has occurred, probably by faeces of wild-livinganimals. This hypothesis is supported by the fact that the relative removal of faecal streptococci was thelowest, because these organisms occur in relatively high numbers in animal faeces. Obviously, also somaticcoliphages and reoviruses, but not FRNA-phages are introduced into the water by animal faeces.

, II IJ 11 17 I'

ENTEROVIRUSES

lOUr,-------------,

" /-- '""'" .,/' ""•.14-~'":':..:":.~.":':..~.~.'::,,:"""".~.~.~'~.~II~,.::. :""_,~,.....J,,

THERMOTOLERANT COLIFORMS

J •••", L..-~~""''''''~'''''''''''':':"''.o.:-'':"':':'':':''•••••,., .. 111 J'" 11 11 1I"1f

F-SPECIPIC IlNA PHAGES

I •••• ,.----..",..."....---,;------,

I£...,.~~.......<...-~~.....~~...........-..•••, ., n •• It I J , , • II II II U 11

REOVIRUSES

1••".------------

,,,I ~.-_ ..~

...........' ~

,/' ...."./ ,,!II L.o..~.....~~.....~~.........".~...........

4•• , •• ., .. til I J J ,

FAECAL STREPTOCOCCI

,......------------,

,L.o..~.....~~.....~~.....~.........J...... '" •• II • I • , • II II II II "

SOMATIC COLIPHAGES

.....~l ....Pr"~~• ']A'/ ' ~

.~ V4' •• U .... II , J , , • 11 It II 17 I'

FigW'C 1. Reduction of viruses and indicator organisms by storage of river water in the Biesbosch reservoirs.

Removal and inactiValioD of viruses S9

Overall reduction of the enteroviruses was relatively low in comparison to the total residence time. Thisindicates that short-circuiting in the reservoirs is important in limiting the effect of storage. Since theinvestigations reported here. the production of the reservoirs has increased, reducing the total averageretention time to 5 months. Nevertheless. measurements in the winter of '93-'94 showed a DR value forenteroviruses of 2.55. which is very close to the results of ten years before (Hans Theunissen. unpublishedresults). The effect of short-circuiting is also evident from the fact that the peaks in virus concentration occuralmost simultaneously in all reservoirs. The effect of storage is thus enhanced if several reservoirs are usedin series. rather than if one large reservoir is used.

River bank filtration

The concentration of micro-organisms in the rivers Rijn and Oude Mans before river bank filtration (Fig. 2)was higher than in the river Meuse (see above). indicating a higher degree of sewage contamination. Also inthese samples, Coxsackie B viruses were the dominant type of enteroviruses (92%). 17 strains of polioviruswere isolated. among which 1 wild type strain. The river bank filtration process was extremely effective inreducing the concentration of all micro-organisms studied. Entero· and reoviruses were reduced by morethan 4 log-units. the reduction of FRNA-phages indicates that actual virus reduction was between 5 and 6log-units. Likewise, faecal indicator bacteria and Aeronwnas were efficiently reduced and usually notdetectable in I litre samples of river bank filtrate. This effect is greater than would be expected on the basisof the relatively short residence times and travelling distances. It is assumed that the major factorcontributing to the reduction of micro-organisms from river water by river bank filtration is the efficientremoval of suspended solids. to which the majority of the micro-organisms are adsorbed.

Coagulation

The faecal contamination of the Lekkanaal and the Amsterdam-Rijnkanaal was roughly equivalent, whichwas expected because both are side-arms of the river Rijn (Table 2). The data were also of the same order ofmagnitude as the river Meuse (see above). The Drentsche Aa is a local river. which is less extensively usedfor sewage discharges. This results in a relatively low concentration of viruses and FRNA-phages. Thedifference in the concentration of faecal bacteria is less. which may be related to agricultural activities in thisarea and resulting input from animal faecal sources. The concentration of viruses was particularly low inFebruary 1991, when the river was completely covered with ice. Colony counts and turbidity were similarfor all three raw water sources.

Table 2. Geometric mean concentration (per litre) of viruses and indicator organisms in river water beforecoagulation

ENTVIR 7.5 x 10'\ 3.0 X 10'\ 2 X 10.2

REOVIR 10 8.8 x 10.1 4 X 10.2

FRNAPH 5.0 x 10J 1.2 X 10J 1.1 X 102

TOTCOL 3.8 x 104 2.2 X 104 4.7 X 10JTHCOL 4.8 x 10J 4.4 X 10J 1.8 X 10JFSTREP 1.3 x leY 8.3 x 102 9.0 X 102

SSRC 4.1 x 10J 3.2x 10J 2.6x IOJ

COLCOUNT 37 8.0 x lOs 3.6 x lOs 3.0 x lOsCOLCOUNT 22 4.5 x 106 2.8 X 106 5.4 X 106

TURB (FTE) 11 12 19

Parameter WRK Lekkanaal' GWA Amsterdam- GWG Drentsche Aa"Rijnkanaal •

• Winter seasons '87-'88 and '88·'89: "Winter season '89·'90. mixed 1:1with seepage water; 11 Winter season '90-'91

AI,.. - Rhenen Oude Mioas - Zw'"aecht 2l

107

- I I10

6

~ I I J. I I 1 Ia: 105 1 I 1 1

~ 10' I I I I I 1 I I !~ 10' 1 I 1 1 I! ::: 1..1 .II 1.....1L ~i 10-' ~S 10-2

...1 ~10 -3 .... "f'. • I ~ .... " ...

10-"ENTV FR-IA TOTC Tl-C FSTR SSRC AER CC22 CC37 ENTV FRNA TOTC THe FSTR SSRC AER CC22 CC37

Figure 2: Reduction ofmicroorganisms by river bank filtration of river water(bars indicate mean and range before (thick) and after (thin) filtration; dashed lines are limits ofdetection.

ENTV = "enteric \'iruses" = entero and rem'iruses. Data from van Olphen d ai, 1993)

Removal and inactivation of viruses 61

The reduction of micro-organisms by the coagulation processes is shown in Figure 3. It is clear that theeffect of the coagulation process at OWA was least for all organisms studied. This is related to the type ofsedimentation, which takes place in a basin. subject to wind and other external influences. Furthermore. theseepage water which is mixed with the river water before coagulation has a very high concentration ofhumic acids. which negatively influences the coagulation process. Mter passage of a reservoir, the water isled through rapid sand filters. which gives a marked improvement of the microbiological quality. Because ofshort-circuiting effects. these data cannot accurately be correlated to the inflowing water. however. Note thevery high removal of reoviruses in comparison to the other organisms. The effect of thecoagulation/sedimentation at GWG was clearly better. Average decimal reduction values mnged from 0.94for COLCOUNT 22 to 1.82 for FSTREP. In the following rapid sand flltration step. a further reduction wasfound. The concentration of ENTVIR and REOVIR was usually below the detection limit (approx. 0.001•0.002 per litre). Likewise. concentrations of model organisms and turbidity were low after filtration.Because of this, the effect of filtration could not be quantified. At WRK. the effect of the process includingfiltration could be quantified because of the higher influent concentrations. Mean DR-values varied from1.34 for COLCOUNT 37 to 2.25 for FSTREP.

COLCOUNT 22 2,5

COlCOUNT 37 2,5

TURS

2,5

ENTVIR

2,5

FSTREP

REOVIR2,5

o GWA (COAGISED)

EI GWG (COAGISED)

• WRK (COAGISED/FILT)

2,5 FRNAPH

2,5 TOTCOl

Figure 3. Mean DR values of viruses, model organisms and turbidity by coagulation/sedimentation/filtration atthree treaunenl plants.

The average DR-values are compared in Figure 3 to examine the possibility of predicting virus removalfrom model parameters. The mean DR of colony counts were usually the lower than those for viruses. Thismight indicate that bacteria contributing to colony counts are difficult to remove by coagulation. However. itis more likely to be an artefact related to the reintroduction of these bacteria by the addition of coagulationaids. Enteroviruses were reduced at relatively low levels, as were reoviruses at two of the three plants. Theremovals of FRNAPH and SSRC were close to those of the enteroviruses. whereas DR values of TOTCOL.THCOL and FSTREP were considembly higher. DR values for turbidity were similar to those forenteroviruses at two of the three plants. but higher at the WRK plant Correlation analysis of individual datapairs did not reveal any significant and/or consistent relations between the reduction of viruses and modelorganisms or turbidity at individual treatment plants. This is probably related to the fact that DR values arecalculated as the difference between two relatively imprecise measurements. and thus have an inherently lowprecision. If the data from all plants are pooled, significant correlations between DR-values of enterovirusesand all model parameters were found. The highest correlation coefficients were found for turbidity (r =

62 A. H. HAVELAAR t!t al.

0.645; n =31); TOTCOL (r =0.641; n =31); SSRC (r =0.628; n =31) and FRNAPH (r =0.583; n =32).Taking into account these correlations, and the ratio of average DR-values, it can be concluded that SSRcand FRNAPH are the best predictors for enterovirus removal by coagulation processes. The reduction ofturbidity also may give a good estimate of the reduction of enteroviruses.

ACKNOWLEDGEMENTS

This work was carried out on behalf of the Netherlands Waterworks Association and the Directorate Generalof Environmental Protection. We thank Elly van de Baan, Ria de Bruin and Didy van Veenendaal for virusanalysis, and Ria Hogeboom, Marcel During, Reina van der Heide , Roelof Pot and Ciska Schets forbacterial and phage analysis. The field studies and interpretation were carried out in close cooperation withmany people from KIWA and the waterworks companies. We are particularly grateful for the support andadvice of Lambert van Breemen (GWA), Jon Schellart (GWA), Guus Soppe (GWG), Dick van der KOOij,Yvonne Drost and Pieter Stuyfzand (KIWA).

REFERENCES

Havelaar. A. H. (1993). The place for microbiological monitoring in the production of safe drinking water. In: Safety ofdrinlcingwater disinfection: balancing chemical and microbiological risks, G.F. Craun (Ed).ILSI Press, Washington D.C., pp.127·141.

Havelaar. A. H.• During. M. and Versteegh.l. F. M. (1987). AmpicillIn-dextrin agar medium for the enumeration of Aeromonasspecies in water by membrane filtration. J. Appl. Bacteriol.• 62. 279-287.

Havelaar. A. H. and Nieuwstad. Th. J. (1985). Bacteriophages and fecal bacteria as indicators of chlorination efficiency ofbiologically treated wastewater. J. Water Pol/ut. Control Fed.• 57. 1084-1088.

HaveJaar. A. H.• Van Olphen, M. and Van Breemen. L. W. C. A. (1992). Bacteriofagen als model voor verwijdering van virussenin spaarbekkens. H20 20. 556-558.

Havelaar. A. H.• Van Olphen. M. and Drost. Y. C. (1993). F-specific RNA bacteriophages are adequate model organisms forenteric viruses in fresh water. Appl. Environ. Microbiol.• 59. 2956-2962.

Minitab (1991). M/NlTAB Reference Manual. release 8, PC version. MlOitab, State College. PA.Regli. S.• Rose. J. B.• Haas. C. N. and Gerba, C. P. (1991). Modeling the risk from Giardia and viruses in drinking water. Journal

AWWA. 83. 76-84.Van Olphen. M., Van de Baan. E.• Kapsenberg, 1.G. and Van Breemen. L.W.C.A. (1992). Virusverwijdering bij opslag van

Maaswater in spaarbekkens. H20 20. 550-555.Van Olphen. M.• Van de Baan. E. and Havelaar. A. H. (1993). Virusverwijdering bij oeverfiltratie. Hp 21.63-66.