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Virus removal in waste stabilization ponds in India
Water Re~earch VO(. 15. pp. 773 to 778. 1981 Printed in Great Britain. All rights re~rved
0043-1354 81 070773-06S02.00~0 Cop,,right ~ 1981 Pergamon Press Ltd
VIRUS REMOVAL IN WASTE STABILIZATION P O N D S IN INDIA V. CHALAPATI RAO*, S. B. LAKHE and S. V. WAGHMARE National Environmental Engineering Research Institute, Nagpur 440 020. India
(Received June !980) Abstract--Among the low cost waste treatment methods suitable for tropical developing countries, stabilization ponds are shown to be least expensive, simple to construct and operate. A quantitative evaluation of the extent of virus removal in both pilot and full-scale ponds have been made in different seasons. Experimental pilot ponds operated at depths of 3-5 ft at different loading rates indicated an average virus removal in the range of 86-95?/0. Full scale ponds with detention times ranging from 2.7 to 17.2 days showed virus removal efflciencies ranging from 88 to 98~.
acre- t day- t and volume of sewage treated in the various seasons was 15.Smgd in rainy season, 9.0mgd in winter and 7.5 mgd in summer. (b) Bhandak. A small township consisting of personnel of Ordnance Factory is situated 180kin from Nagpur. The ponds are divided into 4 cells, each cell measuring 350 x 150 ft. Two cells are connected in series. The total volume of domestic sewage pumped is 0.3 million gallons per day. Ponds are operated at a depth of 4 ft with a detention period of 17.2 days. (c) Nagpur. Pilot plants in the Institute Campus at Nagpur: i. The pond (1) is single celled with a surface area of 8000ft 2 and a depth of 4.5 ft. The influent to this pond consisted of raw domestic sewage. Detention time was between 3 and l0 days. gODs loading varied from 205 to 742 Ib acre- t day - i. 2. Three one cell ponds (designated 2, 3 and 4) each of 50 x 90 ft with depths of 3, 4 and 5 ft have been constructed and were operated from October 1974 through February 1976. Unsettled domestic raw sewage from a middle income group community was fed simultaneously into these ponds. Loading in these ponds varied between 250-300 Ib B O D ; i acre- t day- l
INTRODUCTION Among the various low cost waste treatment methods suitable for developing countries, stabilization ponds are shown to be least expensive, simple to construct and operate (Arceivala et al., 1970). There are more than 50 installations of ponds systems in India, the majority of which are of the facultative type receiving domestic sewage. A number of reports on the survival or inactivation of viruses in both experimental and full scale waste stabilization ponds in different countries have been published. (England et al., 1967; Malberbe & Strickland-Cholmley, 1967; Christie, 1967; Slanetz et al., 1970). It is evident that there is wide variation in the virus removal efficiency of the various ponds in different geographic areas, perhaps due to different field and experimental conditions and sampling methods. There are no published reports on a quantitative evaluation of the performance of stabilization ponds in virus removal in India. The present paper deals with results obtained on the extent of virus removal in two pilot plants located in the Institute Campus at Nagpur and two full scale plants, one at Bhilai and the other at Bhandak, situated about 280 and 180 km respectively from Nagpur.
MATERIALS AND METHODS
Details of the ponds (a) Bhilai. A steel plant township, located east of Nagpur at 280 km distance. An area of 24 acres was divided into two equal parallel compartments, each measuring 1050 x 525 ft with an operational depth of 4 ft. Detention time in the pond was 2.7 days. Raw sewage influent BOD was in the range of 155-187mgl -~ and effluent BOD 15-25mg[ -z. Raw sewage was fed to the pond continuously for 24h. BODs loading varied from 225-361 Ib * Present address: Department of Virology and Epidemiology, Baylor College of Medicine, Houston, TX 77030, U.S.A. w.m. 15/7--A
Sample collection (l) Samples were collected from Bhilai and Bhandak ponds during January-June 1971. Sampling was renewed at Bhandak in November 1972 and continued through August 1973. Raw sewage and final effluent were collected at hourly intervals round the clock, held on ice, pooled and brought to Nagpur in an ice box and processed. (2) Samples from single cell Institute Pond were collected from November 1970 to June 1971 at hourly intervals from 7 a.m. to 4 p.m. composited and an aliquote processed on the same day. (3) Samples of raw sewage influent and effluent from three one,-cciled ponds of depths of 3, 4 and 5 ft respectively were also of a composite nature and were collected from October 1974 to February 1976. Measurements of temperature, suspended solids, BODs, dissolved oxygen, nitrogen, depth, detention time and algal counts in the pilot ponds have been made by the sewage treatment division and are reported separately.
Sample processing For enumeration of viruses, 80ml raw sewage and 320 ml effluent from the composite sample were processed' by the modified membrane-filter method of Rao er al. 773
V. CHALAPATI RAO e'/"al.
774
Table l, Virus removal at different BOD loading in the single cell pilot stabilization pond (Pond 11 Loading* l. 2. 3. ~. 5. 6. 7. 8. 9.
205 205 210 210 336 355 644 644 742
Average
Virus removed {°,,)
Average °o removal of virus
78 80 95 95 96 96 89 94 91
87
208
346 644
96 92
* Ib BOD~- t acre - l day- i
(1972). The method in brief consists of the following steps: wastewater sample was homogenized for 4 rain and then centrifuged for 30rain at 18000. pH of supernatant was adjusted to 3 and the sample again centrifuged for 30 min at 9230 0. The supernatant w.as filtered through a 47 mm diam. 0.45/am cellulose nitrate millipore membrane filter. Virus adsorbed on the membrane was eluted with 5 ml 33/o beef extract, pH 8.0. Beef extract was retained on the membrane for 30rain and then removed under suction. Using this method, experimentally added poliovirus at levels of 10--84 PFU per 100ml of antoclaved sewage, was recovered with an efficiency of 88-98~.
Isolation and enumeration of viruses Monolayer cultures of primary rhesus monkey kidney cells were prepared by standard procedures in 4 oz. milk dilution bottles. Virus in the beef extract eluates was quantitated by the plaque technique using an overlay medium consisting of 25raM magnesium chloride, neutral red (I :40,000) antibiotics and 1.2Yo Difco agar. Plaques were counted from the third day onwards for 10 days and the total count reported as pfu 1- t of the sample. No attempt was made to identify the plaques. However, plaques were picked up at random and passaged twice in tube cultures for cytopathic effect (CPE) to ascertain that the plaques were produced by viruses.
RESULTS
Single-cell pilot stabilization pond (pond I) Results obtained on the percentage removal of viruses are presented in relation to (i) loading; (ii) BODs removal; and (iii) seasonal fluctuation. (i) Loading. BODs loading in the pilot plant was varied by changing the rate of sewage inflow and it ranged from 205 to 742 lb a c r e - t d a y - i . The pond was to be operating in the 75-853/o efficiency range in the removal of BOD ('Manuel et al., 1974). Results on the percentage removal of virus (Table 1) at different loadings indicate that the rate of loading does not seem to influence the extent of virus removal. When the loading was 205 lb acre-~ day-~, the removal of virus was 78 and 80 for the two observations, and for the loading of 210 tb acre-~ day-~, the 9/ooremovals were considerably high, the values being 95. When the loading of the pond was increased
by about 3 times, from 205 to 644 lb acre-~ d a y mean value for the removal of virus remained at 92.0%. In this pilot plant, the results show that in a loading range of 205--742 lb acre- ~ d a y - 1 the average ~o removal of virus was in the range of 87-96. (ii) BODs removal. Results obtained on the virus removal at different percent removals of B O D s are presented in Table 2. During the course of this study the amount of BOD removal varied from 50 to 853/o, but the a m o u n t of virus removal did not appear to be affected. (iii) Seasonalfluctuation. Data on the extent of virus removal for the two seasons, viz. winter (DecemberFebruary) and summer (March-June) are given in Table 3. Values of the 3/o reduction of virus were 91 for winter and 86 for summer. It appears that the seasonal difference in virus removal is not significant. This may be due to lack of a sharp difference between winter and summer temperature of pond effluent at this location. During the present study, the effluent temperatures during winter varied from 20 to 26°C and during summer from 30 to 35'C. That there was no significant difference in stabilization pond performance between winter and summer with regard to BOD5 removal at Nagpur was reported by Lakshminarayana et al. (1969): In their
Table 2. Relationship between virus removal and BOD5 removal in the single cell (Pilot) stabilization pond (Pond 1) BOD5 removed (%)
Virus removed (%)
50 71 73 77 80 82 85
95 95 78 80 91 90 95
Virus removal in waste stabilization ponds in India
775
Table 3. Removal of virus in the single cell pilot stabilization pond (1) in various months Season
Month
Average
Winter
December 1970 January 1971 February 1971
84 95 92
March 1971 April 1971 May 1971 June 1971
83 83 86 91
Summer
studies, covering a period of 5 years (1965-1969) they reported that the average percentage reduction in B O D s for winter was 86 and for summer 85.
Three one-celled facultatioe ponds of varying depths (ponds 2, 3 and 4) Results obtained on the extent of virus removal in 3 one celled ponds of depth 3, 4 and 5 ft respectively over a period of 16 months (October 1974-February, 1976) are arranged seasonwise and presented in Tables 4 and 5. In fall, summer and rainy seasons, the average ~ removal of viruses ranged from 89 to 98.
Reduction of virus (5/o) Mean 91
Mean 86
However, in the winter season, the virus removal efficiency was rather low for the 3 ponds, the values being 66, 89 and 81 respectively (Table 5). The overall average efficiency in the various seasons including winter at the depths 3, 4 and 5 ft indicate virus removals of 86, 95 and 93~. It may be pertinent to point out that a depth of 3 ft in a single cell pond system appears to give a poor virus removal when compared to depths of 4 and 5 ft. Another point that deserves mention is that increasing the depth of a single-cell pond to 5 ft did not result in improved performance so far as virus removal was concerned.
Table 4. Virus removal in three one-celled facultative stabilization ponds of different depths Season Months
Raw sewage (pfu I - ')
Pond 2 (3 It)
Effluent (pfu I-t) in % Pond 3 % Reduction (4 It) Reduction
Pond 4 {5 It)
% Reduction
12 38 17
99 95 94
99 97 97 97
24 39 --
99.5 97 -97
70 89 94 100
50 120 12 5
Fall Sept. 75 Oct. 74 Oct. 75 Nov. 75
1213 674 290 112 4500 1175 620
0 162 40 32 100 53 27
100 76 86 72 98 96 95 89
0 24 29 . 56 36 15
100 725 63 41 150 237 100 100 263
60 232 36 0 --
40 68 53 100
30 80 4 0
40 43 40
60 57 85 66
--50
--82 89
16 31 41
375 1475 157 750
73 60 6 20
81 96 96 98 93
36 48 8 6
90 97 95 99 95
30 47 8 33
92 97 95 96 95
2825 4100 1002
69 113
98 97 -97
27 66 15
99 98 99 98
78 8 51
97 99 95 97
Mean
100 97 90 .
.
.
Winter Dec. 74 Dec. 75 Jan. 76 Feb. 75 Feb. 76 Mean
---
--
-1
99
8 1
50 84 81 88 95 99 84 69 84 81
Summer March 75 April 75 June 75 Mean
Rainy July 75 Aug. 75 Mean
776
V CH,*LAPATI RAO et a!.
Table 5. Average removal I°o) of enteroviruses at various depths in different seasons Depth
Summer
Rainy
Fall
Winter
O~erall e~ciency
3 4 5
93 95 95
97 98 97
89 97 97
66 89 81
86 95 93
Bhandak ponds
A full scale multicelled pond system operated in series at Bhandak provided a uniformly high removal of viruses entering these ponds. The ~o removal ranged from 88 to 99 with an average value of 95 (Table 6). Bhilai ponds
These ponds are quite extensive in area and are operated in parallel. They could not be visited on more occasions owing to d[stance and difficult terrain, lack of facilities for stay and round the clock collection of samples. However, the results obtained on the four sets of samples collected at monthly intervals showed on an average, 96~ removal of viruses (Table 6).
DISCUSSION
Published data on the extent of virus removal or destruction in waste stabilization ponds showed considerable variation ranging from "'not appreciable" (Malherbe & Coetzee, 1965; Slanetz et al., 1970); "'variable" (Klock, 1971; Shuval, 1970) to "satisfactory" (England et al, 1967, Malherbe & StricklandCholmley, 1967; Nupen, 1970). The above results (with the exception of that of Shuval, 1970) are based on a qualitative evaluation taking into consideration the decrease in the number of positive samples in the
Table 6. Virus removal in full-scale stabilization ponds Raw sewage (pfu 1- t)
Effluent (pfu 1- t I
~ Virus reduction
19/I/71
750
9/3/71 17/4/71 5/6/71 29/11/72 27/12/72 2/2/73 28/2/73 27/3/73 4/5/73 1/8/73
250 210 1675 93 187 447 353 125 114 887
50 8 5 43 3 6 16 13 15 10 10
94 97 97 97 97 97 96 96 88 91 99
333 350 117 1150
9 12 6 60
97 97 95 95
Date Bhandak
Bhitai 19/2/71 30/3/71 27/4/71 27/5/71
effluent when compared to raw sewage influent or by seeding the influent with a model virus and examining the effluent for the number of samples positive to virus• Results reported in this paper were based on a quantitative evaluation of the reduction in virus numbers naturally occurring in raw sewage treated in different ponds. Further, care was taken to obtain a reasonable temporal matching of the influent and effluent samples by assaying a composite sample obtained by collecting grab samples at hourly intervals between 7 a.m. and 4 p.m. in the case of pilot plants and a 24 h composite sample in the case of full-scale plants. The present study revealed that the virus removal efficiency of 92-96~ observed in both pilot and full-scale stabilization ponds of 4-5 ft depth (Table 7) is nearly comparable to the results (94-97'~,, reduction of viruses) in a conventional activated sludge sewage treatment plant studied by Rao et al. (1977) at Bombay. This study, as well as three previous studies conducted under field conditions (England et al., t967: Malherbe & Strickland-Cholmley, 1967; Nupen, 1970) have shown that stabilization ponds can remove viruses satisfactorily. England et al. (1967) reported that treatment of sewage effluent from an activated sludge plant in a stabilization pond covering 16 acres and having a detention time of 30 days resulted ira 81~o reduction in the number of samples that contained virus. However, the efficiency of the pond in removing viruses from raw sewage could not be ascertained since the oxidation pond detention was preceded by activated sludge treatment. Malherbe & Strickland-Cholmley (1967) monitored the influent and effluent from 6 maturation ponds, connected in series and having a depth of 5 ft and a retention time of 7 days. All the 14 influent samples were positive to virus. Of the 17 efltuent samples, only 4 were found positive to virus. In studies conducted by Nupen (1970) biologically treated and settled effluent containing 3600 TCID 501-1 of viruses were fed into a series of 9 maturation ponds at the Windhoek wastewater reclamation plant in South Africa. After a retention time of 14 days, 95% reduction of virus was noticed in the effluent. The most important difference between our studies at Nagpur and the above 3 investigations was that in our pilot as well as full-scale ponds, unsettled raw sewage was used for treatment. Efficiency of virus removal in our full-scale ponds ranged from 88 to 999~o
Virus removal in waste stabilization ponds in India
777
Table 7. Comparative features of the stabilization ponds studied and their relative efficiencies in virus removal
Dimensions Depth, ft Detention time, days Average virus removal, % Number of observations Number of cells
Pilot (experimental) ponds Pond 1 2 3 100 × 80ft 90 x 50ft 90 x 5Oft 4.5 3-10 92 14 I
4 90 x 50ft
3 10.8 86
4 12 95
5 13.5 93
20 I
19 1
21 1
and is comparable to the values reported in the above studies. Removal and/or inactivation of the indigenous entero viruses from wastewater in stabilization ponds can be attributed to the combined effect of factors like temperature, sludge activity, adsorption to solids, ammonia and algae-bacterial antagonism. Prolonged exposure of viruses to temperatures in the range of 20-40°C is considered conducive for considerable viral inactivation (Berg, 1966). Experimental studies by Kruse (1971) in a model oxidation lagoon indicated a 4 log reduction of viruses at 28°C in 7 or 8 days. Since the pond water temperatures during our study varied between 20 and 35°C, it may be assumed that temperature was one of the factors contributing to the observed virus reduction. Another factor that can affect virus survival in pond water is the sludge activity in the anaerobic zone. In our pilot pond (no. 1) of 4.5 ft depth, Manuel et al. (1974) have shown that the anaerobic zone extends for 3 ft from the bottom during night when the loading was 2001b BOD~ ~ acre-1 day-1. The pond was completely anaerobic at night at a loading of 600 Ib BOD~-~ acre- t d a y - ~. In tropics, sludge activity increases in the anaerobic zone of these ponds resulting in frequent sludge uplift which in turn results in more thorough removal of colloidal organics and viruses, thereby reducing viruses in the pond effluent. Virus removal by adsorption to suspended solids and their subsequent settling has been demonstrated (Schaub & Sorber, 1976). During the present study, inorganic suspended solids in the raw sewage have been removed to the extent of 63% in the experimental ponds (unpublished data) and it is likely that those viruses adsorbed on or enmeshed in solids in the influent may have been removed by mere settling along with the solids in the pond. Ammonia (NH3) is reported to be an excellent virucide (Ward, 1977) and its activity is highest at pH values greater than 8 (Ward & Ashley, 1977). In view of the fact that free ammonia concentration in the pilot pond effluents varied from 1.5 to 22 mg 1- l and the pH of the water always remained above 8.0
Full scale ponds Bhandak Bhilai 350 x 150ft 1050 x 525ft each cell each cell 4 17.2 95 11 2 x 2 cells operated in series
4 2.7 96 4 2 cells operated in parallel
(unpublished data) significant virus inactivation may be attributed to ammonia in the pond water. Stabilization ponds are inhabited by a variety of algae-bacterial populations. Experimental evidence suggests that when poliovirus I was inoculated into algae-bacterial cultures and incubated at room temperature, virus reduction of 99.999% was noticed in 7 days (Sobsey & Cooper, 1971). It is likely that in our stabilization ponds, considerable algae-bacterial virucidal activity may be taking place constantly and thereby reducing the quantity of virus in the final effluent. CONCLUSIONS It has been shown that domestic waste water can be treated effectively in stabilization ponds to reduce the numbers of human enteric viruses. A facultative pond of one or many cells having a depth of 4-5 ft can remove the virus load by at least 90%. Acknowledgements--Our thanks are due to the Sewage Treatment and Engineering Consultancy Division, NEERI for cooperation and help in the collection of samples from the pilot plants operated by them and for supplying data on some physical and chemical parameters. Appreciation is expressed to the authorities of the Ordnance Factory at Bhandak and Steel Plant at Bhilai for permission and facilities provided for collecting samples from full scale ponds. Technical assistance rendered by Ms Pushpa Dube in the processing of samples is gratefully acknowledged. We are grateful to the Director, National Environmental Engineering Research Institute, Nagpur for providing facilities for carrying out the work.
REFERENCES
Arceivala S. J., Lakshminarayana J. S. S., Alagaraswamy S. R. & Sastry C. A. (1970) Waste stabilization ponds: design, construction and operation in India. CPHERI, Nagpur, India. Berg G. (1966) Virus transmission by the Water Vehicl¢---II. Virus removal by sewage treatment procedures. Hlth Lab. Sci. 3, 90-100. Christie A. E. (1967) Virus reduction in the oxidation lagoon. Wat. Pollut. Control 105, 45, 50-51, 53--54. England B., Leach R. E., Adame B. & Shiosaki R. (1967) Virologic assessment of sewage treatment at Santee, California. Transmission of Viruses by the Water Route
?~S
V. CHALAPAT! RAO et ai.
(Edited by Berg G.). pp. 401-417. Interscience. New York. Klock J. W. (1971) Survival of coliform bacteria in waste water treatment lagoons. J. 14"at. Pollut. Control Fed. 43, 2071-2083. Kruse C. W. (1971) Discussion: Laboratory studies on the survival of poliovirus in algal-bacterial waste water treatment systems by Sobsey, M. D. and Cooper, R. C. Proc. 13th Water Quality Conf., pp. 145--147. University of Illinois at Urbana, Champaign, Urbana. IL. Lakshminarayana J. S. S., Parabrahman M., Khan A. N.. Ratnaparkhi D. Y., Choudhari K. W. & Manuel A. C. (1969) Performance studies on stabilization ponds at Bhandewadi. Nagpur. In Symposium on low cost waste treatment. Vol. 3. 27-29 Oct. CPHERI, Nagpur, India. Malfaerbe H. H. & Coetzee O. J. (1965) The survival of type 2 poliovirus in a model system of stabilization ponds. CSIR Res. Rept. No. 242, National Inst. for Water Research, Pretoria, South Africa. Malherbe H. H. & Strickland-Cholmley M. (1967) Survival of viruses in the presence of algae. Transmission of Viruses by the Water Route (Edited by Berg G.), pp. 440-458. Interscience, New York). Manuel A. C., Ratnaparkhi D. Y. & Siddique R. H. (1974) Anaerobic reactions in facultative stabilization ponds and aerated lagoons in tropical climate. Ind. J. encir. Hlth 16, 213-221. Nupen R. M. (1970) Virus studies on the windhoek wastewater reclamation plant (South West Africa). Water Res. 4, 661-672. Rao V. Chalapati. Chandorkar U.. Rao N. U.. Kumaran P.
& Lakhe S. B. i19721 A simple method for concentrat:n~ and detecting viruses in ;,,astev, ater. W~z~er Re~ 6, 1565-1576. Rao V. Chalapati, Lakhe S. B., Waghmare S '¢ & Dube P (19771 Virus removal in acti*ated sludge sewage treatment. Prog. War. Technol. 9, 113-127. Schaub S. A. & Sorber C. A. (1976) Viruses on solids in water. In Viruses in Water (Edited by Berg, G. et al.), pp. 128-138. American Public Health Association, Washington. DC. Shuval H. 1. (1970) Detection and control of enteroviruses in the water environment. Decelopment in water quality research, pp. 47-71 Ann Arbor Humprey Science, Ann Arbor, MI. Slanetz L. W.. Bartley C. H., Metcalf T. G. & Nesman R. (1970) Survival of enteric bacteria and viruses in municipal sewage lagoon. Proc. 2nd International Symposium on Waste Treatment Lagoons, University of Kansas, Lawrence, KS. Sobsey M. D. & Cooper R. C. (1971) Laboratory studies on the survival of poliovirus in algal-bacterial wastewater treatment systems. Proc. 13th Water Q,ml. Conf, pp. 137-143. University of Illinois at Urbana-Champaign, Urbana, IL. Ward R. L. (1977) Inactivation of enteric viruses in wastewater sludge. Sludge management disposal and utilization. Proc. of the 3rd National Conf. on Sludge Managment Disposal and Utilization, pp, 138-141. Information Transfer Inc., Rockville. MD. Ward R. L. & Ashley C. S. (1977) Identification of the virucidal agent in wastewater sludge. Appl. encir. Microbiol. 33, 860-864.
0043-1354 81 070773-06S02.00~0 Cop,,right ~ 1981 Pergamon Press Ltd
VIRUS REMOVAL IN WASTE STABILIZATION P O N D S IN INDIA V. CHALAPATI RAO*, S. B. LAKHE and S. V. WAGHMARE National Environmental Engineering Research Institute, Nagpur 440 020. India
(Received June !980) Abstract--Among the low cost waste treatment methods suitable for tropical developing countries, stabilization ponds are shown to be least expensive, simple to construct and operate. A quantitative evaluation of the extent of virus removal in both pilot and full-scale ponds have been made in different seasons. Experimental pilot ponds operated at depths of 3-5 ft at different loading rates indicated an average virus removal in the range of 86-95?/0. Full scale ponds with detention times ranging from 2.7 to 17.2 days showed virus removal efflciencies ranging from 88 to 98~.
acre- t day- t and volume of sewage treated in the various seasons was 15.Smgd in rainy season, 9.0mgd in winter and 7.5 mgd in summer. (b) Bhandak. A small township consisting of personnel of Ordnance Factory is situated 180kin from Nagpur. The ponds are divided into 4 cells, each cell measuring 350 x 150 ft. Two cells are connected in series. The total volume of domestic sewage pumped is 0.3 million gallons per day. Ponds are operated at a depth of 4 ft with a detention period of 17.2 days. (c) Nagpur. Pilot plants in the Institute Campus at Nagpur: i. The pond (1) is single celled with a surface area of 8000ft 2 and a depth of 4.5 ft. The influent to this pond consisted of raw domestic sewage. Detention time was between 3 and l0 days. gODs loading varied from 205 to 742 Ib acre- t day - i. 2. Three one cell ponds (designated 2, 3 and 4) each of 50 x 90 ft with depths of 3, 4 and 5 ft have been constructed and were operated from October 1974 through February 1976. Unsettled domestic raw sewage from a middle income group community was fed simultaneously into these ponds. Loading in these ponds varied between 250-300 Ib B O D ; i acre- t day- l
INTRODUCTION Among the various low cost waste treatment methods suitable for developing countries, stabilization ponds are shown to be least expensive, simple to construct and operate (Arceivala et al., 1970). There are more than 50 installations of ponds systems in India, the majority of which are of the facultative type receiving domestic sewage. A number of reports on the survival or inactivation of viruses in both experimental and full scale waste stabilization ponds in different countries have been published. (England et al., 1967; Malberbe & Strickland-Cholmley, 1967; Christie, 1967; Slanetz et al., 1970). It is evident that there is wide variation in the virus removal efficiency of the various ponds in different geographic areas, perhaps due to different field and experimental conditions and sampling methods. There are no published reports on a quantitative evaluation of the performance of stabilization ponds in virus removal in India. The present paper deals with results obtained on the extent of virus removal in two pilot plants located in the Institute Campus at Nagpur and two full scale plants, one at Bhilai and the other at Bhandak, situated about 280 and 180 km respectively from Nagpur.
MATERIALS AND METHODS
Details of the ponds (a) Bhilai. A steel plant township, located east of Nagpur at 280 km distance. An area of 24 acres was divided into two equal parallel compartments, each measuring 1050 x 525 ft with an operational depth of 4 ft. Detention time in the pond was 2.7 days. Raw sewage influent BOD was in the range of 155-187mgl -~ and effluent BOD 15-25mg[ -z. Raw sewage was fed to the pond continuously for 24h. BODs loading varied from 225-361 Ib * Present address: Department of Virology and Epidemiology, Baylor College of Medicine, Houston, TX 77030, U.S.A. w.m. 15/7--A
Sample collection (l) Samples were collected from Bhilai and Bhandak ponds during January-June 1971. Sampling was renewed at Bhandak in November 1972 and continued through August 1973. Raw sewage and final effluent were collected at hourly intervals round the clock, held on ice, pooled and brought to Nagpur in an ice box and processed. (2) Samples from single cell Institute Pond were collected from November 1970 to June 1971 at hourly intervals from 7 a.m. to 4 p.m. composited and an aliquote processed on the same day. (3) Samples of raw sewage influent and effluent from three one,-cciled ponds of depths of 3, 4 and 5 ft respectively were also of a composite nature and were collected from October 1974 to February 1976. Measurements of temperature, suspended solids, BODs, dissolved oxygen, nitrogen, depth, detention time and algal counts in the pilot ponds have been made by the sewage treatment division and are reported separately.
Sample processing For enumeration of viruses, 80ml raw sewage and 320 ml effluent from the composite sample were processed' by the modified membrane-filter method of Rao er al. 773
V. CHALAPATI RAO e'/"al.
774
Table l, Virus removal at different BOD loading in the single cell pilot stabilization pond (Pond 11 Loading* l. 2. 3. ~. 5. 6. 7. 8. 9.
205 205 210 210 336 355 644 644 742
Average
Virus removed {°,,)
Average °o removal of virus
78 80 95 95 96 96 89 94 91
87
208
346 644
96 92
* Ib BOD~- t acre - l day- i
(1972). The method in brief consists of the following steps: wastewater sample was homogenized for 4 rain and then centrifuged for 30rain at 18000. pH of supernatant was adjusted to 3 and the sample again centrifuged for 30 min at 9230 0. The supernatant w.as filtered through a 47 mm diam. 0.45/am cellulose nitrate millipore membrane filter. Virus adsorbed on the membrane was eluted with 5 ml 33/o beef extract, pH 8.0. Beef extract was retained on the membrane for 30rain and then removed under suction. Using this method, experimentally added poliovirus at levels of 10--84 PFU per 100ml of antoclaved sewage, was recovered with an efficiency of 88-98~.
Isolation and enumeration of viruses Monolayer cultures of primary rhesus monkey kidney cells were prepared by standard procedures in 4 oz. milk dilution bottles. Virus in the beef extract eluates was quantitated by the plaque technique using an overlay medium consisting of 25raM magnesium chloride, neutral red (I :40,000) antibiotics and 1.2Yo Difco agar. Plaques were counted from the third day onwards for 10 days and the total count reported as pfu 1- t of the sample. No attempt was made to identify the plaques. However, plaques were picked up at random and passaged twice in tube cultures for cytopathic effect (CPE) to ascertain that the plaques were produced by viruses.
RESULTS
Single-cell pilot stabilization pond (pond I) Results obtained on the percentage removal of viruses are presented in relation to (i) loading; (ii) BODs removal; and (iii) seasonal fluctuation. (i) Loading. BODs loading in the pilot plant was varied by changing the rate of sewage inflow and it ranged from 205 to 742 lb a c r e - t d a y - i . The pond was to be operating in the 75-853/o efficiency range in the removal of BOD ('Manuel et al., 1974). Results on the percentage removal of virus (Table 1) at different loadings indicate that the rate of loading does not seem to influence the extent of virus removal. When the loading was 205 lb acre-~ day-~, the removal of virus was 78 and 80 for the two observations, and for the loading of 210 tb acre-~ day-~, the 9/ooremovals were considerably high, the values being 95. When the loading of the pond was increased
by about 3 times, from 205 to 644 lb acre-~ d a y mean value for the removal of virus remained at 92.0%. In this pilot plant, the results show that in a loading range of 205--742 lb acre- ~ d a y - 1 the average ~o removal of virus was in the range of 87-96. (ii) BODs removal. Results obtained on the virus removal at different percent removals of B O D s are presented in Table 2. During the course of this study the amount of BOD removal varied from 50 to 853/o, but the a m o u n t of virus removal did not appear to be affected. (iii) Seasonalfluctuation. Data on the extent of virus removal for the two seasons, viz. winter (DecemberFebruary) and summer (March-June) are given in Table 3. Values of the 3/o reduction of virus were 91 for winter and 86 for summer. It appears that the seasonal difference in virus removal is not significant. This may be due to lack of a sharp difference between winter and summer temperature of pond effluent at this location. During the present study, the effluent temperatures during winter varied from 20 to 26°C and during summer from 30 to 35'C. That there was no significant difference in stabilization pond performance between winter and summer with regard to BOD5 removal at Nagpur was reported by Lakshminarayana et al. (1969): In their
Table 2. Relationship between virus removal and BOD5 removal in the single cell (Pilot) stabilization pond (Pond 1) BOD5 removed (%)
Virus removed (%)
50 71 73 77 80 82 85
95 95 78 80 91 90 95
Virus removal in waste stabilization ponds in India
775
Table 3. Removal of virus in the single cell pilot stabilization pond (1) in various months Season
Month
Average
Winter
December 1970 January 1971 February 1971
84 95 92
March 1971 April 1971 May 1971 June 1971
83 83 86 91
Summer
studies, covering a period of 5 years (1965-1969) they reported that the average percentage reduction in B O D s for winter was 86 and for summer 85.
Three one-celled facultatioe ponds of varying depths (ponds 2, 3 and 4) Results obtained on the extent of virus removal in 3 one celled ponds of depth 3, 4 and 5 ft respectively over a period of 16 months (October 1974-February, 1976) are arranged seasonwise and presented in Tables 4 and 5. In fall, summer and rainy seasons, the average ~ removal of viruses ranged from 89 to 98.
Reduction of virus (5/o) Mean 91
Mean 86
However, in the winter season, the virus removal efficiency was rather low for the 3 ponds, the values being 66, 89 and 81 respectively (Table 5). The overall average efficiency in the various seasons including winter at the depths 3, 4 and 5 ft indicate virus removals of 86, 95 and 93~. It may be pertinent to point out that a depth of 3 ft in a single cell pond system appears to give a poor virus removal when compared to depths of 4 and 5 ft. Another point that deserves mention is that increasing the depth of a single-cell pond to 5 ft did not result in improved performance so far as virus removal was concerned.
Table 4. Virus removal in three one-celled facultative stabilization ponds of different depths Season Months
Raw sewage (pfu I - ')
Pond 2 (3 It)
Effluent (pfu I-t) in % Pond 3 % Reduction (4 It) Reduction
Pond 4 {5 It)
% Reduction
12 38 17
99 95 94
99 97 97 97
24 39 --
99.5 97 -97
70 89 94 100
50 120 12 5
Fall Sept. 75 Oct. 74 Oct. 75 Nov. 75
1213 674 290 112 4500 1175 620
0 162 40 32 100 53 27
100 76 86 72 98 96 95 89
0 24 29 . 56 36 15
100 725 63 41 150 237 100 100 263
60 232 36 0 --
40 68 53 100
30 80 4 0
40 43 40
60 57 85 66
--50
--82 89
16 31 41
375 1475 157 750
73 60 6 20
81 96 96 98 93
36 48 8 6
90 97 95 99 95
30 47 8 33
92 97 95 96 95
2825 4100 1002
69 113
98 97 -97
27 66 15
99 98 99 98
78 8 51
97 99 95 97
Mean
100 97 90 .
.
.
Winter Dec. 74 Dec. 75 Jan. 76 Feb. 75 Feb. 76 Mean
---
--
-1
99
8 1
50 84 81 88 95 99 84 69 84 81
Summer March 75 April 75 June 75 Mean
Rainy July 75 Aug. 75 Mean
776
V CH,*LAPATI RAO et a!.
Table 5. Average removal I°o) of enteroviruses at various depths in different seasons Depth
Summer
Rainy
Fall
Winter
O~erall e~ciency
3 4 5
93 95 95
97 98 97
89 97 97
66 89 81
86 95 93
Bhandak ponds
A full scale multicelled pond system operated in series at Bhandak provided a uniformly high removal of viruses entering these ponds. The ~o removal ranged from 88 to 99 with an average value of 95 (Table 6). Bhilai ponds
These ponds are quite extensive in area and are operated in parallel. They could not be visited on more occasions owing to d[stance and difficult terrain, lack of facilities for stay and round the clock collection of samples. However, the results obtained on the four sets of samples collected at monthly intervals showed on an average, 96~ removal of viruses (Table 6).
DISCUSSION
Published data on the extent of virus removal or destruction in waste stabilization ponds showed considerable variation ranging from "'not appreciable" (Malherbe & Coetzee, 1965; Slanetz et al., 1970); "'variable" (Klock, 1971; Shuval, 1970) to "satisfactory" (England et al, 1967, Malherbe & StricklandCholmley, 1967; Nupen, 1970). The above results (with the exception of that of Shuval, 1970) are based on a qualitative evaluation taking into consideration the decrease in the number of positive samples in the
Table 6. Virus removal in full-scale stabilization ponds Raw sewage (pfu 1- t)
Effluent (pfu 1- t I
~ Virus reduction
19/I/71
750
9/3/71 17/4/71 5/6/71 29/11/72 27/12/72 2/2/73 28/2/73 27/3/73 4/5/73 1/8/73
250 210 1675 93 187 447 353 125 114 887
50 8 5 43 3 6 16 13 15 10 10
94 97 97 97 97 97 96 96 88 91 99
333 350 117 1150
9 12 6 60
97 97 95 95
Date Bhandak
Bhitai 19/2/71 30/3/71 27/4/71 27/5/71
effluent when compared to raw sewage influent or by seeding the influent with a model virus and examining the effluent for the number of samples positive to virus• Results reported in this paper were based on a quantitative evaluation of the reduction in virus numbers naturally occurring in raw sewage treated in different ponds. Further, care was taken to obtain a reasonable temporal matching of the influent and effluent samples by assaying a composite sample obtained by collecting grab samples at hourly intervals between 7 a.m. and 4 p.m. in the case of pilot plants and a 24 h composite sample in the case of full-scale plants. The present study revealed that the virus removal efficiency of 92-96~ observed in both pilot and full-scale stabilization ponds of 4-5 ft depth (Table 7) is nearly comparable to the results (94-97'~,, reduction of viruses) in a conventional activated sludge sewage treatment plant studied by Rao et al. (1977) at Bombay. This study, as well as three previous studies conducted under field conditions (England et al., t967: Malherbe & Strickland-Cholmley, 1967; Nupen, 1970) have shown that stabilization ponds can remove viruses satisfactorily. England et al. (1967) reported that treatment of sewage effluent from an activated sludge plant in a stabilization pond covering 16 acres and having a detention time of 30 days resulted ira 81~o reduction in the number of samples that contained virus. However, the efficiency of the pond in removing viruses from raw sewage could not be ascertained since the oxidation pond detention was preceded by activated sludge treatment. Malherbe & Strickland-Cholmley (1967) monitored the influent and effluent from 6 maturation ponds, connected in series and having a depth of 5 ft and a retention time of 7 days. All the 14 influent samples were positive to virus. Of the 17 efltuent samples, only 4 were found positive to virus. In studies conducted by Nupen (1970) biologically treated and settled effluent containing 3600 TCID 501-1 of viruses were fed into a series of 9 maturation ponds at the Windhoek wastewater reclamation plant in South Africa. After a retention time of 14 days, 95% reduction of virus was noticed in the effluent. The most important difference between our studies at Nagpur and the above 3 investigations was that in our pilot as well as full-scale ponds, unsettled raw sewage was used for treatment. Efficiency of virus removal in our full-scale ponds ranged from 88 to 999~o
Virus removal in waste stabilization ponds in India
777
Table 7. Comparative features of the stabilization ponds studied and their relative efficiencies in virus removal
Dimensions Depth, ft Detention time, days Average virus removal, % Number of observations Number of cells
Pilot (experimental) ponds Pond 1 2 3 100 × 80ft 90 x 50ft 90 x 5Oft 4.5 3-10 92 14 I
4 90 x 50ft
3 10.8 86
4 12 95
5 13.5 93
20 I
19 1
21 1
and is comparable to the values reported in the above studies. Removal and/or inactivation of the indigenous entero viruses from wastewater in stabilization ponds can be attributed to the combined effect of factors like temperature, sludge activity, adsorption to solids, ammonia and algae-bacterial antagonism. Prolonged exposure of viruses to temperatures in the range of 20-40°C is considered conducive for considerable viral inactivation (Berg, 1966). Experimental studies by Kruse (1971) in a model oxidation lagoon indicated a 4 log reduction of viruses at 28°C in 7 or 8 days. Since the pond water temperatures during our study varied between 20 and 35°C, it may be assumed that temperature was one of the factors contributing to the observed virus reduction. Another factor that can affect virus survival in pond water is the sludge activity in the anaerobic zone. In our pilot pond (no. 1) of 4.5 ft depth, Manuel et al. (1974) have shown that the anaerobic zone extends for 3 ft from the bottom during night when the loading was 2001b BOD~ ~ acre-1 day-1. The pond was completely anaerobic at night at a loading of 600 Ib BOD~-~ acre- t d a y - ~. In tropics, sludge activity increases in the anaerobic zone of these ponds resulting in frequent sludge uplift which in turn results in more thorough removal of colloidal organics and viruses, thereby reducing viruses in the pond effluent. Virus removal by adsorption to suspended solids and their subsequent settling has been demonstrated (Schaub & Sorber, 1976). During the present study, inorganic suspended solids in the raw sewage have been removed to the extent of 63% in the experimental ponds (unpublished data) and it is likely that those viruses adsorbed on or enmeshed in solids in the influent may have been removed by mere settling along with the solids in the pond. Ammonia (NH3) is reported to be an excellent virucide (Ward, 1977) and its activity is highest at pH values greater than 8 (Ward & Ashley, 1977). In view of the fact that free ammonia concentration in the pilot pond effluents varied from 1.5 to 22 mg 1- l and the pH of the water always remained above 8.0
Full scale ponds Bhandak Bhilai 350 x 150ft 1050 x 525ft each cell each cell 4 17.2 95 11 2 x 2 cells operated in series
4 2.7 96 4 2 cells operated in parallel
(unpublished data) significant virus inactivation may be attributed to ammonia in the pond water. Stabilization ponds are inhabited by a variety of algae-bacterial populations. Experimental evidence suggests that when poliovirus I was inoculated into algae-bacterial cultures and incubated at room temperature, virus reduction of 99.999% was noticed in 7 days (Sobsey & Cooper, 1971). It is likely that in our stabilization ponds, considerable algae-bacterial virucidal activity may be taking place constantly and thereby reducing the quantity of virus in the final effluent. CONCLUSIONS It has been shown that domestic waste water can be treated effectively in stabilization ponds to reduce the numbers of human enteric viruses. A facultative pond of one or many cells having a depth of 4-5 ft can remove the virus load by at least 90%. Acknowledgements--Our thanks are due to the Sewage Treatment and Engineering Consultancy Division, NEERI for cooperation and help in the collection of samples from the pilot plants operated by them and for supplying data on some physical and chemical parameters. Appreciation is expressed to the authorities of the Ordnance Factory at Bhandak and Steel Plant at Bhilai for permission and facilities provided for collecting samples from full scale ponds. Technical assistance rendered by Ms Pushpa Dube in the processing of samples is gratefully acknowledged. We are grateful to the Director, National Environmental Engineering Research Institute, Nagpur for providing facilities for carrying out the work.
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Arceivala S. J., Lakshminarayana J. S. S., Alagaraswamy S. R. & Sastry C. A. (1970) Waste stabilization ponds: design, construction and operation in India. CPHERI, Nagpur, India. Berg G. (1966) Virus transmission by the Water Vehicl¢---II. Virus removal by sewage treatment procedures. Hlth Lab. Sci. 3, 90-100. Christie A. E. (1967) Virus reduction in the oxidation lagoon. Wat. Pollut. Control 105, 45, 50-51, 53--54. England B., Leach R. E., Adame B. & Shiosaki R. (1967) Virologic assessment of sewage treatment at Santee, California. Transmission of Viruses by the Water Route
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(Edited by Berg G.). pp. 401-417. Interscience. New York. Klock J. W. (1971) Survival of coliform bacteria in waste water treatment lagoons. J. 14"at. Pollut. Control Fed. 43, 2071-2083. Kruse C. W. (1971) Discussion: Laboratory studies on the survival of poliovirus in algal-bacterial waste water treatment systems by Sobsey, M. D. and Cooper, R. C. Proc. 13th Water Quality Conf., pp. 145--147. University of Illinois at Urbana, Champaign, Urbana. IL. Lakshminarayana J. S. S., Parabrahman M., Khan A. N.. Ratnaparkhi D. Y., Choudhari K. W. & Manuel A. C. (1969) Performance studies on stabilization ponds at Bhandewadi. Nagpur. In Symposium on low cost waste treatment. Vol. 3. 27-29 Oct. CPHERI, Nagpur, India. Malfaerbe H. H. & Coetzee O. J. (1965) The survival of type 2 poliovirus in a model system of stabilization ponds. CSIR Res. Rept. No. 242, National Inst. for Water Research, Pretoria, South Africa. Malherbe H. H. & Strickland-Cholmley M. (1967) Survival of viruses in the presence of algae. Transmission of Viruses by the Water Route (Edited by Berg G.), pp. 440-458. Interscience, New York). Manuel A. C., Ratnaparkhi D. Y. & Siddique R. H. (1974) Anaerobic reactions in facultative stabilization ponds and aerated lagoons in tropical climate. Ind. J. encir. Hlth 16, 213-221. Nupen R. M. (1970) Virus studies on the windhoek wastewater reclamation plant (South West Africa). Water Res. 4, 661-672. Rao V. Chalapati. Chandorkar U.. Rao N. U.. Kumaran P.
& Lakhe S. B. i19721 A simple method for concentrat:n~ and detecting viruses in ;,,astev, ater. W~z~er Re~ 6, 1565-1576. Rao V. Chalapati, Lakhe S. B., Waghmare S '¢ & Dube P (19771 Virus removal in acti*ated sludge sewage treatment. Prog. War. Technol. 9, 113-127. Schaub S. A. & Sorber C. A. (1976) Viruses on solids in water. In Viruses in Water (Edited by Berg, G. et al.), pp. 128-138. American Public Health Association, Washington. DC. Shuval H. 1. (1970) Detection and control of enteroviruses in the water environment. Decelopment in water quality research, pp. 47-71 Ann Arbor Humprey Science, Ann Arbor, MI. Slanetz L. W.. Bartley C. H., Metcalf T. G. & Nesman R. (1970) Survival of enteric bacteria and viruses in municipal sewage lagoon. Proc. 2nd International Symposium on Waste Treatment Lagoons, University of Kansas, Lawrence, KS. Sobsey M. D. & Cooper R. C. (1971) Laboratory studies on the survival of poliovirus in algal-bacterial wastewater treatment systems. Proc. 13th Water Q,ml. Conf, pp. 137-143. University of Illinois at Urbana-Champaign, Urbana, IL. Ward R. L. (1977) Inactivation of enteric viruses in wastewater sludge. Sludge management disposal and utilization. Proc. of the 3rd National Conf. on Sludge Managment Disposal and Utilization, pp, 138-141. Information Transfer Inc., Rockville. MD. Ward R. L. & Ashley C. S. (1977) Identification of the virucidal agent in wastewater sludge. Appl. encir. Microbiol. 33, 860-864.