APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 1993, p. 2956-29620099-2240/93/092956-07$02.00/0Copyright X) 1993, American Society for Microbiology

F-Specific RNA Bacteriophages Are Adequate ModelOrganisms for Enteric Viruses in Fresh WaterARIE H. HAVELAAR,1* MARJA vAN OLPHEN,1'2 AND YVONNE C. DROST2

Laboratory of Water and Food Microbiology, National Institute of Public Health and EnvironmentalProtection, P.O. Box 1, 3720 BA Bilthoven, 1 and KIWA NJVResearch and Consultancy,

3430 BB Nieuwegein,2 The Netherlands

Received 24 February 1993/Accepted 17 June 1993

Culturable enteroviruses were detected by applying concentration techniques and by inoculating theconcentrates on the BGM cell line. Samples were obtained from a wide variety of environments, including rawsewage, secondary effluent, coagulated effluent, chlorinated and UV-irradiated effluents, river water, coagu-lated river water, and lake water. The virus concentrations varied widely between 0.001 and 570/liter. Thesame cell line also supported growth of reoviruses, which were abundant in winter (up to 95% of the virusesdetected) and scarce in summer (less than 15%). The concentrations of three groups of model organisms inrelation to virus concentrations were also studied. The concentrations of bacteria (thermotolerant coliformsand fecal streptococci) were significantly correlated with virus concentrations in river water and coagulatedsecondary effluent, but were relatively low in disinfected effluents and relatively high in surface water open tononhuman fecal pollution. The concentrations of F-specific RNA bacteriophages (FRNA phages) were highlycorrelated with virus concentrations in all environments studied except raw and biologically treated sewage.Numerical relationships were consistent over the whole range of environments; the regression equations forFRNA phages on viruses in river water and lake water were statistically equivalent. These relationships supportthe possibility that enteric virus concentrations can be predicted from FRNA phage data.

The presence of human enteric viruses in water used fordrinking, recreation, or growing shellfish may pose a risk tohuman health. Treatment processes and watershed manage-ment strategies designed on the basis of bacteriologicalcriteria do not necessarily protect against virus infectionbecause viruses are generally more persistent in the waterenvironment and are not removed as well by treatmentprocesses. More than 100 types of human pathogenic virusesmay be present in fecally polluted water, but only a smallnumber can be detected by currently available methods. Ofthese methods, only those based on concentration determi-nations by adsorption-elution methods and subsequent de-tection by cell culture have gained widespread acceptance.Many methods readily detect enteroviruses and reoviruses;methods for detecting adeno- and rotaviruses by cell cultureare also available but are less widely used. As a conse-quence, protection of water quality has come to rely heavilyon the detection of culturable entero- and reoviruses. Theseviruses occur abundantly in fecally polluted environmentsand are relatively resistant to inactivation by natural ortreatment processes, and it is assumed that processes capa-ble of removing or inactivating the entero- and reoviruses arealso capable of controlling the wider range of waterborneviruses. The methods are relatively laborious, require spe-cialized personnel, and are therefore not well suited formonitoring purposes (2). For monitoring purposes, modelorganisms that behave like waterborne viruses but arereadily detectable by simple, rapid, and inexpensive meth-ods should be selected. Bacteriophages, in particular theF-specific RNA bacteriophages (FRNA phages), appear tobe well suited for this purpose (6).

In the past decade we have studied the fate of humanviruses and model organisms in a variety of environments

* Corresponding author.

and treatment processes, including river water stored inopen reservoirs (11), bank infiltration (10), coagulation,flocculation, and filtration of drinking water (unpublisheddata) and secondary effluent (8), disinfection of secondaryeffluent by chlorination (5) and UV radiation (7), recreationalwaters, and other surface waters (unpublished data). In thispaper we summarize the data from these studies and specif-ically address the relationship between virus concentrationsand FRNA phage concentrations. For comparison, data forthermotolerant coliforms and fecal streptococci are alsopresented below.

MATERMILS AND METHODS

Sampling points. Samples were obtained between January1985 and March 1991 from a variety of sources in theNetherlands, including raw and treated wastewater, raw andpartially treated drinking water, and surface and recreationalwaters. A summary of the sampling points used is given inTable 1. Samples were classified into eight categories asindicated in Table 1.

Detection and enumeration of viruses. Viruses from rawsewage (RS), secondary effluent (SE), coagulated (Fe3")effluent (CE), chlorinated effluent (CIE), and UV-irradiatedeffluent (UVE) were recovered from 10-liter grab samples byusing an adsorption-elution technique followed by ultrafiltra-tion, as described elsewhere (8); river water (RW), coagu-lated river water (CR), and lake water (LW) were alsopartially examined by using 10-liter grab samples, but largersamples (10 to 1,000 liters) were collected by a field methodin which an automated concentration device was used.Briefly, the water to be examined was continuously dosedwith an Mg2" solution to a final concentration of 0.05 M andadjusted to pH 3.8 with hydrochloric acid, and the water waspassed through Milligard type CW30012C3 pleated filtercartridges (pore size, 3 ,um). The filters were eluted in the

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TABLE 1. Sampling points and periods of sampling (see Materials and Methods for explanation of abbreviations)

Type of Description No. of Sampling periodsample samples

RS Pilot plant, Technical University, Delft' 11 October 1985-January 1986SE Pilot plant, Technical University, Delft' 11 October 1985-January 1986

Pilot plant, Technical University, Delfth' 13 November 1988-February 1989Sewage treatment plant, Leerdam 5 July 1987-September 1987

CE Simultaneous precipitationa 11 October 1985-January 1986Simultaneous precipitation with filtrationa 11 October 1985-January 1986Postprecipitationa 11 October 1985-January 1986

ClE Sewage treatment plant, Zwaanshoek 5 July 1987-September 1987UVE Medium-pressure lampsb 10 December 1988-February 1989

Low-pressure lampsb 10 December 1988-February 1989RW Linge 14 July 1987-September 1987

Haarlemmer Ringvaart 15 July 1987-September 1987Rhine, Meuse, and side armsc 22 October 1986-May 1987Rhine, Meuse, and side arms 10 November 1987-March 1988Rhine, Meuse, and side arms 7 August 1989-September 1990Rhine, Meuse, and side arms 12 November 1989-March 1990Drentse Aa 12 December 1990-March 1991

CR Watertransportmij Rijn-Kennemerland, Nieuwegein 10 November 1987-March 1989Gemeentewaterleidingen, Amsterdam, Loenen 12 November 1989-March 1990GWG De Punt, mixed 1:1 with groundwater 12 December 1990-March 1991

LW Various recreational waters 32 June 1989-September 1990a See reference 8.b See reference 7.c See reference 10.

field with ca. 1,500 ml of a 3% beef extract solution (pH 9.0),and the eluate was further concentrated in the laboratory byultrafiltration, using cellulose acetate membranes (typeNMWL 10,000). Viruses were isolated from concentratedsamples by using the BGM cell line and an agar overlaymethod. After incubation for 10 days at 37°C, plaques werecounted and differentiated into entero- and reoviruses on thebasis of plaque morphology. The virus type was frequentlyverified by subculturing and serological typing of isolatedvirus strains. Concentrations are reported below for entero-viruses and enteric viruses (the total concentrations ofentero- and reoviruses).

Detection and enumeration ofFRNA phages. FRNA phageswere measured as the concentration of plaque-forming par-ticles on host strain WG49 [Salmonella typhimurium phagetype 3 Nalr (F' 42 lac::TnS)] as described by Havelaar andHogeboom (4). If the concentration of plaque-forming parti-cles was expected to be greater than 5/ml, samples ordilutions of samples were directly examined by using theconventional double agar layer method; at expected concen-trations between 0.1 and 5/ml a modified double agar layermethod was performed with 5-ml samples and 14-cm petridishes was used. At lower expected phage concentrations, a

part of the eluate from the first step of the virus concentra-tion procedure was examined by the double agar layermethod.

Detection and enumeration of fecal indicator bacteria. Ther-motolerant coliforms and fecal streptococci were determinedby measuring concentrations of colony-forming particles byusing membrane filtration methods described previously (5),except that sodium lauryl sulfate (10 g/liter) was used insteadof Teepol 610 as the surfactant in the selective growthmedium for thermotolerant coliforms. In samples of second-ary effluent collected between November 1988 and February1989 and in all samples of UV-irradiated effluent, thermotol-erant coliform counts were replaced by Eschenchia colicounts by using the direct plating method (3).

Statistical methods. Table 2 shows the numbers of valuesin the data set that were either missing or below thedetection limit. For statistical analysis, most values belowthe detection limit were set equal to this value. The excep-tions were the values for enteroviruses in UV-irradiatedeffluent, for which all concentrations were below the detec-tion limit; these values were excluded from the analysis.Pairs with missing values were also excluded from theanalysis. All data were log transformed before statisticalanalysis. Because variables on both axes in the regressionanalysis are subject to error, model II regression analysiswas used instead of the regular (model I) regression analysis,which only accounts for errors on the y axis. The type ofmodel II regression used was the so-called reduced majoraxis regression or geometric mean regression (9). The regres-sion coefficient was calculated as vy, = sI,Js., where v, isequivalent to b (the slope of the regression line) in the modelI regression and s, and sy are the standard deviations of the

TABLE 2. Number of samples with values below detection limitsand with missing values

No. of samples withvalues below the No. of samples with

Type of No. of detection limit for:sample samples Thermo- FecalEntero- Enteric FRNA FRNA tolerant strepto-viruses viruses phages phages coliforms cocci

RS 11 0 0 0 3 0 0SE 29 2 0 0 3 0 5CE 33 15 4 2 9 0 0CIE 5 0 0 0 0 0 5UVE 20 20 2 0 0 1 0RW 92 3 0 0 0 1 30CR 34 10 6 0 0 1 1LW 32 11 2 8 0 0 0

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x and y data, respectively. The sign of vy, can be obtainedfrom the correlation coefficient. The intercept (a) was calcu-lated as follows: a = y-v x. Because the number ofsamples varied so much among water types, causing differ-ences in variance, a regression analysis was first performedseparately for each water type. The log concentrations of themodel organisms were regressed on the log concentrations ofthe viruses. The resulting prediction formulae could be usedto estimate the log concentration of enteroviruses or entericviruses from a given log concentration of a model organism.The prediction errors (which were higher than the errors ofthe regression!) were calculated by using the followingformula:

Sp= S.2 l{ + (l/n) + [(Xp - X)2Is2x (n - 1)]}

where 52 is the unexplained mean square which measuredthe resid'ual variation (error) of the regression, Xp is the logconcentration of the model organisms from which the logconcentration of the viruses was predicted, sx2 is the vari-ance of the observed log concentrations of the model organ-isms, xi is the mean, and n is the sample size. With theprediction error, 95% prediction limits (Lu per and Llower)were calculated for a range of possible vaFues for the logconcentrations of model organisms:

Lupper = predicted value + t(o.05,n-1)Sp (1)

Llower = predicted value - t(O.o5,n01lSp (2)

where t(oo05,n-1) is the value from a two-tailed t table for a =

0.05 and n - 1 degrees of freedom. For large sample sizes twas the value of 1.96.To determine whether data obtained from different water

types could be combined, a regression analysis was per-formed on combined data from all sampling points. Subse-quently, it was determined with a Kruskal-Wallis testwhether the residual values for the various water types weredifferent from each other. If there was a significant differ-ence, the most deviant water type was deleted from the dataset and the regression analysis and Kruskal-Wallis test wererepeated for the remaining water set. This procedure wasrepeated if necessary.

RESULTS

The BGM cell line used in this study is sensitive to bothentero- and reoviruses. By maintaining cell cultures for aperiod of 10 days and by optimizing the sensitivity of thecells to viral infection, we routinely obtained high yields ofreoviruses. A distinct seasonal variation in the relativenumbers of the two groups of viruses was observed; primar-ily enteroviruses were found in the summer, and relativelyhigh proportions of reoviruses were found in the winter (Fig.1). This pattern was detectable in the total data set, and alsoin sample groups that were assayed in both seasons, such asthe RW and SE samples. A summary of the range of virusconcentrations found in the different types of samples isshown in Table 3. The virus concentrations were highlyvariable in all of the sample types examined, which may

have been related to the different locations where sampleswere obtained and also to temporal variations in virusconcentrations at fixed sampling stations. The virus concen-trations spanned almost 7 orders of magnitude, with theconcentrations of plaque-forming particles of enterovirusesranging between <0.001 and 570/liter and the concentrationsof enteric viruses ranging between <0.001 and 8,700/liter.

100

90

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0

co

D

co

0

t-

80

70

60

50

40

30

20

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J F M A M J J A S o N DMonth

FIG. 1. Seasonal variation in enterovirus concentration as apercentage of the total enteric virus concentration (combined datafrom all samples).

Similarly, there were wide variations in the concentrationsof the model organisms, which spanned 6 to 7 orders ofmagnitude (Table 4). The concentrations of plaque-formingparticles of FRNA phages ranged between 0.0003 and 3,100/ml; the concentrations of colony-forming particles of ther-motolerant coliforms were between 0.005 and 34,000/ml, andthe concentrations of fecal streptococci were between 0.006and 7,800/ml.The correlations between model organism and virus con-

centrations are shown in Table 5. FRNA phage concentra-tions were significantly correlated with enteric virus concen-trations in five of eight water types examined and withenterovirus concentrations in four of six sample types. Thecorrelation between FRNA phage and virus concentrationswas relatively poor in samples of raw or secondary treatedsewage, but very good in (partially treated) surface waters.Of the model bacteria, the concentrations of thermotolerantcoliforms correlated better than the FRNA phage concentra-tions with enterovirus concentrations but not with entericvirus concentrations in raw and treated sewage. In surfacewater, the correlations were not as good; the absence of asignificant correlation in LW samples should be particularlynoted. The pattern of correlations observed between fecalstreptococcal concentrations and virus concentrations was

TABLE 3. Virus concentrations in various sample groups

Median concn (range) of plaque-forming particlesType of No. of per litersample samples

Enteroviruses Enteric viruses

RS 11 200 (<0.1-570) 1,100 (400-8,700)SE 29 1.3 (<0.1-15) 18 (1.1-1,900)CE 33 0.2 (<0.1-9.8) 15 (<0.1-1,300)CIE 5 4.1 (2.8-14) 6.9 (3.4-23)UVE 20 <0.1 0.7 (<0.1-16)RW 92 0.38 (<0.001-10) 1.6 (0.0028-38)CR 34 0.02 (<0.001-0.71) 0.042 (<0.001-1.4)LW 32 0.006 (<0.005-1.3) 0.052 (<0.005-11)

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TABLE 4. Concentrations of model organisms in various sample groups

Type of No. of Median concn (range) of plaque- or colony-forming particles per mlsample samples FRNA phages Thermotolerant coliforms Fecal streptococci

RS 11 1,800 (360-3,100) 20,000 (13,000-34,000) 3,300 (1,100-7,800)SE 29 39 (3.1-170) 580 (24-10,000) 140 (36-720)CE 33 13 (<0.1-96) 63 (2-3,100) 5.1 (0.1-250)CIE 5 30 (7.6-180) 10 (2-3,000) NDaUVE 20 6.2 (0.06-32) 2.3 (0.064-180) 6.1 (0.13-310)RW 92 1.5 (0.018-52) 6.4 (0.36-600) 1.2 (0.23-26)CR 34 0.16 (0.0003-1.7) 0.086 (0.005-2.3) 0.18 (0.006-0.95)LW 32 0.028 (<0.004-1.3) 2.5 (0.38-89) 0.40 (0.06-76)a ND, not done.

similar to the pattern observed between thermotolerantcoliform concentrations and virus concentrations.

If a relationship between viruses and model organisms isto be of practical use, it is important that there is a goodcorrelation for individual types of water samples and alsothat the relationship between the organisms is consistent fordifferent types of water samples. This was examined byplotting the predicted concentrations of the viruses from theregression equations against the concentrations of modelorganisms, as shown in Fig. 2 and 3. Figures 2 and 3 clearlyshow that there was a fairly consistent relationship betweenvirus and FRNA concentrations. The relationship betweenthermotolerant coliform and fecal streptococcal concentra-tions and virus concentrations was more variable. There wasa fair degree of similarity among the different regressionequations for FRNA phages and viruses (Table 6). Statisti-

TABLE 5. Correlations between concentrations of modelorganisms and viruses in different types of water samples

(log-transformed data)Enteroviruses Enteric viruses

Model ognssType oflorganisms sample No. of Correlation No. of Correlationsamples coefficient samples coefficient

FRNA phages RS 8 -0.072 8 0.504SE 26 -0.118 26 0.250CE 24 0.475a 24 0.764bCIE NDC 5 0.515UVE ND 20 0.508aRW 92 0.591b 92 0.625bCR 34 0.726b 34 0.708bLW 32 0.468b 32 0.461b

Thermotolerant RS 11 0.428 11 -0.135coliforms SE 29 0.423a 29 0.242

CE 33 0.607k 33 0.755kCIE ND 5 0.223UVE ND 20 0.336RW 91 0.431b 91 0.4791CR 33 0.545b 33 0.368aLW 32 -0.033 32 0.281

Fecal streptococci RS 11 0.181 11 -0.252SE 24 0.323 24 0.172CE 33 0.645k 33 0.744bCIE ND NDUVE ND 20 0.255RW 62 0.463b 62 0.515kCR 33 0.433k 33 0.230LW 32 -0.356a 32 0.162

a Correlation significant at P < 5%.b Correlation significant at P < 1%.c ND, not done.

cally, it was permitted to regress data for FRNA phages oneither enteroviruses or enteric viruses for RW and LWsamples combined. Also, data for RS, SE, CE, and CRsamples could be combined. The prediction lines for thecombined RW-LW data set, along with upper and lowerprediction limits and the actual, measured data, are shown inFig. 4. These graphs can be used to estimate the virusconcentration at a given concentration of FRNA phages orvice versa.

DISCUSSION

The results of this study indicate that there is a generaltendency of virus concentrations to correlate with the con-centrations of all three types of model organisms in sometypes of samples, such as RW samples. This means that forthese types of water samples, an acceptable estimate of thevirus concentration can be derived from data for conven-tional bacteriological water quality parameters, such as theconcentrations of thermotolerant coliforms or fecal strepto-cocci. It must be realized, however, that such an estimate isnot universally applicable and may be wrong by severalorders of magnitude in other types of water samples. Bacte-ria are generally more susceptible to disinfectants, and theirnumbers are relatively low in disinfected waters. Data usedin this study indeed confirmed this expectation for CIE andUVE samples (5, 7). On the other hand, nonhuman fecalsources contribute to counts of thermotolerant coliforms andfecal streptococci in the environment in the absence ofhuman enteroviruses. The data presented for the LW sam-ples clearly reflect this effect, making bacterial standardsless effective for recreational waters, for example. In all ofthese cases, FRNA phage concentrations were found to bebetter predictors of virus concentrations. The correlationcoefficients were higher, and also the behavior of FRNAphages in individual water treatment processes was moresimilar to the behavior of viruses (5, 7, 8, 10, 11). Theseresults therefore confirm the effectiveness of FRNA phagesas model organisms for human viruses in a wide range ofenvironments and treatment processes. Despite this, therewere exceptions to the rule, particularly in raw or biologi-cally treated sewage and with some samples obtained fromrelatively unpolluted environments. The poor relationshipbetween FRNA phage and virus concentrations in sewagemay be due to the inherent variability of virus concentrationsin sewage samples in general, because these concentrationsdepend strongly on the effects of a limited number of activevirus carriers in the population. This explanation is sup-ported by the fact that there was also no correlation with theconcentration of fecal streptococci and only a weak corre-

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2960 HAVELAAR ET AL.

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FIG. 2. Predicted concentrations of enteroviruses in relation toconcentrations of FRNA phages (A), thermotolerant coliforms (B),and fecal streptococci (C) in different types of water samples. Theline thickness is directly related to the significance of the correlation(see Table 5). CENTERO, concentration of plaque-forming particles ofenteroviruses; CFRNA, concentration of plaque-forming particles ofF-specific RNA phages; CITHCOL, concentration of colony-formingparticles of thermotolerant coliforms; CFSTREP, concentration ofcolony-forming particles of fecal streptococci.

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FIG. 3. Predicted concentrations of enteric viruses in relation toconcentrations of FRNA phages (A), thermotolerant coliforms (B),and fecal streptococci (C) in different types of water samples. Theline thickness is directly related to the significance of the correlation(see Table 5). CENTERIC, concentration of plaque-forming particlesof enteric viruses. For an explanation of other abbreviations see thelegend to Fig. 2.

lation with the concentration of thermotolerant coliforms inone case.

In several LW samples FRNA phages were detected in theabsence of enteroviruses (Fig. 4A). A plausible explanation

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FRNA PHAGES AS MODEL VIRUSES IN WATER 2961

TABLE 6. Regression equations of the logarithms of viruses (y)on model organisms (x): log (y) = a + v: log (x)

(significant correlations only)

Enteroiruses EntericModel Type(s) of Enteroviruses viruses

organisms samplea VY, a V:

FRNA phages CE -1.09 0.82 -0.25 1.24UVE NDa -0.56 0.84RW -0.56 0.86 0.10 1.05CR -0.80 0.96 -0.27 1.10LW -0.23 0.99 0.80 1.25RW+LW -0.53 0.85 0.17 0.98RS+SE+CE+CR -0.98 0.93 -0.23 1.13

Thermotolerant SE -3.21 1.20 NSbcoliforms CE -1.89 0.84 -1.47 1.27

RW -1.59 1.28 -1.16 1.56CR -0.96 1.06 -0.46 1.21

Fecal streptococci CE -1.05 0.79 -0.20 1.20RW -0.80 1.48 -0.20 1.81CR -0.07 1.20 0.57 1.37LW -2.01 -0.91 NS

a ND, not done.b NS, not significant.

is that these samples were affected by small-scale sewagedischarges that were not affected by an active virus carrier.In this case, the potential for virus contamination waspresent, and FRNA phage counts would have provided amargin of safety. On the other hand, we also isolatedenteroviruses in the absence of FRNA phages. This is amore serious shortcoming of the phages as model organismsand may be related to direct fecal input of active viruscarriers (e.g., from recreational activities such as bathing orswimming). However, these cases were exceptional, and thevirus concentrations in these samples were less than 0.1particle per liter, the limit currently considered acceptablefor recreational waters in the European Communities (1).The strong relationship between virus and FRNA phage

concentrations in surface waters makes the latter organismsa suitable alternative for direct detection of viruses inrecreational waters. In applying the prediction equationsgiven in this paper, it must be realized that they wereobtained in a limited geographical region and by using onlysamples from fresh waters. More data are needed before thesuitability of FRNA phages as virus models can be assessedon an international basis. These data should also include theoccurrence of FRNA phages in animal feces. To date,FRNA phages have been detected only infrequently inanimal feces, but studies have concentrated on domestic,farm, and zoo animals. No data are available on wild animalsthat may contribute to the contamination of surface waters.

It is interesting to note that in certain cases, reoviruseswere detected in recreational waters in the absence ofenteroviruses, even in the summer when the relative abun-dance of reoviruses was small. This presumably indicatesthat there were animal sources of reoviruses in surfacewaters, making reoviruses less suitable for setting health-related standards for recreational waters. Because of theirhigh numbers and relatively high resistance, reoviruses areuseful organisms in the study of the effectiveness of watertreatment processes.

ACKNOWLEDGMENTSThis work was carried out on behalf of the Directorate-General of

Environmental Protection, Ministry of Housing, Physical Planning

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prediction limits (-- - -) for enterovirus (A) and enteric virus (B)concentrations in relation to concentrations of FRNA phages in RWand LW samples. See Table 6 for the regression equation. Forexplanations of abbreviations see the legends to Fig. 2 and 3.

and Environmental Protection, and the Netherlands WaterworksAssociation. We thank Elly van de Baan, Ria de Bruin, and Didyvan Veenendaal for virus analysis and Ria Hogeboom, MarcelDuring, Reina van der Heide, Roelof Pot, Ciska Schets, and JackSchijven for bacterial and phage analyses.

REFERENCES

1. European Communities. 1975. Council directive on the quality ofbathing water. Off. J. Eur. Commun. L31:1-7.

2. Havelaar, A. H. The place for microbiological monitoring in theproduction of safe drinking water. In First International Con-ference on the Safety of Water Disinfection-Balancing Chem-ical and Microbiological Risks, in press. ILSI Press, Washing-ton, D.C.

3. Havelaar, A. H., and M. During. 1988. Evaluation of theAnderson Baird-Parker direct plating method for enumeratingEscherichia coli in water. J. Appl. Bacteriol. 64:89-98.

4. Havelaar, A. H., and W. M. Hogeboom. 1984. A method for theenumeration of male-specific bacteriophages in sewage. J. Appl.Bacteriol. 56:439-447.

5. Havelaar, A. H., and T. J. Nieuwstad. 1985. Bacteriophages andfecal bacteria as indicators of chlorination efficiency of biolog-ically treated wastewater. J. Water Pollut. Control Fed. 57:1084-1088.

6. IAWPRC Study Group on Health Related Water Microbiology.1991. Bacteriophages as model viruses in water quality control.

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Hydraulic and microbiological characterization of reactors forultraviolet disinfection of secondary wastewater effluent. WaterRes. 25:775-783.

8. Nieuwstad, T. J., E. P. Mulder, A. H. Havelaar, and M. vanOlphen. 1988. Elimination of micro-organisms from wastewaterby tertiary precipitation and simultaneous precipitation fol-lowed by filtration. Water Res. 22:1389-1397.

9. Sokal, R. R., and F. J. Rohlf. 1981. Biometry: the principles andpractice of statistics in biological research, 2nd ed. W. H.Freeman, New York.

10. van Olphen, M., E. van de Baan, and A. H. Havelaar. 1993.Virusverwijdering bij oeverinfiltratie. H20 26:63-66.

11. van Olphen, M., E. van de Baan, J. G. Kapsenberg, andL. W. C. A. Van Breemen. 1992. Virusverwijdering bij opslagvan Maaswater in spaarbekkens. H20 25:550-555.

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