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Virus removal and recovery in the drinking water treatment train
War. Res. Vol. 26, No. 6, pp. 727-731. 1992 Pnnted in Great Britain
0043-1354:92$5.00+ 0,00 PergamonPress Ltd
VIRUS REMOVAL AND RECOVERY IN THE DRINKING WATER TREATMENT TRAIN RONALD E. STETLER*,STEVENC. WALTRIPand CHRISTONJ. HURST~ United States Environmental Protection Agency, Cincinnati, OH 45268, U.S.A. (First received September 1990: accepted in revised form December 1991)
Abstract--Paired water samples were collected at four points in the drinking water treatment train. One sample of each pair was used to determine levels of indigenous enteroviruses and bacteriophages at each stage. The other sample was seeded with an enterovirus and two different coliphages to determine if. in the treatment process train, the water was affected in such a way that the efficiencyof the chosen method for viral detection would be adversely influenced. Recovery efficiencieswith seeded enterovirus varied from 40 to 68%. Coliphage recoveries were considerably lower, ranging from 4 to 12%. Recovery of seeded q~XI74 from the water samples demonstrated a statistically significant inverse correlation with water temperature. The results indicate the importance of demonstrating the satisfactory performance for recovering particular viruses by the method selected. Key words--enterovirus, bacteriophage, virus detection
ing viruses from matched water samples collected at different stages of the treatment train.
INTRODUCTION Human enteroviruses have been found at various points in the treatment train of drinking water treatment plants including the finished water (Deetz et al., 1984: Payment el al., 1985; Rose et al., 1986; Stetler et al., 1984). In the study by Payment et al. (1985) in-depth information was provided on virus removal through drinking water treatmen~ trains at seven different plants but no information was obtained on the efficiency of the virus concentration-recovery procedure at any treatment point. On the other hand, Sobsey and Glass (1984) presented data on seeded virus recovery from 1.3-1. samples of raw, finished and granular activated carbon-treated waters using two different filter types and four different enteric viruses. However, that study did not examine other points within the treatment train. Stetler et al. (1984) reported on the relative efficiency of available methods for recovering viruses from an operating water treatment plant but efficiency of the concentration-recovery procedure was limited to seeded finished water. Dahling and Wright 0984) have recommended that, to maximize virus recovery by the chosen virus conccntration method and establish a baseline value for virus recovery efficiency of the monitoring system, a course of pretesting bc performed with environmental samples containing seeded virus. The purpose of the present study was 2 fold, measuring virus removal through the course of the treatment processes at an operating full-seale drinking water treatment plant and establishing the efficiency of the detection methodology for recover-
*Author to whom all correspondence should be addressed.
MATERIALS AND METHODS
Physico-chemical analysis Several analyses were performed on-site. These were: determination of turbidity using a Hach 2100A Turbidimeter (Hach Co., Loveland, Colo.), conductivity using a Hach 16300-00 Conductivity meter (Hach Co., Loveland, Colo.), temperature and pH. Values for total and suspended solids content were determined at an off-site laboratory using the methods described in Standard Methods for the Analysis of Water and Wastewater (APHA. 1985). Dissolved solids were calculated as the differencebetween the total and suspended solids. Bacterial analysis Bacterial analyses of grab samples were performed by membrane filtration. Fecal coliforms, total coliforms and heterotrophic bacteria analyses were performed according to methods described in Standard Methods (APHA, 1985). Enterococci analysis was performed according to a published US Environmental Protection Agency method (US EPA, 1985). CIostridium perfringens analysis was performed by the method of Bisson and Cab¢lli (1979), as modified for use with anaerobic incubation using GasPak pouches (BBL Microbiology Systems, Cockeysville, Md). Virus stocks and hosts Bacterial viruses and their respective host organisms used in this study were: coliphage [2 (ATCC 15766-BI) with its host Escherichia coil A-19, an Hfr strain of E. coil from R. L. Ward (Christ Hospital Institute of Medical Research, Cincinnati, Ohio) and coliphage ~XI74 (ATCC 13706-B1) with its host E. coil C (ATCC 13706). The method of Ward and Mahler (1982) was used to prepare stock suspensions of coliphages [2 and ~XI74 and to perform plaque assays of these viruses. Bacteriophages recovered from water samples were assayed as referenced above using as host strains E. coil A-19 and C. The laboratory strain of enterovirus used in this study was human coxsackievirus B5 (CB5). BGM cells, a continuous
727
728
RONALDE. STETLER et al.
River a
b
Hocculation
c
d
Filter Clear
Pump l Sedimentati°n IIL~
~Filter
C12
Well
C12
(Routine
(For Study)
Fig. 1. Treatment plan diagram, Letters a-d are sampling sites used throughout the study. (a) Source water; (b) post-sedimentation; (c) post-sand filtration; (d) finished water. CI 2 (routine) is the usual chlorination point. CI, (for study) was the chlorination point during study sampling periods. cell line originating from African green monkey kidney (Dahling et aL. 1974), were used for the cultivation and recovery of CB5 and for the recovery of enteroviruses concentrated from the water samples collected during this study. The procedure of Benton and Hurst (1986) was used for plaque assays. ! "irus sampling
At the study site. described previously by Stctlcr et uL (1984), chlorine was added post-sand liltration 24 h prior to and during the time perkvd when sltmples were collected (Fig. I). Samples were collected in (I) May, (2) August and (3) November 1987 and (4) February 1988. Two 189-1. (50-gal.) water samples, one for detection of indigenous enteroviruscs and coliphages, the other to be seeded with coxsackicvirus CB5 and coliphages t"2 and ~XI74 were collected simultaneously in 208-1. (55-gal.) plastic barrels from four sites in the treatment process. These samples were: source water taken at the plant intake, post-sedimentation. post-sand filtration and finished tap water (chlorinated, post sand-filtration). Samples were collected sequentially from source water to finished water in a manner that was intended to coincide timewise with the flow of water through the treatment regimen of the plant. The tap water was immediately dechlorinated by addition of Na,SzO 3 to a final concentration of 0.3 raM. Each of the water samples was supplemented with AICI3 to a final concentration of 0.5 mM. 25 ml of concentrated acetic acid and sufficient concentrated HC1 was added to the salted samples to bring the water sample to pH 3.5. Prior to the virus concentration process. one water sample from each pair was seeded with CB5 at an average input of 1.6 x 101 pfu/ml, t"2 at an average input of I.I x 10" pfu/ml and ~XI74 at an average input of 2.0 x l0 s pfu/ml. Each sample was filtered through a 0.45#m Filterite 10-inch cartridge (Duo-Fine Series, Filterite Corp., Timonium, Md). After filtration of the water sample, excess water was drained from each filter and filter holder and the filter was eluted with 1600ml of 3% beef extract, 1.5% w/'v of both Lab Lemco Powder (Oxoid Ltd, Long. U.K.) and Beef extract V (BBL Microbiology Systcms. Cockeysville, Md), at pH 9.5 plus 0.1% w/v antifoam C (Dow Chemical, Midland. Mich.). The elution procedure has been described by Dahling and Wright (1984). A 20-ml volume of each filter eluate was removed, adjusted to pH 7.0 and used in bacteriophage assays. The remaining eluate was concentrated by the method of Katzenelson et al. (1976) and frozen at - 7 0 C until assayed for enteroviruses on BGM cells. RESULTS The physico-chcmical characteristics o f the water collected at different stages in the treatment train
are reported in Table I. The removal o f turbidity and suspended solids during the treatment process from source to finished water averaged 98 and 93%, respectivcly. Only a slight decrease in conductivity was obscrved between the source and finished water. Characterization of the bacterial content at different stagcs in the treatment train showed that enterococcus and C. p e r f r i n g e n s were rapidly reduced by post-sedimentation and post-sand filtration stages (Table 2). Fccal and total coliforms, as well as hetcrotrophic bacteria, usually showed significant penetration through the point of post-sand filtration. F r o m source to post-sand filtration, bacterial counts were reduced at least 93% for all tested bacterial populations except the heterotrophic bacteria which were reduced 88% or greater. Table I. Physico-chcmicalcharacteristics of the water samples* Parameter
Sampling site
Mean (range)
pH (standard units)
Source Post-sedimentation Post-sand filtration Finished
7.8 (7.6-8.0) 7.1 (6.9-7.2) 7.1 (7.1-7.1) 7.1 (7.1-7.1)
Temperature (C)
Source Post-sedimentation Post-sand filtration Finished Source Post-sedimentation Post-sand filtration Finished Source
12.0 ( I. 1-23.0) 12.0 ( I. 1-23.0) 12.0 (I.I-23.0) 12.0 (I.I-23.0) 15.4 (5.8-31.0) 3.5 (2.3-5.0) 0:3 (0.3-0.4) 0.3 (0.2-0.4) 650 (600 -700)
Turbidity (NTU)
Conductivity
(~umhos/cm:) Total solids (rag/l)
Suspended solids
(rag/I) Dissolved solids (rag/I)
Post-sedimentation
635 (510-700)
Post-sand filtration Finished
615 (500-680) 607 (500-700)
Source Post-sedimentation Post-sand filtration Finished
517.5 (440.0-553.3) 521.7 (440.0-566.7) 513.7 (446.7-580.0) 541.0 (469.0-666.7)
Source
Post-sedimentation Post-sand filtration Finished Source
15.7 (3.0-37.0)
7.3 (3.5-14.5) 0.9 (0.2-1.5) I.I (I.1-1.4)
491.7 (436.9-528.8) Post-sedlmentation 514.4(433.0-563.2) Post-sand filtration 513.0(446.5-579.2) Finished 540.0 (468.0-665.3)
*Reported values are averages of duplicate samples.
729
Virus removal from drinking water Table 2. Bacterial content of waters collected from the treatment train* Sample
Sampling site
Total coliform
Fecal coliform
Emerococcus
Clostriditun perfrmgens
Hcterotropic plate count
I
Source Post-sedimentation Post-sand filtration Finished
1800 500 40 < I
140 70 I0 < I
100 80 < I < I
233 13 < I < I
49000 31000 --t < I
2
Source Post-sedimentation Post-sand filtration Finished
2000 400 45 < I
400 I I0 13 < I
600 30 2 < I
108 33 < I < I
13000 1800 1500 < I
3
Source Post-sedimentation Post-sand filtration Finished
1050 260 l0 < 1
330 5 I < I
< < < <
I I I I
300 < I < I < 1
3000 430 170 < I
4
Source Post-sedimentation Post-sand filtration Finished
725 49 < I < I
685 5 < I < I
6 < I < I < I
56 3 < I < I
1300 30 < I
"Values reported are the averages of duplicate assays expressed as colony forming units (cfu) per I00 ml except for the heterotropic plate count (HPC) which is expressed as cfu per | ml. tSample lost.
Table 3. Percent recovery of seeded virus from source water*
Table 5. Percent recovery of seeded virus from post-sand filtration water*
Coliphage
Coliphage
Sample
Enterovirus CB 5
~
~ X 174
I 2t 3 4
53.6 56.1 47.5 ~.5
3.2 23.1 9.1 13.9
2.5 0.7 II.0 23.3
50.4±5.3**
12.3±8.4.*
9.4±10.3.*
*Reported values are the average of duplicate assays. fSample size was 170 I, (45 gal.). **Overall mean of percent recovery for that virus type + ISD.
Sample
Enterovirus CB5
t"2
~ X 174
I 2 3 4
70.7 76.9 89.1 35.1
I.I 17.2 1.8 4.8
3.8 0.1 22.8 17,1
67.9±23.2?
6.2 ± 7.5t
I1.0 ± 10.8t
*Reported values are the average of duplicate assays. tOverall mean for that virus type ± I SD.
Tables 3-6 summarize recovery efficiencies for seeded enterovirus and coliphage from water samples collected at different points in the treatment train. Average enterovirus recovery varied from 39.9% in finished water to 67.9% in post-sand filtration samples. Enterovirus recovery efficiency varied the least with samples collected from the source water. With post-sedimentation and post-sand filtration samples, a single low recovery value from sampling period four greatly influenced the average enterovirus recovery value. The finished water sample varied greatly due to a very low recovery in sampling period three. Analysis of variance (ANOVA) indicated no significant differences in relative recovery of the seeded CB5 among the four treatment stages
(P=0.3900) nor among the sampling periods (P --- 0.0795). Recovery of 1'2 ranged from a low of 4.0% for post-sedimentation to a high of 12.3% for source water samples. Recovery of ~X174 ranged from a low of 6.7% for finished water to a high of 11.0% for post-sand filtration samples. A wide variation was observed in the recovery of each coliphage type, particularly when comparing the recovery data for each stage of the water treatment train. However, ANOVA results indicated there were no significant differences in relative recovery of the seeded coliphages among the four treatment stages (P = 0.1215 for 1'2 and 0.7448 for ~XI74). Among sampling periods though there were significantly higher relative
Table 4. Percent recovery of seeded virus from postsedimentation water"
Table 6. Percent recovery of seeded virus from finished water*
Coliphage
Coliphage
Sample
Enterovirus CB5
t2.
~ X 174
Sample
Enterovirus CB 5
I 2 3 4
~.4 53.0 56.5 10.4
2.2 9.4 3.4* 1.1*
4.4 1.2 15.2* 8.8
I 2 3 4
39.3 7,3 86.4 26.9
16.6 15.5 5.2 7.1
3.1 0.2 7.0 16.5
46.0 _+ 24.2**
4.0 ± 3.7**
7.4 -t- 6.1.*
39.9 ± 33.6t
I1.1 ± 5.8t
6.7 ± 7. It
*Reported values are the average of duplicate assays. $Based on a single assay. **Overall mean of percent recovery for that virus type ±1 SD.
t"2
#~X 174
*Reported values are the average of duplicate assays. tOverall mean for that virus type -t- I SD.
730
ROSALDE. S'nETLr~ et al.
recoveries of f2 in the second sampling period than in the first and third sampling periods (P --0.0262). For ~XI74, relative recoveries were significantly different between all sampling periods except the last two (P = 0.0001). Indigenous enteroviruses and coliphages were detected in source, post-sedimentation and post-sand filtration water samples, but never in post-chlorinated (finished) water samples (Table 7). The highest enterovirus recovery occurred in sampling period one whereas the highest coliphage recovery using both E. coil hosts, occurred in sampling period three. Enteroviruses were detected in the absence of coliphages in sampling period one. In sampling periods two and three, the reverse was observed with coliphages detected but not enteroviruses. In sampling period one. enterovirus and, in sampling periods three and four, coliphages penetrated through the sand filtration stage of the water treatment train. Only sampling periods one and four yielded sufficient indigenous enteroviruses to enable comparisons among the treatment stages and each analysis had to be done separately. At P > 0.05 log recoveries were not significantly different among stages for either sampling period. Analysis of coltphage recovery on E. colt A-19 in sampling periods three and fi)ur indicated only in sampling period three were there statistically significant differences in recovery among treatment stages. In this sampling period significantly fewer coliphage were found in the post-sand filtration sample than in either the source or post-sedimentation samples (P =0.0164). Coltphage recovery on E. colt C between the source and post-sedimentation stages showed no significant difference for sampling periods two and three. However, in sampling period three, post-sand filtration coliphage recovery was significantly lower than either of the two prior treatment stages. In sampling period four, recovery differences were highly significant. Sourcc sample recovery was significantly lower than
both the post-sedimentation and post-sand filtration samples which is counterintuitive to expected results. The primary reason for the high significance level of these results is not as much due to the magnitude of the differences as it is to the unusually low variance in this sampling period compared to the other results (mean square error =0.00053). The low variance is reflected by the consistency among the duplicate assays during the fourth sampling period. DISCUSSION
This study was conducted at the same treatment plant previously used to determine what effect moving the chlorination point to reduce trihalomethane formation would have upon the levels of enteroviruses, indicator bacteria and coliphages (Stetler et al., 1984: Stetler, 1984). The turbidity values for source to finished water were considerably lower in this study than those reported in the previous study but the level of turbidity removal was nearly identical. The removal of fecal and total coliforms and heterotrophic bacteria was very similar to the earlier study with the reduction of fecal coliforms being slightly higher (Stetler et al., 1984). It is difficult to compare the reduction of enteroviruses between this study and the previous ones, because of the lesser number of samples collected. Based on the two virus-containing samples, this limited data indicated a much lower reduction than in the previous study. The limited coliphage data permitted only a generalized comparison of their reduction with the previous study where average reduction of coliphages on both E. coil A-19 and C was 85 and 68%, respectively (Stetler, 1984). In this study the average reduction was 74 and 75% on E. colt A-19 and C, respectively. Recovery of seeded enterovirus ranged from 39.0 to 67.9% with the source sample being the least variable. Both the post-sedimentation and post-sand
Tahlc 7. Enterovirus and coliphage levels detected at different treatment stages" Coliphage assayed on: Sample
Sampling site
Enterovirus
E. coil A-19
E. colt C
l
Source Post-sedimentation Post-sand filtration Finished
0.11 0.33 0.09 0.0
0.0 0.0 0.0 0.0
0.0 0.0 0.0 0.0
2
Source¢ Post-sedimentation Post-sand filtration Finished
0.0 0.0 0.0 0.0
0r0 0.0 0.0 0.0
I 2.6 4.2 0.0 0.0
3
Source Post-sedtmcntation Post-sand filtration Finished
0.0 0.0 0.0 0.0
49.7 23.1 5.5 0.0
311.7 339.8 78.5 0.0
4
Source Post-sedimentation Post-sand filtration Finished
0.06 0.03 0.0 0.0
5.4 2.2 2.4 0.0
1.4 26.2 32.7 0.0
*Values reported arc averages of duplicate assays expressed as pfu/I. tSample volume 170 I. (45 gal.).
Virus removal from drinking water filtration samples had one low recovery in sampling period four which altered an otherwise fairly consistent recovery. Since the source water sample assay remained consistent with previously obtained values, the assay system would not appear to be the source of the low recovery value for that particular sampling period. Rather, interference in the elution or reconcentration process would seem responsible for the low recoveries. Virus recovery from finished water was the most variable and interference in the ¢lution or reconcentration process would also be a likely exp!anation. Dahling and Wright 0984) found that AICI3-conditioned Cincinnati finished water gave lower recoveries than did MgCl:-conditioned water due to substantial amounts of sulfate in the water. This finished water was not chemically analyzed so it is unknown if interfering levels of sulfate were present. However, AI:(SO,)~ formation would not seem to be a good explanation because the post-sand filtration water should have been equally affected. As anticipated, recovery of seeded coliphage was low. The low pH conditions of the membrane adsorption-clution method of enterovirus recovery are detrimental to some bacteriophage (Sabatino and Maier, 1980). Nevertheless, an approximate 10% recovery was obtained: therefore, when only small numbers of bacteriophage are present, their detection would be very unlikely. Payment et al. (1988) published a method using MgCl:-conditioned water at pH 6 with a Filterite cartridge which provided more accurate data on coliphage recoveries from seeded samples as well as for coliphage numbers in environmental samples. Analyses of variance for the log-transformed recovery data were performed to determine whether there were signiticant differences in relative recovery (I) among the four stages of the treatment process and (2) among the four samples taken at different periods at each treatment stage. Data were analyzed with respect to each of the three seed viruses. ANOVA results indicated no evidence that recovery efficiency differed among samples taken at different stages in the treatment process. Such a difference would be of concern in comparing virus survival at these stages. While recovery efficiency did differ among the four sampling periods for the two coliphages, this was not an important concern since the experiment was balanced with respect to sampling periods in assessing virus survival at each successive stage. An effort was made to determine whether the recovery efficiencies for the seeded viruses correlated with the various physical or chemical characteristics of the water samples which include temperature, pH, conductivity, turbidity, total solids, suspended solids or dissolved solids. Recovery of ~ X 174 demonstrated a correlation with water temperature (r = - 0.80838 as determined by Pearson's test, P =0.00015). No other correlations existed between recovery of the
731
seeded viruses and the water characteristics. The information obtained in this study indicated the importance of a pre-examination of environmental samples to determine the satisfactory performance of the method applied to monitoring a water system especially when monitoring is done over a period of time. Acknowledgement--The authors are grateful to Larry
Wymer of Computer ScienceCorporation for the statistical analysis of the data. REFERENCES
APHA (1985) Standard Methods fi~r the Examination o f Water and Wastewater, 16th edition, pp. 860-896. American Public Health Association, Washington. D.C. Benton W. H. and Hurst C. J. (1986) Evaluation of mixed types and 5-iodo-2'-deoxyuridine treatment upon plaque assay titers of human enteric viruses. AppL em'ir. MicrobioL 51, 1036-1040. Bisson J. W. and CabeUi V. J. (1979) Membrane filter enumeration methods for CIostridium perfringens. Appl. em'ir. Microbiol. 37, 55-66. Dahling D. R. and Wright B, A. (1984) Processing and transport of environmental virus samples. Appl. enrir. Microbiol. 47, 1272-1276. Dahling D. R., Berg G. and Berman D. (19741 BGM, a continuous cell line more sensitive than primary Rhesus and African green kidney cells for the recovery of viruses from water, tilth Sci. II, 275 282. Dcetz T. R., Smith E. M., Goyal S. M., Gerba C. P.. Vollet J. J., Tsai L.. DuPont I!. L. and Keswick B. H. (19841 Occurrence of rota- and enteroviruses in drinking and environmental water in a deveh)pingnation, lrat. Res. 18, 567- 57 I. Katzenelson E., Fattal B. and Ih)stovesky T. (1976) Organic fiocculation: an efficient second-step concentration method for the detection of viruses in tap water. Appl. em'ir. MicrobioL 32, 638-639. Payment P., Morin E. and Trudel M. (1988) Coliphage and enteric virus in the particulate phase of river water. Can. J. MicrobioL 34, 907-910. Payment P., Trudel M. and Plante R. (1985) Elimination of viruses and indicator bacteria at each step of treatment during preparation of drinking water at se','en water treatment plants. Appl. em'ir. Microbiol. 49, 1418-1428. Rose J. B., Gerba C. P., Singh S. N., Toranzos G. A. and Keswick B. (1986) Isolating viruses from finished water. J. Am. Wat. Wks Ass. 78, 56-61. Sabatino C. M. and Maier S. (1980) Differentialinactivation of 3 bacteriophages by acid and alkaline pH used in the membrane adsorption-elution method of virus recovery. Can. J. Microbiol. 26, 403-407. Sobsey M. D. and Glass J. C. (1984) Influence of water quality on enteric virus concentration by microporous filter methods. Appl. era,Jr. MicrobioL 47, 956-960. Stetler R. E. (1984) Coliphages as indicators of enteroviruses. AppL em~ir. MicrobioL 48, 668-670. Stetler R. E., Ward R. L. and Waltrip S. C. (1984) Enteric virus and indicator bacteria levels in a water treatment system modified to reduce trihalomethane production. Appl. enrir. Microbiol. 47, 319-324. USEPA (1985) Test methods for Escherichia coli and Enterococcus in water by the membrane filter procedure. EPA 00/4-85/076. US EPA, Cincinnati, Ohio. Ward R. L. and Mahler R. J. (1982) Uptake of bacteriophage 1"2,through plant roots. AppL era'it. Microbiol. 43, 1098-1103.
0043-1354:92$5.00+ 0,00 PergamonPress Ltd
VIRUS REMOVAL AND RECOVERY IN THE DRINKING WATER TREATMENT TRAIN RONALD E. STETLER*,STEVENC. WALTRIPand CHRISTONJ. HURST~ United States Environmental Protection Agency, Cincinnati, OH 45268, U.S.A. (First received September 1990: accepted in revised form December 1991)
Abstract--Paired water samples were collected at four points in the drinking water treatment train. One sample of each pair was used to determine levels of indigenous enteroviruses and bacteriophages at each stage. The other sample was seeded with an enterovirus and two different coliphages to determine if. in the treatment process train, the water was affected in such a way that the efficiencyof the chosen method for viral detection would be adversely influenced. Recovery efficiencieswith seeded enterovirus varied from 40 to 68%. Coliphage recoveries were considerably lower, ranging from 4 to 12%. Recovery of seeded q~XI74 from the water samples demonstrated a statistically significant inverse correlation with water temperature. The results indicate the importance of demonstrating the satisfactory performance for recovering particular viruses by the method selected. Key words--enterovirus, bacteriophage, virus detection
ing viruses from matched water samples collected at different stages of the treatment train.
INTRODUCTION Human enteroviruses have been found at various points in the treatment train of drinking water treatment plants including the finished water (Deetz et al., 1984: Payment el al., 1985; Rose et al., 1986; Stetler et al., 1984). In the study by Payment et al. (1985) in-depth information was provided on virus removal through drinking water treatmen~ trains at seven different plants but no information was obtained on the efficiency of the virus concentration-recovery procedure at any treatment point. On the other hand, Sobsey and Glass (1984) presented data on seeded virus recovery from 1.3-1. samples of raw, finished and granular activated carbon-treated waters using two different filter types and four different enteric viruses. However, that study did not examine other points within the treatment train. Stetler et al. (1984) reported on the relative efficiency of available methods for recovering viruses from an operating water treatment plant but efficiency of the concentration-recovery procedure was limited to seeded finished water. Dahling and Wright 0984) have recommended that, to maximize virus recovery by the chosen virus conccntration method and establish a baseline value for virus recovery efficiency of the monitoring system, a course of pretesting bc performed with environmental samples containing seeded virus. The purpose of the present study was 2 fold, measuring virus removal through the course of the treatment processes at an operating full-seale drinking water treatment plant and establishing the efficiency of the detection methodology for recover-
*Author to whom all correspondence should be addressed.
MATERIALS AND METHODS
Physico-chemical analysis Several analyses were performed on-site. These were: determination of turbidity using a Hach 2100A Turbidimeter (Hach Co., Loveland, Colo.), conductivity using a Hach 16300-00 Conductivity meter (Hach Co., Loveland, Colo.), temperature and pH. Values for total and suspended solids content were determined at an off-site laboratory using the methods described in Standard Methods for the Analysis of Water and Wastewater (APHA. 1985). Dissolved solids were calculated as the differencebetween the total and suspended solids. Bacterial analysis Bacterial analyses of grab samples were performed by membrane filtration. Fecal coliforms, total coliforms and heterotrophic bacteria analyses were performed according to methods described in Standard Methods (APHA, 1985). Enterococci analysis was performed according to a published US Environmental Protection Agency method (US EPA, 1985). CIostridium perfringens analysis was performed by the method of Bisson and Cab¢lli (1979), as modified for use with anaerobic incubation using GasPak pouches (BBL Microbiology Systems, Cockeysville, Md). Virus stocks and hosts Bacterial viruses and their respective host organisms used in this study were: coliphage [2 (ATCC 15766-BI) with its host Escherichia coil A-19, an Hfr strain of E. coil from R. L. Ward (Christ Hospital Institute of Medical Research, Cincinnati, Ohio) and coliphage ~XI74 (ATCC 13706-B1) with its host E. coil C (ATCC 13706). The method of Ward and Mahler (1982) was used to prepare stock suspensions of coliphages [2 and ~XI74 and to perform plaque assays of these viruses. Bacteriophages recovered from water samples were assayed as referenced above using as host strains E. coil A-19 and C. The laboratory strain of enterovirus used in this study was human coxsackievirus B5 (CB5). BGM cells, a continuous
727
728
RONALDE. STETLER et al.
River a
b
Hocculation
c
d
Filter Clear
Pump l Sedimentati°n IIL~
~Filter
C12
Well
C12
(Routine
(For Study)
Fig. 1. Treatment plan diagram, Letters a-d are sampling sites used throughout the study. (a) Source water; (b) post-sedimentation; (c) post-sand filtration; (d) finished water. CI 2 (routine) is the usual chlorination point. CI, (for study) was the chlorination point during study sampling periods. cell line originating from African green monkey kidney (Dahling et aL. 1974), were used for the cultivation and recovery of CB5 and for the recovery of enteroviruses concentrated from the water samples collected during this study. The procedure of Benton and Hurst (1986) was used for plaque assays. ! "irus sampling
At the study site. described previously by Stctlcr et uL (1984), chlorine was added post-sand liltration 24 h prior to and during the time perkvd when sltmples were collected (Fig. I). Samples were collected in (I) May, (2) August and (3) November 1987 and (4) February 1988. Two 189-1. (50-gal.) water samples, one for detection of indigenous enteroviruscs and coliphages, the other to be seeded with coxsackicvirus CB5 and coliphages t"2 and ~XI74 were collected simultaneously in 208-1. (55-gal.) plastic barrels from four sites in the treatment process. These samples were: source water taken at the plant intake, post-sedimentation. post-sand filtration and finished tap water (chlorinated, post sand-filtration). Samples were collected sequentially from source water to finished water in a manner that was intended to coincide timewise with the flow of water through the treatment regimen of the plant. The tap water was immediately dechlorinated by addition of Na,SzO 3 to a final concentration of 0.3 raM. Each of the water samples was supplemented with AICI3 to a final concentration of 0.5 mM. 25 ml of concentrated acetic acid and sufficient concentrated HC1 was added to the salted samples to bring the water sample to pH 3.5. Prior to the virus concentration process. one water sample from each pair was seeded with CB5 at an average input of 1.6 x 101 pfu/ml, t"2 at an average input of I.I x 10" pfu/ml and ~XI74 at an average input of 2.0 x l0 s pfu/ml. Each sample was filtered through a 0.45#m Filterite 10-inch cartridge (Duo-Fine Series, Filterite Corp., Timonium, Md). After filtration of the water sample, excess water was drained from each filter and filter holder and the filter was eluted with 1600ml of 3% beef extract, 1.5% w/'v of both Lab Lemco Powder (Oxoid Ltd, Long. U.K.) and Beef extract V (BBL Microbiology Systcms. Cockeysville, Md), at pH 9.5 plus 0.1% w/v antifoam C (Dow Chemical, Midland. Mich.). The elution procedure has been described by Dahling and Wright (1984). A 20-ml volume of each filter eluate was removed, adjusted to pH 7.0 and used in bacteriophage assays. The remaining eluate was concentrated by the method of Katzenelson et al. (1976) and frozen at - 7 0 C until assayed for enteroviruses on BGM cells. RESULTS The physico-chcmical characteristics o f the water collected at different stages in the treatment train
are reported in Table I. The removal o f turbidity and suspended solids during the treatment process from source to finished water averaged 98 and 93%, respectivcly. Only a slight decrease in conductivity was obscrved between the source and finished water. Characterization of the bacterial content at different stagcs in the treatment train showed that enterococcus and C. p e r f r i n g e n s were rapidly reduced by post-sedimentation and post-sand filtration stages (Table 2). Fccal and total coliforms, as well as hetcrotrophic bacteria, usually showed significant penetration through the point of post-sand filtration. F r o m source to post-sand filtration, bacterial counts were reduced at least 93% for all tested bacterial populations except the heterotrophic bacteria which were reduced 88% or greater. Table I. Physico-chcmicalcharacteristics of the water samples* Parameter
Sampling site
Mean (range)
pH (standard units)
Source Post-sedimentation Post-sand filtration Finished
7.8 (7.6-8.0) 7.1 (6.9-7.2) 7.1 (7.1-7.1) 7.1 (7.1-7.1)
Temperature (C)
Source Post-sedimentation Post-sand filtration Finished Source Post-sedimentation Post-sand filtration Finished Source
12.0 ( I. 1-23.0) 12.0 ( I. 1-23.0) 12.0 (I.I-23.0) 12.0 (I.I-23.0) 15.4 (5.8-31.0) 3.5 (2.3-5.0) 0:3 (0.3-0.4) 0.3 (0.2-0.4) 650 (600 -700)
Turbidity (NTU)
Conductivity
(~umhos/cm:) Total solids (rag/l)
Suspended solids
(rag/I) Dissolved solids (rag/I)
Post-sedimentation
635 (510-700)
Post-sand filtration Finished
615 (500-680) 607 (500-700)
Source Post-sedimentation Post-sand filtration Finished
517.5 (440.0-553.3) 521.7 (440.0-566.7) 513.7 (446.7-580.0) 541.0 (469.0-666.7)
Source
Post-sedimentation Post-sand filtration Finished Source
15.7 (3.0-37.0)
7.3 (3.5-14.5) 0.9 (0.2-1.5) I.I (I.1-1.4)
491.7 (436.9-528.8) Post-sedlmentation 514.4(433.0-563.2) Post-sand filtration 513.0(446.5-579.2) Finished 540.0 (468.0-665.3)
*Reported values are averages of duplicate samples.
729
Virus removal from drinking water Table 2. Bacterial content of waters collected from the treatment train* Sample
Sampling site
Total coliform
Fecal coliform
Emerococcus
Clostriditun perfrmgens
Hcterotropic plate count
I
Source Post-sedimentation Post-sand filtration Finished
1800 500 40 < I
140 70 I0 < I
100 80 < I < I
233 13 < I < I
49000 31000 --t < I
2
Source Post-sedimentation Post-sand filtration Finished
2000 400 45 < I
400 I I0 13 < I
600 30 2 < I
108 33 < I < I
13000 1800 1500 < I
3
Source Post-sedimentation Post-sand filtration Finished
1050 260 l0 < 1
330 5 I < I
< < < <
I I I I
300 < I < I < 1
3000 430 170 < I
4
Source Post-sedimentation Post-sand filtration Finished
725 49 < I < I
685 5 < I < I
6 < I < I < I
56 3 < I < I
1300 30 < I
"Values reported are the averages of duplicate assays expressed as colony forming units (cfu) per I00 ml except for the heterotropic plate count (HPC) which is expressed as cfu per | ml. tSample lost.
Table 3. Percent recovery of seeded virus from source water*
Table 5. Percent recovery of seeded virus from post-sand filtration water*
Coliphage
Coliphage
Sample
Enterovirus CB 5
~
~ X 174
I 2t 3 4
53.6 56.1 47.5 ~.5
3.2 23.1 9.1 13.9
2.5 0.7 II.0 23.3
50.4±5.3**
12.3±8.4.*
9.4±10.3.*
*Reported values are the average of duplicate assays. fSample size was 170 I, (45 gal.). **Overall mean of percent recovery for that virus type + ISD.
Sample
Enterovirus CB5
t"2
~ X 174
I 2 3 4
70.7 76.9 89.1 35.1
I.I 17.2 1.8 4.8
3.8 0.1 22.8 17,1
67.9±23.2?
6.2 ± 7.5t
I1.0 ± 10.8t
*Reported values are the average of duplicate assays. tOverall mean for that virus type ± I SD.
Tables 3-6 summarize recovery efficiencies for seeded enterovirus and coliphage from water samples collected at different points in the treatment train. Average enterovirus recovery varied from 39.9% in finished water to 67.9% in post-sand filtration samples. Enterovirus recovery efficiency varied the least with samples collected from the source water. With post-sedimentation and post-sand filtration samples, a single low recovery value from sampling period four greatly influenced the average enterovirus recovery value. The finished water sample varied greatly due to a very low recovery in sampling period three. Analysis of variance (ANOVA) indicated no significant differences in relative recovery of the seeded CB5 among the four treatment stages
(P=0.3900) nor among the sampling periods (P --- 0.0795). Recovery of 1'2 ranged from a low of 4.0% for post-sedimentation to a high of 12.3% for source water samples. Recovery of ~X174 ranged from a low of 6.7% for finished water to a high of 11.0% for post-sand filtration samples. A wide variation was observed in the recovery of each coliphage type, particularly when comparing the recovery data for each stage of the water treatment train. However, ANOVA results indicated there were no significant differences in relative recovery of the seeded coliphages among the four treatment stages (P = 0.1215 for 1'2 and 0.7448 for ~XI74). Among sampling periods though there were significantly higher relative
Table 4. Percent recovery of seeded virus from postsedimentation water"
Table 6. Percent recovery of seeded virus from finished water*
Coliphage
Coliphage
Sample
Enterovirus CB5
t2.
~ X 174
Sample
Enterovirus CB 5
I 2 3 4
~.4 53.0 56.5 10.4
2.2 9.4 3.4* 1.1*
4.4 1.2 15.2* 8.8
I 2 3 4
39.3 7,3 86.4 26.9
16.6 15.5 5.2 7.1
3.1 0.2 7.0 16.5
46.0 _+ 24.2**
4.0 ± 3.7**
7.4 -t- 6.1.*
39.9 ± 33.6t
I1.1 ± 5.8t
6.7 ± 7. It
*Reported values are the average of duplicate assays. $Based on a single assay. **Overall mean of percent recovery for that virus type ±1 SD.
t"2
#~X 174
*Reported values are the average of duplicate assays. tOverall mean for that virus type -t- I SD.
730
ROSALDE. S'nETLr~ et al.
recoveries of f2 in the second sampling period than in the first and third sampling periods (P --0.0262). For ~XI74, relative recoveries were significantly different between all sampling periods except the last two (P = 0.0001). Indigenous enteroviruses and coliphages were detected in source, post-sedimentation and post-sand filtration water samples, but never in post-chlorinated (finished) water samples (Table 7). The highest enterovirus recovery occurred in sampling period one whereas the highest coliphage recovery using both E. coil hosts, occurred in sampling period three. Enteroviruses were detected in the absence of coliphages in sampling period one. In sampling periods two and three, the reverse was observed with coliphages detected but not enteroviruses. In sampling period one. enterovirus and, in sampling periods three and four, coliphages penetrated through the sand filtration stage of the water treatment train. Only sampling periods one and four yielded sufficient indigenous enteroviruses to enable comparisons among the treatment stages and each analysis had to be done separately. At P > 0.05 log recoveries were not significantly different among stages for either sampling period. Analysis of coltphage recovery on E. colt A-19 in sampling periods three and fi)ur indicated only in sampling period three were there statistically significant differences in recovery among treatment stages. In this sampling period significantly fewer coliphage were found in the post-sand filtration sample than in either the source or post-sedimentation samples (P =0.0164). Coltphage recovery on E. colt C between the source and post-sedimentation stages showed no significant difference for sampling periods two and three. However, in sampling period three, post-sand filtration coliphage recovery was significantly lower than either of the two prior treatment stages. In sampling period four, recovery differences were highly significant. Sourcc sample recovery was significantly lower than
both the post-sedimentation and post-sand filtration samples which is counterintuitive to expected results. The primary reason for the high significance level of these results is not as much due to the magnitude of the differences as it is to the unusually low variance in this sampling period compared to the other results (mean square error =0.00053). The low variance is reflected by the consistency among the duplicate assays during the fourth sampling period. DISCUSSION
This study was conducted at the same treatment plant previously used to determine what effect moving the chlorination point to reduce trihalomethane formation would have upon the levels of enteroviruses, indicator bacteria and coliphages (Stetler et al., 1984: Stetler, 1984). The turbidity values for source to finished water were considerably lower in this study than those reported in the previous study but the level of turbidity removal was nearly identical. The removal of fecal and total coliforms and heterotrophic bacteria was very similar to the earlier study with the reduction of fecal coliforms being slightly higher (Stetler et al., 1984). It is difficult to compare the reduction of enteroviruses between this study and the previous ones, because of the lesser number of samples collected. Based on the two virus-containing samples, this limited data indicated a much lower reduction than in the previous study. The limited coliphage data permitted only a generalized comparison of their reduction with the previous study where average reduction of coliphages on both E. coil A-19 and C was 85 and 68%, respectively (Stetler, 1984). In this study the average reduction was 74 and 75% on E. colt A-19 and C, respectively. Recovery of seeded enterovirus ranged from 39.0 to 67.9% with the source sample being the least variable. Both the post-sedimentation and post-sand
Tahlc 7. Enterovirus and coliphage levels detected at different treatment stages" Coliphage assayed on: Sample
Sampling site
Enterovirus
E. coil A-19
E. colt C
l
Source Post-sedimentation Post-sand filtration Finished
0.11 0.33 0.09 0.0
0.0 0.0 0.0 0.0
0.0 0.0 0.0 0.0
2
Source¢ Post-sedimentation Post-sand filtration Finished
0.0 0.0 0.0 0.0
0r0 0.0 0.0 0.0
I 2.6 4.2 0.0 0.0
3
Source Post-sedtmcntation Post-sand filtration Finished
0.0 0.0 0.0 0.0
49.7 23.1 5.5 0.0
311.7 339.8 78.5 0.0
4
Source Post-sedimentation Post-sand filtration Finished
0.06 0.03 0.0 0.0
5.4 2.2 2.4 0.0
1.4 26.2 32.7 0.0
*Values reported arc averages of duplicate assays expressed as pfu/I. tSample volume 170 I. (45 gal.).
Virus removal from drinking water filtration samples had one low recovery in sampling period four which altered an otherwise fairly consistent recovery. Since the source water sample assay remained consistent with previously obtained values, the assay system would not appear to be the source of the low recovery value for that particular sampling period. Rather, interference in the elution or reconcentration process would seem responsible for the low recoveries. Virus recovery from finished water was the most variable and interference in the ¢lution or reconcentration process would also be a likely exp!anation. Dahling and Wright 0984) found that AICI3-conditioned Cincinnati finished water gave lower recoveries than did MgCl:-conditioned water due to substantial amounts of sulfate in the water. This finished water was not chemically analyzed so it is unknown if interfering levels of sulfate were present. However, AI:(SO,)~ formation would not seem to be a good explanation because the post-sand filtration water should have been equally affected. As anticipated, recovery of seeded coliphage was low. The low pH conditions of the membrane adsorption-clution method of enterovirus recovery are detrimental to some bacteriophage (Sabatino and Maier, 1980). Nevertheless, an approximate 10% recovery was obtained: therefore, when only small numbers of bacteriophage are present, their detection would be very unlikely. Payment et al. (1988) published a method using MgCl:-conditioned water at pH 6 with a Filterite cartridge which provided more accurate data on coliphage recoveries from seeded samples as well as for coliphage numbers in environmental samples. Analyses of variance for the log-transformed recovery data were performed to determine whether there were signiticant differences in relative recovery (I) among the four stages of the treatment process and (2) among the four samples taken at different periods at each treatment stage. Data were analyzed with respect to each of the three seed viruses. ANOVA results indicated no evidence that recovery efficiency differed among samples taken at different stages in the treatment process. Such a difference would be of concern in comparing virus survival at these stages. While recovery efficiency did differ among the four sampling periods for the two coliphages, this was not an important concern since the experiment was balanced with respect to sampling periods in assessing virus survival at each successive stage. An effort was made to determine whether the recovery efficiencies for the seeded viruses correlated with the various physical or chemical characteristics of the water samples which include temperature, pH, conductivity, turbidity, total solids, suspended solids or dissolved solids. Recovery of ~ X 174 demonstrated a correlation with water temperature (r = - 0.80838 as determined by Pearson's test, P =0.00015). No other correlations existed between recovery of the
731
seeded viruses and the water characteristics. The information obtained in this study indicated the importance of a pre-examination of environmental samples to determine the satisfactory performance of the method applied to monitoring a water system especially when monitoring is done over a period of time. Acknowledgement--The authors are grateful to Larry
Wymer of Computer ScienceCorporation for the statistical analysis of the data. REFERENCES
APHA (1985) Standard Methods fi~r the Examination o f Water and Wastewater, 16th edition, pp. 860-896. American Public Health Association, Washington. D.C. Benton W. H. and Hurst C. J. (1986) Evaluation of mixed types and 5-iodo-2'-deoxyuridine treatment upon plaque assay titers of human enteric viruses. AppL em'ir. MicrobioL 51, 1036-1040. Bisson J. W. and CabeUi V. J. (1979) Membrane filter enumeration methods for CIostridium perfringens. Appl. em'ir. Microbiol. 37, 55-66. Dahling D. R. and Wright B, A. (1984) Processing and transport of environmental virus samples. Appl. enrir. Microbiol. 47, 1272-1276. Dahling D. R., Berg G. and Berman D. (19741 BGM, a continuous cell line more sensitive than primary Rhesus and African green kidney cells for the recovery of viruses from water, tilth Sci. II, 275 282. Dcetz T. R., Smith E. M., Goyal S. M., Gerba C. P.. Vollet J. J., Tsai L.. DuPont I!. L. and Keswick B. H. (19841 Occurrence of rota- and enteroviruses in drinking and environmental water in a deveh)pingnation, lrat. Res. 18, 567- 57 I. Katzenelson E., Fattal B. and Ih)stovesky T. (1976) Organic fiocculation: an efficient second-step concentration method for the detection of viruses in tap water. Appl. em'ir. MicrobioL 32, 638-639. Payment P., Morin E. and Trudel M. (1988) Coliphage and enteric virus in the particulate phase of river water. Can. J. MicrobioL 34, 907-910. Payment P., Trudel M. and Plante R. (1985) Elimination of viruses and indicator bacteria at each step of treatment during preparation of drinking water at se','en water treatment plants. Appl. em'ir. Microbiol. 49, 1418-1428. Rose J. B., Gerba C. P., Singh S. N., Toranzos G. A. and Keswick B. (1986) Isolating viruses from finished water. J. Am. Wat. Wks Ass. 78, 56-61. Sabatino C. M. and Maier S. (1980) Differentialinactivation of 3 bacteriophages by acid and alkaline pH used in the membrane adsorption-elution method of virus recovery. Can. J. Microbiol. 26, 403-407. Sobsey M. D. and Glass J. C. (1984) Influence of water quality on enteric virus concentration by microporous filter methods. Appl. era,Jr. MicrobioL 47, 956-960. Stetler R. E. (1984) Coliphages as indicators of enteroviruses. AppL em~ir. MicrobioL 48, 668-670. Stetler R. E., Ward R. L. and Waltrip S. C. (1984) Enteric virus and indicator bacteria levels in a water treatment system modified to reduce trihalomethane production. Appl. enrir. Microbiol. 47, 319-324. USEPA (1985) Test methods for Escherichia coli and Enterococcus in water by the membrane filter procedure. EPA 00/4-85/076. US EPA, Cincinnati, Ohio. Ward R. L. and Mahler R. J. (1982) Uptake of bacteriophage 1"2,through plant roots. AppL era'it. Microbiol. 43, 1098-1103.