- Email: [email protected]
Evaluation of the viral population in two wastewater treatment plants
Water Res. Vol. 19, No. 11. pp. 1353-1356, 1985 Pnnted in Great Britain
0043-1354 85 53.00+0.00 Pergamon Press Ltd
EVALUATION OF THE VIRAL POPULATION IN TWO WASTEWATER TREATMENT PLANTS STUDY
OF
DIFFERENT
SAMPLING
TECHNIQUES
L. SCHWARTZBRODI, PH. VILAGINES2, J. SCHWARTZBRODI, B. SARRETTE", R. VILAGIN~:S2 and J. COLLOMBt ~Universit~ de Nancy I, Facult~ des Sciences Pharmaceutiques et Biologiques, 5 Rue Albert Lebrun 41 and :Service de Contr61e des Eaux de la Ville de Paris, 144 Avenue P.V. Couturier, 75014 Paris, France ( Receired June 1984) A b s t r a c t - - A l-year survey was undertaken on two biological wastewater treatment plants by two different laboratories, in order to gather information on the enteric viral population concentration and on the removal efficiency of the treatment. Different sampling techniques were compared. Results were comparable and showed that enteric viruses were present all year round, their numbers fluctuating randomly within a day. Composite sampling over a period of at least 15 h gave a good representation of the average virus population.
Key words---enteroviruses, wastewater, sampling
INTRODUCTION It is well established that wastewaters constitute a reservoir for very large numbers of potentially pathogenic human enteric viruses. Therefore, the increasing demand for water sources, resulting in their direct or indirect reuse for drinking, recreational or industrial purposes, poses a potential risk for the health of the population. Thus, information on the occurrence of enteric viruses and their removal by conventional wastewater treatment processes is of major concern. Many studies have been reported from other countries (Buras, 1976; Irving and Smith, 1981; Ruiter and Fujioka, 1978) but few investigations providing such information have been carried out in France. The present study was undertaken to evaluate the extent of viral pollution in wastewaters and its elimination by the activated sludge process. It was performed by two different laboratories on two geographically different wastewater treatment plants (Nancy and Paris). Over a 1-year period, at monthly intervals, raw sewage and secondary effluent samples were collected every 3 h (6 a.m.-9 p.m.) to determine the diurnal variation in virus load. In addition, two sampling methods: composite samples over a 3 and 15 h period and grab samples, were compared in order to determine the sampling method most representative of virus pollution and wastewater treatment process efficiency. MATERIALS AND METHODS
Sampling sites Sewage and treated effluent samples were collected from two activated sludge treatment plants situated at Nancy and in the Hauts de Seine (Paris suburb). Treatment consists in primary sedimentation, aeration with activated sludge and
secondary sedimentation. The Nancy treatment plant processes 150,000 m ~ of domestic and industrial wastewater per day. The estimated average transit time is 6 h. The Hauts de Seine treatment plant processes part of the wastewater drained from the Paris area (130,000 m 3day-~). The pilot used in this study treated 17,000 m3 day -t. The average transit time is approx. 4 h. Samples Samples of 201. were collected at the station's intake just after rough screening for raw water and at secondary clarifier's outlet for treated water. Three types of samples were tested: Grab samples collected every 3 h from 6a.m. to 9 p.m. Composite samples obtained with an automatic sampler Isco 2000 3 h composite samples from 9a.m. to 12p.m. from 3 p.m. to 6 p.m.
Nancy
from 8 a.m. to 11 a.m. from 3 p.m. to 6 p.m.
Hauts de Seine
15h composite samples from 6a.m. to 9p.m. in both plants. Concentration procedure Adsorption-elution on glass powder fluidized bed (Sarrette et al., 1977) was the concentration technique used by both laboratories, but with the apparatus described by Schwartzbrod and Gutierrez (1978). Adsorption on glass powder is performed at pH = 3.5, with aluminium chloride added to a 5.10~M final concentration. Elution is conducted at pH = 11.5. Specific experimental conditions of adsorption were chosen after a preliminary study by Dr Hartemann's laboratory (Laboratoire d'Hygi~ne et de Recherche en Sant~ publique, Facult6 de M~decine, Route de Maron, 54500 Vandoeuvre, France): 150 g glass powder with a 1001h -~ flow rate. Elution was performed with 50 mM glycine buffer (pH 11.5) with phenol red (0.02 mg 1-~). After immediate neutralization, the concentrates (approx. 35 ml) were decontaminated with chloroform using Buras" technique (1974).
1353
L. SCHW~.RTZBROD 2 t ¢.~l'.
t35a
Table I Virus concentra:~on tPFU I - } detected betore and after biological treatment at Nanc) with dltI'ert.'n: ~ampiing technique,; t\la': 198 t-April 1982I Sampling
Ma,,
June
July
Aug.
Sept.
Oct.
Nov.
Dec.
Jan.
Feb,
Mar
Apt
Average
5.9 5.7 6.4 21.0 8.9 9.8 96 3.4 12.2
0 10.2 12.5 -{a I_ ._ 6.1 10.2 8.7 14.6 26.8
35 12.6 11.3 10.7 6.0 6.1 8.4 17.6 47.3
39 10.6 9.5 6.3 22.8 3.6 9.4 19.4 12,8
6.'~ ,,a.i 2/1 1 56 11.3 69 13.4 0
l0 -~0 58 1.2." 1.5 7~ 14.2 30
5" 124 119 12.a 12.5 9.8 10.8 15N 23 (1
I) 0 0.6 0.6 1.2 0 0.4 0 0
0 2.1 0 10.7 12.6 4.6 5.0 4.3 20.3
0.8 0.8 0.8 1.4 3.1 0.5 1.2 0.6 7.1
0.5 0.5 2.2 1.5 3.2 3.5 1,9 4.1 3.3
2.6 1.0 1.1 2.0 5.0 2.9 2.4 1.6 4.6
1l 1.6 0.9 3.1 4.7 3.2 2.4 2.1 5.3
R a w ;*awe
P 6a.m P 9 am. P 12 p.m. P 3pm. P 6p.m. P 9p.m. Average S:~ (6 a.m.-9 p.m.) S~ ~9 a.m.-12 p.m.)
9.4 15 3 16.5 5.1 2t.4 15.9 13.9 12.0 39.2
10.4 29.2 51.7 47.4 33.0 10.8 30.4 -63.8
15.2 /4.5 11.2 94 13.2 10.7 1,2.4 26.9 32.0
3.0 8.2 37 a7 8.3 11.5 6.6 18.4 88
0 8.4 1.3 2. l 9.3 4.6 4.3 7,3 6.1
P P P P P P
0 1.8 2.1 7.5 6.9 3.0 3.6 2.0 12.5
4.9 10.9 0 1.6 5.5 10.8 5.6 1.9 2.6
0.7 0.0 3.0 3.4 4.1 2.6 2.3 2.3 2.9
0 0.9 0 0.8 1.7 5.8 1.5 3.6 0
0 1.2 0 0 0 1.1 0.4 0 0
0 20.7 t0.8 20. -7 2.5 11.4 I 1.0 28.2 31.2
Treated water
6 a.m 9 a.m. 12 p.m. 3 p.m. 6p.m 9p.m.
Average S:~ (6 a.m.-9 p.m.) 53 (3 p.m.-6 p.m.)
0.8 0 1.7 3.6 9.5 3.2 3.1 0 6.7
3.0 0 0 3.8 3.7 0 1.8 4.4 3.4
P = grab sample; S = composite samples (3 and 15 h).
Viral assays
These were done using the BGM continuous cell line (Baron et al., 1970) cultivated in Eagle's Minimum Essential Medium (MEM) supplemented with 20 mM glutamine, 1~o Non Essential Amino Acids and 7% fetal calf serum. Maintenance modium was the same but with 2% fetal calf serum. After decontamination, eluates were brought to isotonicity by adding MEM 4 x with antibiotics. Aliquots of l ml were inoculated to BGM monolayers grown in 60 mm dia Petri dishes (Coming). After a 1 h incubation at 36'C the inoculum was eliminated and the cells were overlaid with agar. Plaques were counted after 3 and 6 days incubation at 36°C. RESULTS
Data obtained after a l-year survey are summarized in Table I for Nancy and Table 2 for the Hauts de Seine plant.
In raw water, the average virus recovery for all types of sampling was 17 and 18 PFU I- *, for Nancy and the Hauts de Seine plant, respectively. At Nancy, 4 samples were virus-free (3 of them were the grab samples from 6 a.m.) and the highest concentration observed was 6 4 P F U I -t. At the Hauts de Seine plant, all samples were positive and virus levels varied from 1.95 to 61 PFU I-~. At the outlet of the secondary decantation tank at Nancy the average virus concentration was 3 PFU l(with 21 negative out of 96 samples) and the highest concentration was 2 0 . 3 P F U I - L At the Hauts de Seine plant, viruses were detected in all samples but one; their concentration varied from 0.95 to 40 PFU I-~, the average being 10 PFU I-*. Virus removal efficiency through biological treatment was calculated from data provided by grab
Table 2. Virus concentration (PFU I- ) detected before and after biological treatment at the Hauts de Seine ttw-atment plant with different sarapl!at t~hniques (Nov. [981-Oct. [982) Sampling P 6a.m
Nov.
P 9p.m.
[l.l
1.9
Average Sis (6 a.m.-9 p.m.)
11.7 13.6 5.2 14.8
9.6 7.3 16.7 5.4
6.4 5.8 3.9 1.6 0.9 2.7 3.6 1.5 3.8 0.3
4.8 2.1 2.2 3.4 1.7 0 2.4 1.9 4,7 0.7
43.2 37.2 21.6 11.4 11.8 12.7 23.0 17.2 29.7 12.6
P 12 a.m. P 3p.m. P 6 p.m.
S3 ( S a . m . - I I a.m.) S 3 (3 p,m.-6 p.m.) P P P P P P
6a.m. 9a.m. 12 a.m. 3p.m. 6p.m. 9p,m.
Average S~5 (6 a.m.-9 p.m.) S~ (8 a.m.-I 1 a,m.) S~ (3 p,m.-6 p.m.)
13.9 16.4 7.3 9.0 9.2
Jan. 10.8 8,2 8.9 61.2 17,7 43.0 25.0 26.9 7.2 21.7
P 9a.m.
10.5 10.5 7.2 15.8 15.2
Dec.
Feb. 20,9 3.4 30.2 42.3 33.6 32.0 27.1 30.1 7.2 25,2 ll.0 10.8 9.8 4.8 4.8 9,4 8.4 9.1 3.6 4.1
P = grab samples; S = composite samples (3 and 15 h).
Mar.
Apr.
Raw w a t e r 34.0 9.3 31.4 5.9 21.6 12.8 5.9 16.4 18.8 15.4 10.6 8,8
20.4 18.8 47.3 33.3
May
June
July
Aug.
SepL
Oct.
Average
18.7 15.7 6.7 19.3 7.0 15.3 13.8 13.4 9.6 13.4
42.2 53.3 43.7 25,5 25.4 39.9 38.3 31.5 56.2 28.3
[2.4 16.3 9.4 15.6 19.0 17.2 15.0 31.0 4,7 25
6.1 6.5 5.9 19.0 16.0 10.0 10.6 13.4 4.8 17.3
92 7.3 16.8 20.6 22,5 18.2 15.8 26.2 13.8 tl.6
17,0 18.5 29.5 18.6 17.3 21.8 20.5 24.8 16.6 23.7
17.1 16.1 16.7 22,4 18.1 t9.1 18.3 19.9 16.8 19.l
8.2 32.1 20,4 18.4 20.3 7,4 t7,8 7.5 27.9 39,8
7.6 6.6 4.8 8,2 9.1 8.6 7.5 10.7 6.2 8.0
8.0 10.0 5.5 10.5 12.9 16.6 10.6 12.1 7.1 10.3
14.6 15.9 10.4 9.3 15.1 14.5 13.3 12.6 11.3 8.2
1.5 2.3 1.2 0.9 1.7 6.5 2.3 1.7 1.9 1.4
12.0 13.5 7.7 7.0 7.0 9.3 9.6 8.5 10.0 10.3
11.5 2.2 12.7 9.6 Treated water 18.4 18.5 1.9 20,6 16.4 2.8 4,0 7.3 1.4 3.1"9.6 3.1 8.3 3.6 5.5 8.3 14.6 10,6 10.4 11.7 4.2 18.9 2.8 6.0 10.5 12.0 2.0 26.2 9.I 2,7
Evaluation of the viral population in two wastewater treatment plants Table 3. Virus removal efficiencyat the Nancy treatment plant Average PFU detected* (PFU I - ~) Virus removal Raw water Treated water efficiency Date 6 a.m.-3 p.m. 12 a.m.-9 p.m. 1°o) May June July August September October November December January February March April Average
11.6 34.6 12.6 4.9 3.0 13. I 9.7 9.0 9.5 7.6 6.2 5.6
4.8 4.5 3.3 2.0 0.3 4.5 1.9 0.6 6.9 1.4 2.6 2.7
59 87 74 59 90 66 80 93 27 81.5 58 52 69
*Calculated from data obtained with grab samples.
samples, taking into account the stations average retention time (6 h at Nancy, 4 h at the Hauts de Seine plant). At Nancy, an average o f 6 8 ~ of viruses were removed by treatment and, if the exceptionally low removal efficiency observed in January is ignored, the observed efficiency is over 70~o (Table 3). At the Hauts de Seine plant (Table 4), even if the retention time of 4 h leads to approximate results, the average virus removal efficiency was lower than at Nancy, 489/o, with very large fluctuations: from no purification in January, August and September to results over 6 0 ~ in half of the samples. Statistical analysis of the results was possible because of the large amount of data obtained. Significance of possible daily, monthly and or seasonal variations was tested along with the differences between the different sampling methods. The results are summarized in Table 5. Raw sewage showed no significant daily variations but both plants showed significant monthly variations in viral pollution of raw sewage. Treated wastewater also had significant monthly variations at both plants. Daily variations were also significant; at the Hauts de Seine plant, virus concentration was significantly higher in the Table 4. Virus removal efficiencyat the Hauts de Seine treatment plant Average PFU detected (PFU 1-~) Virus removal Raw water Treated water efficiency Date 6 a.m.-3 p.m. 9 a.m.-6 p.m. (~) November December January" February March April May June July August September October Average
11.0 11.7 22.3 24.2 23.3 l 1.1 15.1 41.2 13.4 9.4 13.5 20.9
3.1 2.3 20.5 7.6 9.0 9.2 3.2 22.8 7.2 9.7 12.7 1.5
*Calculated from data obtained with grab samples.
72 80 8 69 67 17 79 0,5 47 0 6 93 48
1355
Table 5. Comparison by variance analysis of hourly and monthb average virus concentrations detected in grab samples Nancy Hauts de Seine Hourly Monthy NS S 0.119 0.000 Treated water S (p.m.) S 0.015 0.005 NS--not significant: S---significant. Raw water
Houri)• NS 0.358 S (a.m.) 0.031
Monthly S 0.000 S 0.000
morning while at Nancy this occurred in the afternoon. Statistical comparison (Wilcoxon test) of virus concentration for different sampling techniques did not give the same results for both treatment plants. At the Hauts de Seine treatment plant, the sampling method did not significantly affect virus concentration, neither for raw sewage nor for treated effluent. In contrast, a significant difference was found between grab samples and composite samples (3 and 15 h) for raw sewage virus concentration at Nancy (Table 6). Virus concentration in treated water at Nancy was significantly different for grab samples and 3 h composite samples, although at the limit of the 95~o confidence interval; grab samples and 15 h composite samples for treated water did not differ significantly. DISCUSSION When considering the direct or indirect reuse of treated wastewater as a reservoir for drinking purposes, special consideration should be given to the viral population present in secondary effluents. The present study was particularly concerned with the virus population going through two different plants both using biological purification. Between the two methods most often employed in France for virus concentration, adsorption-elution on either microfibre glass filters (Balston) or on glass powder fluidized bed, we chose the latter. A comparison of these methods made by Joret et al. (1980) showed that the glass powder method has several advantages such as not clogging and a better concentration of indigenous viruses from wastewater, even under conditions which are not the best (Vilagines et al., 1983). Thus, virus concentrations for the same volume of raw or treated water could be compared at Nancy and at the Hauts de Seine plants. Although the hydraulic parameters were not identical, virus concentrations were of the same order of magnitude. Table 6. Statistical comparison (Wilcoxon test) of virus detection by different sampling techniques at Nancy P/Ssh P/Stsh S3hlS,~h Raw water S S NS 0.01 0.00 0.17 Treated water S NS NS 0.04 0.21 0.07 P--grab samples; S3h--composite sample over 3 h: S,~h--composite sample over 15 h; S--significant; NS--not significant.
135~
k. ScH,*.t~rzugoo et ai.
The levels found were considerably lower than certain authors have reported (Rao eta/., 1977: Buras, 1976) but were in agreement with other reports (Cli~er, 1975: Payment et at., 1979: Hugues et al., 1974). Virus removal efficienc? by biological treatment in these plants was similar to values reported by others (Kelly et al., 1961: England et al., 1967, Malina et al., 1974). The difference between Nancy's (80",o) and the Hauts de Seine plant's (50°0) virus removal efficiency could possibly be due to the different retention times. 6 and 4 h, respectively. tn raw wastewater at the Hauts de Seine plant. hourly variations of virus concentration were observed but, as maxima occurred at different hours from one group of experiments to another, it resulted in no detectable variation in the hourly average concentration. This lack of variability was confirmed by statistical variance analysis. At Nancy, more or less the same phenomenon was observed, except that the m i n i m u m in virus concentration occurred most of the time in the 6a.m. samples, this being corroborated by the average of hourly virus concentration (5.8 and 12.5 P F U 1-~) but not confirmed by statistical analysis. In treated wastewater, hourly variations in virus concentration were statistically different in both plants. Maxima occurred at the Hauts de Seine plant in the morning and at Nancy in the afternoon, this being related to (after considering the respective retention times) a maximum virus load in raw water around 2 a.m. at the Hauts de Seine plant (compatible with the long and variable route of sewer in a big city) and between 9 a.m. and 12 p.m. at Nancy (corroborated by the higher viral concentration always detected in the 3 h composite samples). In all cases, viral pollution was significantly different from one group of experiments to another. Since they were done at monthly intervals, it is tempting to attribute this difference to monthly or seasonal variations, but this could only be confirmed by more experiments within the same months over several years. In addition to obtaining information about the viral pollution of a biological wastewater treatment plant, the aim of this study was to compare results obtained with different sampling techniques in order to indicate the one giving the best estimation of viral pollution in such a system and applicable anywhere. Results obtained from the plants were different. At the Hauts de Seine plant, all techniques led to equal detection. At Nancy, composite samples always yielded higher virus concentrations compared to the average obtained with grab samples. Consequently, and logically, automatic composite sampling ov.er a period of at least 15 h and, even better 24 h, as long as it is refrigerated and in accordance with the flow rate, gives rather good information on viral concentration. Nevertheless, the sampling time must be
adjusted to take into consideration the retenuon time in the station, in order to e,,aluate ~tru-~ ~"cmo~al e~cienc.~.
REFERENCES Baron A. L., Otshevesky C. and Cohen M. N. (1970) Characteristics of the BGM line of ceils from African Green Monkey Kidney. Arch. ges. Viru~rsch. 32, 389-392. Buras N. (1974) Recovery of viruses from wastewater and efltuent by direct inoculation method, lKater Res. 8, 19-22. Buras N. (1976) Concentration of enteric viruses in wastewater and effluent. A two year survey. Water Res. 10, 295-298. Ctiver D. O. (1975) Virus association with wastewater solids. Ent+ir. Lett. 10, 215-223. England B., Leach R. E., Adame B. and Shiosaki R. (t967) Virologic assessment of sewage treatment at Santee, California. In Transmission of Viruses by the Water Route (Edited by Berg G.). Interscience, New York. Hugues B., Plissier M., Andre M., Pagliardini A. and Laurent D. (1979) Evaluation de la charge virale darts les eaux d'une station d'~puration biologique au moyen de deux m6thodes de concentration de virus par adsorption elution sur poudre de verre ou sur microfibre de verre. Water Res. 13, 1117-1123. Irving L. G. and Smith F. A. (1981) One year survey of enteric viruses, Adenoviruses and Reoviruses isolated from an effluent of an activated sludge purification plant. Appl. envir. Microbiol. 41, 51-59. Joret J. C., Block J. C., Gutierrez F., Schwartzbrod L., Hugues B. and Plissier M. (1980) Virus concentration from secondary wastewater. Comparative study between epoxy fiberglass and glass powder adsorbantsEur. J. appL Microbiol. BiotechnoL 10, 245--252. Kelly S. M., Sanderson W. W. and Neide C. (t961) Removal of enteroviruses from sewage by activated sludge. 3". War. Pollut. Control Fed. 33, 1056-1062. Malina J. F. Jr, Ranganathan K. R., Moore B. E. and Sagik B. P. (1974) Poliovirus inactivation by activated sludges. In Virus Surt'ival in Water and Wastewater Systems (Edited by Malina J. F. and Sagik B. P.), pp. 95-106. Center for Research in Water Resources, University of Texas at Austin, Austin, "rex. Payment P., Larose Y. and Trudel M. (1979) Poliovirus and other enteroviruses in urban sewage from Laval (Canada): presence of non vaccinal strains of Poliovirus. Can. J. Microbiol. 25, 1305-1309. Rao V. C., Lahke S. B., Waghmare S. V. and Dube P+ (1977) Virus removal in activated-sludge sewage treatment. Prog. Wat. Technol. 9, 113-127. Ruiter G. G. and Fujioka R. S. (1978) Human enteric viruses in sewage and their discharge into the ocean. Wat. Air Soil Pollut. 10, 95-103. Sarrette B., Danglot C. and Vilagines R. (1977) A new and simple method for recuperation of enteroviruses from water. Water Res. 11, 355-358. Schwartzbrod L. and Gutierrez F. (1978) Concentration des virus dans les eaux par adsorption sur poudre de verre: proposition d'un appareillage simplifi~. Mierobia 4, 55-68. Vilagines Ph,, Sarrette B.. Husson G. and Vilagines R. (1983) Concentration of waterborne viruses on glass powder fluidized bed adapted to the analysis of wastewater. In Det'elopments in Ecology and Environmental Quality, Vol. II. Second International Conference of Ecology and Environment.
0043-1354 85 53.00+0.00 Pergamon Press Ltd
EVALUATION OF THE VIRAL POPULATION IN TWO WASTEWATER TREATMENT PLANTS STUDY
OF
DIFFERENT
SAMPLING
TECHNIQUES
L. SCHWARTZBRODI, PH. VILAGINES2, J. SCHWARTZBRODI, B. SARRETTE", R. VILAGIN~:S2 and J. COLLOMBt ~Universit~ de Nancy I, Facult~ des Sciences Pharmaceutiques et Biologiques, 5 Rue Albert Lebrun 41 and :Service de Contr61e des Eaux de la Ville de Paris, 144 Avenue P.V. Couturier, 75014 Paris, France ( Receired June 1984) A b s t r a c t - - A l-year survey was undertaken on two biological wastewater treatment plants by two different laboratories, in order to gather information on the enteric viral population concentration and on the removal efficiency of the treatment. Different sampling techniques were compared. Results were comparable and showed that enteric viruses were present all year round, their numbers fluctuating randomly within a day. Composite sampling over a period of at least 15 h gave a good representation of the average virus population.
Key words---enteroviruses, wastewater, sampling
INTRODUCTION It is well established that wastewaters constitute a reservoir for very large numbers of potentially pathogenic human enteric viruses. Therefore, the increasing demand for water sources, resulting in their direct or indirect reuse for drinking, recreational or industrial purposes, poses a potential risk for the health of the population. Thus, information on the occurrence of enteric viruses and their removal by conventional wastewater treatment processes is of major concern. Many studies have been reported from other countries (Buras, 1976; Irving and Smith, 1981; Ruiter and Fujioka, 1978) but few investigations providing such information have been carried out in France. The present study was undertaken to evaluate the extent of viral pollution in wastewaters and its elimination by the activated sludge process. It was performed by two different laboratories on two geographically different wastewater treatment plants (Nancy and Paris). Over a 1-year period, at monthly intervals, raw sewage and secondary effluent samples were collected every 3 h (6 a.m.-9 p.m.) to determine the diurnal variation in virus load. In addition, two sampling methods: composite samples over a 3 and 15 h period and grab samples, were compared in order to determine the sampling method most representative of virus pollution and wastewater treatment process efficiency. MATERIALS AND METHODS
Sampling sites Sewage and treated effluent samples were collected from two activated sludge treatment plants situated at Nancy and in the Hauts de Seine (Paris suburb). Treatment consists in primary sedimentation, aeration with activated sludge and
secondary sedimentation. The Nancy treatment plant processes 150,000 m ~ of domestic and industrial wastewater per day. The estimated average transit time is 6 h. The Hauts de Seine treatment plant processes part of the wastewater drained from the Paris area (130,000 m 3day-~). The pilot used in this study treated 17,000 m3 day -t. The average transit time is approx. 4 h. Samples Samples of 201. were collected at the station's intake just after rough screening for raw water and at secondary clarifier's outlet for treated water. Three types of samples were tested: Grab samples collected every 3 h from 6a.m. to 9 p.m. Composite samples obtained with an automatic sampler Isco 2000 3 h composite samples from 9a.m. to 12p.m. from 3 p.m. to 6 p.m.
Nancy
from 8 a.m. to 11 a.m. from 3 p.m. to 6 p.m.
Hauts de Seine
15h composite samples from 6a.m. to 9p.m. in both plants. Concentration procedure Adsorption-elution on glass powder fluidized bed (Sarrette et al., 1977) was the concentration technique used by both laboratories, but with the apparatus described by Schwartzbrod and Gutierrez (1978). Adsorption on glass powder is performed at pH = 3.5, with aluminium chloride added to a 5.10~M final concentration. Elution is conducted at pH = 11.5. Specific experimental conditions of adsorption were chosen after a preliminary study by Dr Hartemann's laboratory (Laboratoire d'Hygi~ne et de Recherche en Sant~ publique, Facult6 de M~decine, Route de Maron, 54500 Vandoeuvre, France): 150 g glass powder with a 1001h -~ flow rate. Elution was performed with 50 mM glycine buffer (pH 11.5) with phenol red (0.02 mg 1-~). After immediate neutralization, the concentrates (approx. 35 ml) were decontaminated with chloroform using Buras" technique (1974).
1353
L. SCHW~.RTZBROD 2 t ¢.~l'.
t35a
Table I Virus concentra:~on tPFU I - } detected betore and after biological treatment at Nanc) with dltI'ert.'n: ~ampiing technique,; t\la': 198 t-April 1982I Sampling
Ma,,
June
July
Aug.
Sept.
Oct.
Nov.
Dec.
Jan.
Feb,
Mar
Apt
Average
5.9 5.7 6.4 21.0 8.9 9.8 96 3.4 12.2
0 10.2 12.5 -{a I_ ._ 6.1 10.2 8.7 14.6 26.8
35 12.6 11.3 10.7 6.0 6.1 8.4 17.6 47.3
39 10.6 9.5 6.3 22.8 3.6 9.4 19.4 12,8
6.'~ ,,a.i 2/1 1 56 11.3 69 13.4 0
l0 -~0 58 1.2." 1.5 7~ 14.2 30
5" 124 119 12.a 12.5 9.8 10.8 15N 23 (1
I) 0 0.6 0.6 1.2 0 0.4 0 0
0 2.1 0 10.7 12.6 4.6 5.0 4.3 20.3
0.8 0.8 0.8 1.4 3.1 0.5 1.2 0.6 7.1
0.5 0.5 2.2 1.5 3.2 3.5 1,9 4.1 3.3
2.6 1.0 1.1 2.0 5.0 2.9 2.4 1.6 4.6
1l 1.6 0.9 3.1 4.7 3.2 2.4 2.1 5.3
R a w ;*awe
P 6a.m P 9 am. P 12 p.m. P 3pm. P 6p.m. P 9p.m. Average S:~ (6 a.m.-9 p.m.) S~ ~9 a.m.-12 p.m.)
9.4 15 3 16.5 5.1 2t.4 15.9 13.9 12.0 39.2
10.4 29.2 51.7 47.4 33.0 10.8 30.4 -63.8
15.2 /4.5 11.2 94 13.2 10.7 1,2.4 26.9 32.0
3.0 8.2 37 a7 8.3 11.5 6.6 18.4 88
0 8.4 1.3 2. l 9.3 4.6 4.3 7,3 6.1
P P P P P P
0 1.8 2.1 7.5 6.9 3.0 3.6 2.0 12.5
4.9 10.9 0 1.6 5.5 10.8 5.6 1.9 2.6
0.7 0.0 3.0 3.4 4.1 2.6 2.3 2.3 2.9
0 0.9 0 0.8 1.7 5.8 1.5 3.6 0
0 1.2 0 0 0 1.1 0.4 0 0
0 20.7 t0.8 20. -7 2.5 11.4 I 1.0 28.2 31.2
Treated water
6 a.m 9 a.m. 12 p.m. 3 p.m. 6p.m 9p.m.
Average S:~ (6 a.m.-9 p.m.) 53 (3 p.m.-6 p.m.)
0.8 0 1.7 3.6 9.5 3.2 3.1 0 6.7
3.0 0 0 3.8 3.7 0 1.8 4.4 3.4
P = grab sample; S = composite samples (3 and 15 h).
Viral assays
These were done using the BGM continuous cell line (Baron et al., 1970) cultivated in Eagle's Minimum Essential Medium (MEM) supplemented with 20 mM glutamine, 1~o Non Essential Amino Acids and 7% fetal calf serum. Maintenance modium was the same but with 2% fetal calf serum. After decontamination, eluates were brought to isotonicity by adding MEM 4 x with antibiotics. Aliquots of l ml were inoculated to BGM monolayers grown in 60 mm dia Petri dishes (Coming). After a 1 h incubation at 36'C the inoculum was eliminated and the cells were overlaid with agar. Plaques were counted after 3 and 6 days incubation at 36°C. RESULTS
Data obtained after a l-year survey are summarized in Table I for Nancy and Table 2 for the Hauts de Seine plant.
In raw water, the average virus recovery for all types of sampling was 17 and 18 PFU I- *, for Nancy and the Hauts de Seine plant, respectively. At Nancy, 4 samples were virus-free (3 of them were the grab samples from 6 a.m.) and the highest concentration observed was 6 4 P F U I -t. At the Hauts de Seine plant, all samples were positive and virus levels varied from 1.95 to 61 PFU I-~. At the outlet of the secondary decantation tank at Nancy the average virus concentration was 3 PFU l(with 21 negative out of 96 samples) and the highest concentration was 2 0 . 3 P F U I - L At the Hauts de Seine plant, viruses were detected in all samples but one; their concentration varied from 0.95 to 40 PFU I-~, the average being 10 PFU I-*. Virus removal efficiency through biological treatment was calculated from data provided by grab
Table 2. Virus concentration (PFU I- ) detected before and after biological treatment at the Hauts de Seine ttw-atment plant with different sarapl!at t~hniques (Nov. [981-Oct. [982) Sampling P 6a.m
Nov.
P 9p.m.
[l.l
1.9
Average Sis (6 a.m.-9 p.m.)
11.7 13.6 5.2 14.8
9.6 7.3 16.7 5.4
6.4 5.8 3.9 1.6 0.9 2.7 3.6 1.5 3.8 0.3
4.8 2.1 2.2 3.4 1.7 0 2.4 1.9 4,7 0.7
43.2 37.2 21.6 11.4 11.8 12.7 23.0 17.2 29.7 12.6
P 12 a.m. P 3p.m. P 6 p.m.
S3 ( S a . m . - I I a.m.) S 3 (3 p,m.-6 p.m.) P P P P P P
6a.m. 9a.m. 12 a.m. 3p.m. 6p.m. 9p,m.
Average S~5 (6 a.m.-9 p.m.) S~ (8 a.m.-I 1 a,m.) S~ (3 p,m.-6 p.m.)
13.9 16.4 7.3 9.0 9.2
Jan. 10.8 8,2 8.9 61.2 17,7 43.0 25.0 26.9 7.2 21.7
P 9a.m.
10.5 10.5 7.2 15.8 15.2
Dec.
Feb. 20,9 3.4 30.2 42.3 33.6 32.0 27.1 30.1 7.2 25,2 ll.0 10.8 9.8 4.8 4.8 9,4 8.4 9.1 3.6 4.1
P = grab samples; S = composite samples (3 and 15 h).
Mar.
Apr.
Raw w a t e r 34.0 9.3 31.4 5.9 21.6 12.8 5.9 16.4 18.8 15.4 10.6 8,8
20.4 18.8 47.3 33.3
May
June
July
Aug.
SepL
Oct.
Average
18.7 15.7 6.7 19.3 7.0 15.3 13.8 13.4 9.6 13.4
42.2 53.3 43.7 25,5 25.4 39.9 38.3 31.5 56.2 28.3
[2.4 16.3 9.4 15.6 19.0 17.2 15.0 31.0 4,7 25
6.1 6.5 5.9 19.0 16.0 10.0 10.6 13.4 4.8 17.3
92 7.3 16.8 20.6 22,5 18.2 15.8 26.2 13.8 tl.6
17,0 18.5 29.5 18.6 17.3 21.8 20.5 24.8 16.6 23.7
17.1 16.1 16.7 22,4 18.1 t9.1 18.3 19.9 16.8 19.l
8.2 32.1 20,4 18.4 20.3 7,4 t7,8 7.5 27.9 39,8
7.6 6.6 4.8 8,2 9.1 8.6 7.5 10.7 6.2 8.0
8.0 10.0 5.5 10.5 12.9 16.6 10.6 12.1 7.1 10.3
14.6 15.9 10.4 9.3 15.1 14.5 13.3 12.6 11.3 8.2
1.5 2.3 1.2 0.9 1.7 6.5 2.3 1.7 1.9 1.4
12.0 13.5 7.7 7.0 7.0 9.3 9.6 8.5 10.0 10.3
11.5 2.2 12.7 9.6 Treated water 18.4 18.5 1.9 20,6 16.4 2.8 4,0 7.3 1.4 3.1"9.6 3.1 8.3 3.6 5.5 8.3 14.6 10,6 10.4 11.7 4.2 18.9 2.8 6.0 10.5 12.0 2.0 26.2 9.I 2,7
Evaluation of the viral population in two wastewater treatment plants Table 3. Virus removal efficiencyat the Nancy treatment plant Average PFU detected* (PFU I - ~) Virus removal Raw water Treated water efficiency Date 6 a.m.-3 p.m. 12 a.m.-9 p.m. 1°o) May June July August September October November December January February March April Average
11.6 34.6 12.6 4.9 3.0 13. I 9.7 9.0 9.5 7.6 6.2 5.6
4.8 4.5 3.3 2.0 0.3 4.5 1.9 0.6 6.9 1.4 2.6 2.7
59 87 74 59 90 66 80 93 27 81.5 58 52 69
*Calculated from data obtained with grab samples.
samples, taking into account the stations average retention time (6 h at Nancy, 4 h at the Hauts de Seine plant). At Nancy, an average o f 6 8 ~ of viruses were removed by treatment and, if the exceptionally low removal efficiency observed in January is ignored, the observed efficiency is over 70~o (Table 3). At the Hauts de Seine plant (Table 4), even if the retention time of 4 h leads to approximate results, the average virus removal efficiency was lower than at Nancy, 489/o, with very large fluctuations: from no purification in January, August and September to results over 6 0 ~ in half of the samples. Statistical analysis of the results was possible because of the large amount of data obtained. Significance of possible daily, monthly and or seasonal variations was tested along with the differences between the different sampling methods. The results are summarized in Table 5. Raw sewage showed no significant daily variations but both plants showed significant monthly variations in viral pollution of raw sewage. Treated wastewater also had significant monthly variations at both plants. Daily variations were also significant; at the Hauts de Seine plant, virus concentration was significantly higher in the Table 4. Virus removal efficiencyat the Hauts de Seine treatment plant Average PFU detected (PFU 1-~) Virus removal Raw water Treated water efficiency Date 6 a.m.-3 p.m. 9 a.m.-6 p.m. (~) November December January" February March April May June July August September October Average
11.0 11.7 22.3 24.2 23.3 l 1.1 15.1 41.2 13.4 9.4 13.5 20.9
3.1 2.3 20.5 7.6 9.0 9.2 3.2 22.8 7.2 9.7 12.7 1.5
*Calculated from data obtained with grab samples.
72 80 8 69 67 17 79 0,5 47 0 6 93 48
1355
Table 5. Comparison by variance analysis of hourly and monthb average virus concentrations detected in grab samples Nancy Hauts de Seine Hourly Monthy NS S 0.119 0.000 Treated water S (p.m.) S 0.015 0.005 NS--not significant: S---significant. Raw water
Houri)• NS 0.358 S (a.m.) 0.031
Monthly S 0.000 S 0.000
morning while at Nancy this occurred in the afternoon. Statistical comparison (Wilcoxon test) of virus concentration for different sampling techniques did not give the same results for both treatment plants. At the Hauts de Seine treatment plant, the sampling method did not significantly affect virus concentration, neither for raw sewage nor for treated effluent. In contrast, a significant difference was found between grab samples and composite samples (3 and 15 h) for raw sewage virus concentration at Nancy (Table 6). Virus concentration in treated water at Nancy was significantly different for grab samples and 3 h composite samples, although at the limit of the 95~o confidence interval; grab samples and 15 h composite samples for treated water did not differ significantly. DISCUSSION When considering the direct or indirect reuse of treated wastewater as a reservoir for drinking purposes, special consideration should be given to the viral population present in secondary effluents. The present study was particularly concerned with the virus population going through two different plants both using biological purification. Between the two methods most often employed in France for virus concentration, adsorption-elution on either microfibre glass filters (Balston) or on glass powder fluidized bed, we chose the latter. A comparison of these methods made by Joret et al. (1980) showed that the glass powder method has several advantages such as not clogging and a better concentration of indigenous viruses from wastewater, even under conditions which are not the best (Vilagines et al., 1983). Thus, virus concentrations for the same volume of raw or treated water could be compared at Nancy and at the Hauts de Seine plants. Although the hydraulic parameters were not identical, virus concentrations were of the same order of magnitude. Table 6. Statistical comparison (Wilcoxon test) of virus detection by different sampling techniques at Nancy P/Ssh P/Stsh S3hlS,~h Raw water S S NS 0.01 0.00 0.17 Treated water S NS NS 0.04 0.21 0.07 P--grab samples; S3h--composite sample over 3 h: S,~h--composite sample over 15 h; S--significant; NS--not significant.
135~
k. ScH,*.t~rzugoo et ai.
The levels found were considerably lower than certain authors have reported (Rao eta/., 1977: Buras, 1976) but were in agreement with other reports (Cli~er, 1975: Payment et at., 1979: Hugues et al., 1974). Virus removal efficienc? by biological treatment in these plants was similar to values reported by others (Kelly et al., 1961: England et al., 1967, Malina et al., 1974). The difference between Nancy's (80",o) and the Hauts de Seine plant's (50°0) virus removal efficiency could possibly be due to the different retention times. 6 and 4 h, respectively. tn raw wastewater at the Hauts de Seine plant. hourly variations of virus concentration were observed but, as maxima occurred at different hours from one group of experiments to another, it resulted in no detectable variation in the hourly average concentration. This lack of variability was confirmed by statistical variance analysis. At Nancy, more or less the same phenomenon was observed, except that the m i n i m u m in virus concentration occurred most of the time in the 6a.m. samples, this being corroborated by the average of hourly virus concentration (5.8 and 12.5 P F U 1-~) but not confirmed by statistical analysis. In treated wastewater, hourly variations in virus concentration were statistically different in both plants. Maxima occurred at the Hauts de Seine plant in the morning and at Nancy in the afternoon, this being related to (after considering the respective retention times) a maximum virus load in raw water around 2 a.m. at the Hauts de Seine plant (compatible with the long and variable route of sewer in a big city) and between 9 a.m. and 12 p.m. at Nancy (corroborated by the higher viral concentration always detected in the 3 h composite samples). In all cases, viral pollution was significantly different from one group of experiments to another. Since they were done at monthly intervals, it is tempting to attribute this difference to monthly or seasonal variations, but this could only be confirmed by more experiments within the same months over several years. In addition to obtaining information about the viral pollution of a biological wastewater treatment plant, the aim of this study was to compare results obtained with different sampling techniques in order to indicate the one giving the best estimation of viral pollution in such a system and applicable anywhere. Results obtained from the plants were different. At the Hauts de Seine plant, all techniques led to equal detection. At Nancy, composite samples always yielded higher virus concentrations compared to the average obtained with grab samples. Consequently, and logically, automatic composite sampling ov.er a period of at least 15 h and, even better 24 h, as long as it is refrigerated and in accordance with the flow rate, gives rather good information on viral concentration. Nevertheless, the sampling time must be
adjusted to take into consideration the retenuon time in the station, in order to e,,aluate ~tru-~ ~"cmo~al e~cienc.~.
REFERENCES Baron A. L., Otshevesky C. and Cohen M. N. (1970) Characteristics of the BGM line of ceils from African Green Monkey Kidney. Arch. ges. Viru~rsch. 32, 389-392. Buras N. (1974) Recovery of viruses from wastewater and efltuent by direct inoculation method, lKater Res. 8, 19-22. Buras N. (1976) Concentration of enteric viruses in wastewater and effluent. A two year survey. Water Res. 10, 295-298. Ctiver D. O. (1975) Virus association with wastewater solids. Ent+ir. Lett. 10, 215-223. England B., Leach R. E., Adame B. and Shiosaki R. (t967) Virologic assessment of sewage treatment at Santee, California. In Transmission of Viruses by the Water Route (Edited by Berg G.). Interscience, New York. Hugues B., Plissier M., Andre M., Pagliardini A. and Laurent D. (1979) Evaluation de la charge virale darts les eaux d'une station d'~puration biologique au moyen de deux m6thodes de concentration de virus par adsorption elution sur poudre de verre ou sur microfibre de verre. Water Res. 13, 1117-1123. Irving L. G. and Smith F. A. (1981) One year survey of enteric viruses, Adenoviruses and Reoviruses isolated from an effluent of an activated sludge purification plant. Appl. envir. Microbiol. 41, 51-59. Joret J. C., Block J. C., Gutierrez F., Schwartzbrod L., Hugues B. and Plissier M. (1980) Virus concentration from secondary wastewater. Comparative study between epoxy fiberglass and glass powder adsorbantsEur. J. appL Microbiol. BiotechnoL 10, 245--252. Kelly S. M., Sanderson W. W. and Neide C. (t961) Removal of enteroviruses from sewage by activated sludge. 3". War. Pollut. Control Fed. 33, 1056-1062. Malina J. F. Jr, Ranganathan K. R., Moore B. E. and Sagik B. P. (1974) Poliovirus inactivation by activated sludges. In Virus Surt'ival in Water and Wastewater Systems (Edited by Malina J. F. and Sagik B. P.), pp. 95-106. Center for Research in Water Resources, University of Texas at Austin, Austin, "rex. Payment P., Larose Y. and Trudel M. (1979) Poliovirus and other enteroviruses in urban sewage from Laval (Canada): presence of non vaccinal strains of Poliovirus. Can. J. Microbiol. 25, 1305-1309. Rao V. C., Lahke S. B., Waghmare S. V. and Dube P+ (1977) Virus removal in activated-sludge sewage treatment. Prog. Wat. Technol. 9, 113-127. Ruiter G. G. and Fujioka R. S. (1978) Human enteric viruses in sewage and their discharge into the ocean. Wat. Air Soil Pollut. 10, 95-103. Sarrette B., Danglot C. and Vilagines R. (1977) A new and simple method for recuperation of enteroviruses from water. Water Res. 11, 355-358. Schwartzbrod L. and Gutierrez F. (1978) Concentration des virus dans les eaux par adsorption sur poudre de verre: proposition d'un appareillage simplifi~. Mierobia 4, 55-68. Vilagines Ph,, Sarrette B.. Husson G. and Vilagines R. (1983) Concentration of waterborne viruses on glass powder fluidized bed adapted to the analysis of wastewater. In Det'elopments in Ecology and Environmental Quality, Vol. II. Second International Conference of Ecology and Environment.