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Methods for detection of virus in wastewater, applied to samples from small scale treatment systems
~lilter Research Vol. 14. pp. 169 to 173 © Pergamon Press Lid 198{I. Printed in Great Britain
11{)43-135480/0201-0169502.00/0
METHODS FOR DETECTION OF VIRUS IN WASTEWATER, APPLIED TO SAMPLES FROM SMALL SCALE TREATMENT SYSTEMS B. LYDHOLM and A. L. NIELSEN Department of Veterinary Virology and Immunology, The Royal Veterinary and Agricultural University of Copenhagen, Denmark. (Receipted No~emher 19781
Abstract--Two small scale treatment systems were investigated to evaluate their effect on indigenous viruses in wastewater. In the effluent from an emscher tank system, treating a mixture of outlet from toilet, bath and kitchen, virus was demonstrated rather constantly. Samples were taken from a pasteurization plant where outlet from toilets was treated by addition of formaldehyde and exposure to 76°C for 30 min. A few samples from influent were positive, but no virus was found in the effluent. A comparison was made of four methods to detect viruses in wastewater. Alum precipitation followed by tris elution, acidic precipitation followed by elution with beef extract and two-phase concentration all made it possible to isolate indigenous viruses from wastewater and gave comparable results. No virus was demonstrated by direct inoculation. This corresponds well with the findings that the contents of virus was so low as to require concentration to be demonstrable.
INTRODUCTION
Treatment of wastewater can be carried out in several ways. The system chosen will to a large extent depend on the socioeconomic level and the size of the community. For buildings outside urban districts septic and emscher tank systems can be applied. In Denmark 15-20~ of the population use this type of treatment, which has been practised since the beginning of this century. The effluent from such systems is carried off to a seepage area or to a stream. The main purpose of this mechanical treatment is to get a reduction in organic matter, whereas less attention has been given to the content of pathogens. Such considerations are taken in newer developed systems for small communities, especially in systems employing pasteurization. The usefulness of this type of system is however somewhat limited because of high operating expenses. I.t is to be expected, that the reduction of the virus concentration of wastewater in septic tank or emscher tank systems is small, whereas a good reduction presumably will be obtained in a well functioning pasteurization plant. Examination of the effect of these systems on the virus content is difficult because the available methods are not sufficiently quantitative and equally reliable under different conditions. Several methods have been attempted to recover viruses from wastewater. A number of review articles have appeared of these, e.g. Sobsey (1976). Acidic precipitation of the proteins in the wastewater might also trap the viruses and possibly be used as a method of concentration (Lund, 1977). Katzenelson et al. (1976) used this in a secondary concentration after membrane filtration of tapwater. It was found by Lund (1973) that virus might be bound to particles and therefore removal of any part of the sample before a quantitative assay should be
avoided. This was supported by Wellings et al. (1976), who considered the indigenous virus to be both adsorbed to and embedded in the solids. These works throw new light upon earlier methods for virus recovery and show that seeding experiments should be estimated with care. The purpose of this work was to evaluate the efficiency of two small scale treatment systems, a pasteurization and an emscher tank system. Simultaneously a comparison was made of four different methods for the detection of indigenous viruses from wastewater. The methods chosen were direct inoculation, acidic precipitation followed by elution with beef extract, alum precipitation followed by tris-buffer elution, and two-phase separation.
MATERIAL
AND
METHODS
Stud), site
At the Tvind Schools located in the country outside Ulfborg in Jutland wastewater from the 500 inhabitants is treated in two separate small scale systems, each receiving about half of the wastewater. When the schools started in 1972 a traditional emscher tank system (Trix, Nordisk Triclair) was set up to treat the mixed outlet from bath, kitchen and toilet. This system is composed of two separate tanks, both with sedimentation of particles from the sewage in the upper part and a digestion chamber at the bottom, partly separated from the stream of water above. This system is designed to give a 30~o reduction in the B O D before the water is discharged into a brook. In 1975, when the schools were extended, an alternative form of treatment was chosen to purify sewage from the new houses. The toilet outlets from these houses are treated separately. By use of vacuum it is sufficient to use 1 1 of water per flushing. This sewage with high 169
170
B. LYDHOLMand A. L. NIELSEN
amount of total suspended solids is mixed with formaldehyde to give a concentration of 170-200mg/l and is immediately afterwards pasteurized at 76°C for 30 min. The effluent from this system is kept in open basins until land application. Outlet from bathrooms and kitchens is conducted to a seepage area. Rainwater is collected and discharged into the same brook as the emscher tank effluent.
Sample collection Samples of influent and effluent from both the emscher tank system and the pasteurization plant were collected. Samples were also taken from the influent to the seepage area. Samples of raw sewage were collected from the treatment plant at Damhusaaen where part of the domestic and industrial wastewater from Copenhagen is treated. In all cases the volume was I I. Transport of the samples to the laboratory was carried out within 4 h. The samples were left overnight at 4°C and processed the following day.
Virus assay Tube cultures of HeLa cells were grown in Eagles medium with 3~o calf serum, 200 IU benzyl-penicillin per ml and 260 #g streptomycinsulfate per ml. Each tube contained 2 ml of medium, which was renewed twice during the incubation period of 7 days at 37°C. Just before inoculation mycostatin was added to the sample to give a final concentration of 1000 IU per ml. The inoculum was 0.1 ml and each sample was inoculated into four tubes. Three hours after inoculation of the two-phase concentrates and of the direct samples the medium was renewed to avoid cell degeneration because of the harmful effect of these materials. Blind passages were carried out twice on negative cultures (Lund & HedstriSm, 1969). If three or four of the tubes were positive at the primary inoculation serial ten-fold dilutions were carried out in Hanks' balanced salt solution, and each dilution inoculated into three tubes. Virus concentration was calculated according to the 50~o-endpoint method of Reed & Muench (1938). The isolated virus strains were typed by use of neutralization test.
Methods used to isolate virus from wastewater Alum precipitation. 75 mg A!2(SO4)3"18 H20 (Merck) was added to 100 ml of the sample. After shaking for 2 h the mixture was centrifuged at 4080 g for 15 min at 4°C. The precipitate was eluted with tris buffer at pH 9.0 (Lal &Lund, 1975), in the proportion 1:4. After shaking for 2 h the sample was recentrifuged and the eluate neutralized. Acidic precipitation (Lund, 1977). 1 N HCI was added to 200 ml sewage until pH 3.5 was obtained. The sample was mixed by shaking for 1 h and centrifuged at 16,000 g for 30 min at 4°C. The precipitate was mixed with four volumes of a 10~o solution of beef extract (Difco) at pH 7. After mechanical stirring for 2 h centrifugation was repeated and the eluate neutralized. Two-phase concentration (Lund & Hedstri~m, 1966). 20 g of 5 M NaCl, 58 g of 30~ polyethylene glycol (Carbowax 6000), 2.7 g of sodium dextran sulfate (2000 DS, Pharmica) were added to 200 ml sample and pH adjusted to 7.2. After thorough shaking for t h the sample was left overnight at 4°C in a separatory funnel. A bottom-phase and an interphase were collected each giving 2 ml. Direct inoculation. 2 ml wastewater was adjusted to pH 7.2.
RESULTS
In Table 1 a comparison is made between the different methods used to detect viruses in wastewater. The results shown for the two-phase concentration are the values from the bottom-phase isolations as only a few inter-phase samples turned out positive and then in a lower concentration than in the corresponding bottom-phase samples, The two-phase separation then gave a 100-fold concentration based on volume reduction. This factor was used to estimate the virus concentration in the total sample, and cons~uently the lowest detectable virus concentration with this method was 0.05 TCIDso per mi wastewater. Lowest detectable amount per ml for direct inoculation was 4.6 TCIDso. In the two precipitation methods the sensibility of virus detection varies because the quantity of the eluent was dependent on the amount of precipitate. The average amount of total suspended solids in the sewage was 0.2~o for the raw sewage, for the sewage from the vacuum toilets 4Yo and for the emscher tank effluent 0.1~. When direct inoculation was used 33Yo of the samples were harmful to the cell cultures, none of these or any of the other direct inoculated samples gave a positive result in the blind passages. With both acidic Decontamination precipitation and two-phase concentration 5 out of 17 The eluates, the concentrates from the two-phase samples were positive, and with alum precipitation separation and the samples to be inoculated directly virus was detectable in 6 out of 18 samples. A comwere decontaminated by addition of ether in the pro- parison between the methods did not reveal any difportion 1:I (normally 2ml each). After thorough ference in the estimated concentrations of virus. shaking the mixture was allowed to stand for at least Before the examination of the wastewater from the 3 h before most of the ether was pipetted off and the small community at the Tvind Schools two samples of rest was removed by bubbling air through the sample raw wastewater from a municipal treatment plant in for 20 min or more. Copenhagen were tested by means of the different iso-
171
Detection of virus in wastewater Table 1. Comparison of different methods for the isolation of viruses from sewage
Indigenous virus recovered (TCIDs0/ml wastewater) Direct Alum Acidic Two-phase ~ inoculation precipitation precipitation concentration
Type of sample
Date
Raw sewageb Average pH 7.5
6/7/77 7/7/77
0~ 0
0.9 0
0.8 0.8
0 0.3
Influent to the pasteurization plant (before addition of formaldehyde)J Average pH 9.0
28/7/77 19/8/77 31/8/77 14/9/77 20/9/77 27/9/77 4/10/77 31/10/77
0 0 tox t 0 0 0 0 tox
0 0 0 0 0 0 1.3 0
ND ~ 0 0 0 0 0 0 0
1.8 ND 0 0 0 0 0.1 0
Effluent from the emscher tank system~ Average pH 7.5
19/8/77 31/8/77 14/9/77 20/9/77 27/9/77 4/10/77 11/10/77 31/7/77
tox tox 0 tox 0 tox 0 0 0/18
0 0 0.3* 0.6 0.7 0 0 0.2* 6/18
0 0 0 0 0.1 * 0.1 * 0 0.2 5/17
0 0 0 1.8 0 0 0 0.05* 5/17
Samples positive/samples tested
a The value of TCIDs0 per ml sample is calculated on the basis of virus detected in the bottom-phase. b Sewage collected from the influent at the treatment plant at Damhusaaen, located in Copenhagen. c "O" means that no viruses were isolated from the sample by the applied method. For sensibility of detection see the text. d Sewage from two small scale treatment systems at the Tvind Schools. The mean temperature was 13°C in the period of sample collection. ' Not done. f "tox" means that the sample inoculated had a toxic effect on the cell cultures. * Only one out of four inoculated tubes was positive. lation methods. The sewage was expected to contain virus because of the size of the population and the time of the year (July). All the methods except the direct inoculation revealed viruses. The highest concentration detected was 0.9 TCIDs0 per ml. At the Tvind Schools virus was found in 2 out of 8 samples in the influent to the pasteurization plant before the formaldehyde was added. The highest concentration was 1.8 TCIDs0 per ml, estimated from two-phase concentration. Twice it was possible to get samples after addition of formaldehyde, but before pasteurization (4 and 11 October). N o n e of these samples turned out positive, although the corresponding sample without formaldehyde taken on 4 October was positive. On 11 October no sample without formaldehyde was tested. Virus was never detected in the effluent from the pasteurization plant in the examination period (19 August-31 October 1977). In the same way no virus was demonstrated in the influent to the seepage area and to the emscher tank. In the effluent from the emscher tank system virus was isolated 5 out of 8 times in a concentration up to !.8 TCIDs0 per ml. In Table 1 only the wastewater types from which virus was detected are shown. In Table 2 the virus types isolated are given. In the raw municipal sewage only coxsackievirus B5 was
found. This virus was also demonstrated in both systems at the Tvind Schools. Adenovirus 2 was isolated in one sample. This was also the case with echovirus 13, which was found by use of the two-phase concentration method, but only in the interphase. Polioviruses were detected several times after the 20 September in the emscher tank system. DISCUSSION The results of the present report show that the twophase concentration, alum precipitation and acidic precipitation all were applicable for the isolation of viruses from waste-water. The two-phase concentration method has been used in several works, giving good results both on natural and on artificially contaminated samples (Lund & Hcdstr6m, 1966; Shuval et at., 1967; Lal & Lund, 1975). In the present work the method was usable even for the sewage from the pasteurization plant, where the amount of total suspended solids was high. The viruses embedded in the wastewater particles may not have been detected when the two-phase concentration was used, as this method 'does not employ an elution step. Lal & Lund (1975) t6sted the alum precipitation method on artificially contaminated sludge. They obtained a good recovery and a high increase in virus
172
B. LYDHOLMand A. L. NIELSEN Table 2. Virus types isolated from the sewage samples mentioned in Table 1
Type of sample Raw sewage lnfluent to the pasteurization plant (before addition of formaldehyde)
Effluent from the emscher tank system
Date
Direct inoculation
6/7/'77 7/7/77 28/7/77 19/8/77 31/8/77 14/9/77 20/9/77 27/9/77 4/10/77 31i10/77 19/8/77 31/8/77 14/9/77 20/9/77 27/9/77 4/10/77 11/10/77 31/10/77
Alum precipitation
Acidic precipitation
Two-phase concentration
Coxs B5
Coxs B5 Coxs B5
Coxs B5 Coxs B5
~"Echo 13" L Adeno 2
Adeno 2
Coxs B5 Polio 3 Polio 3 Polio 3
Polio 3 Polio 2 Polio 3 Polio 1 Polio 3
Polio 2
" Only isolated from the inter-phase. Coxs = Coxsackievirus concentration and consequently a high degree in sensibility. Although they worked with seeded sewage, their results may be transferred to natural conditions as the viruses embedded in wastewater particles probably will be loosened during several hours' shaking in eluent. In the present work the vigorous stirring for 2 h presumably also had such a loosening effect. The acidic precipitation gave a good recovery of viruses although the concentration might be lower than in the two other concentration methods. Perhaps some inactivation of the viruses occur during the period with low pH. In a single seeding experiment with coxsackievirus B3 added to sewage a 100% recovery was obtained by this method, but the effect of acidification may be different for other virus types. Therefore a shortening of the period with low pH may be recommended. The concentrations estimated by two-phase separation and the precipitation methods were between 0 and 1.8 TCIDs0 per ml wastewater. These values are smaller than the lowest detectable amount for direct inoculation, which is 4.6 TCIDs0 per ml for the used assay system, 0.1 ml inoculated into four tubes per sample. Buras (1974) obtained good results by use of the direct inoculation method on sewage from Israel, which had a higher content of virus (average 1069 PFU per 100 ml) than the Danish sewage tested in the present report. Six samples out of 18 tested were harmful to the cell cultures so even if the concentration in the sewage had been higher this might have interfered with the results. Maybe the viruses embedded in solids were not detected in the direct inoculated samples, as in the case with the two-phase concentration. There seems to be no difference in the virus types
isolated by the different methods except the isolation of echovirus 13 from the inter-phase. The virus assay system used, HeLa cells in tube cultures with one week of incubation and two blind passages, gives a limitation to the types that can be detected. It is possible to isolate adeno-, polio-, echo-, coxsackie B- and a few coxsackie A viruses, but probably not reoviruses. For most coxsackie A types inoculation in new-born mice would be required and the viruses of infectious hepatitis and non-bacterial gastroenteritis cannot be cultivated from wastewater samples. The adenoviruses were detected in blind passages, and would not have appeared if these had not been carried out (Lund & Hedstr6m, 1969). The poliovirus isolated from the emscher tank effluent probably resulted from vaccination of a group of people in the beginning of September. The group belonged to the part of the school connected to the emscher tank system. The treatment system employing pasteurization and addition of formaldehyde is difficult to evaluate because of the few positive samples in the influent. Laboratory studies with artificially contaminated sewage show that the addition of 100mg formaldehyde per 1 at pH from 8 to 10.5 gives at least a 2.4 logt0 reduction in the content of poliovirus type 1 (Sobsey et al., 1974). Foliguet & Doncoeur (1972) obtained a 5 loglo reduction in poliovirus type 1 during heating of raw sludge to 80°C in 10 min. These results indicate that a considerable reduction in the virus concentration will be achieved by addition of 170-200 mg formaldehyde per litre sewage at pH 9 and heating to 76°C for 30 min as used at the Tvind Schools. In the.period of sample collection no positive samples were found in the effluent from the system. The Salmonella content of the wastewater in the open
Detection of virus in wastewater basins after the pasteurization plant has been measured regularly by the municipal laboratory, but has only been demonstrated twice; once because of human errors and once because of overloading. The types isolated were S. indiana, S. montevideo and S. typhimurittm (Faulenborg, 1978). The samples of wastewater from bathroom and kitchen did not contain virus in detectable amounts. Bacteriological examinations have shown that 20-40% of the total amount of fecal colibacteria can be found in wastewater from bath and kitchen (Tullander, 1967). The results of the present work may be due to the low concentration of virus in the sewage. No virus was detected in the emscher tank influent during the sampling period probably because of the heterogenity of the flow and of the composition of the water. The planned time of sedimentation in the tanks was 2 h during which sedimentation took place. Presumably also a homogenization happened from which a liberation of the viruses embedded in fecal matter results, leading to a constant flow of virus from the system. In this water Salmonella was found in three out of five samples; the types isolated were S. heidelberg and S. singapore (Faulenborg, 1978). This way of discharging the effluent must be considered inappropriate especially because of the small flow in the brook. If circumstances (soil structure, groundwater level, available area) allow, it must be recommended to lead the water to a seepage area. Apart from the initial difficulties in the operation of the pasteurization plant the treatment in this system seems to be satisfactory from a virological point of view. As none of the methods used gave 9 positive sampies, which were the total positive number detected, the effectivity of each method was less than 100~o. From the results obtained in the present study none of the concentration methods can be preferred to the others. It cannot be excluded that the same samples tested a number of times employing just one method might have given similar results. Acknowledgements--This work was carried out at the Royal Veterinary and Agricultural University of Copen-
173
hagen, where we were permitted to work during our graduate study of biology at the University of Copenhagen. We wish to express our gratitude to Professor Ebba Lund and her staff at the Department of Veterinary Virology and Immunology for their generous assistance. We also want to thank the Tvind Schools for their kind co-operation.
REFERENCES
Buras N. (1974) Recovery of viruses from wastewater and effluent by direct inoculation method. Water Res. 8, 19-22. Faulenborg G. (1978) Personal communication. Foliguet J. M. & F. Doncoeur. (1972) Inactivation assays of enteroviruses and salmonella in fresh and digested wastewater sludges by pasteurization. Water Res. 6, 1399-1407. Katzenelson E., B. Fattal and T. Hostovesky. (1976) Organic flocculation: an efficient second-step concentration method for the detection of viruses in Tap Water. Applied and Em'ironmental Microbioloqy 32, 638-639. Lal S. M. and E. Lund (1975) Recovery of virus by chemical precipitation followed by elution. Pro#. in Water Technol. 7, 687-693. Lund E. and C. E. Hedstr6m (1966) The use of an aqueous polymer phase system for enterovirus isolations from sewage. Am. J. Epidemiology 84, 287-291. Lund E. and C. E. Hedstr6m. (1969) A study on sampling and isolation methods for detection of virus in sewage. Water Res, 3, 823-832. Lund E. (1973) The effect of pretreatments on the virus contents of sewage samples. Water Res. 7, 873-879. Lund E. (1977) Personal communication. Reed L. J. and H. Muench. (1938) A simple method of estimating fifty percent endpoints. Am. J. Hygiene 27, 493-497. Shuval H. I., S. Cymbalista, B. Fattal and N. Goldblum. (1967) Concentration of enteric viruses in water by hydroextraction and two-phase separation. Transmission of t,irus by the water route. G. Berg (ed.) 45-55. Sobsey M. D., C. Wallis and J. Melnick. (1974) Chemical disinfection of holding-tank sewage. Appl. Microbiol. 28, 861-866. Sobsey M. D. (1976) Methods for detecting enteric viruses in water and wastewater. Viruses in Water. G. Berg (ed.) 89-127. Tullander V. (1967) Hushb.llsavloppsvatten 5. Bakteriologisk Sammens~ittning. Byggforskningen, Informationsblad no. 23. Wellings F. M., A. L. Lewis and C. W. Mountain. (1976) Demonstration of solids-associated virus in wastewater and sludge. Appl. Env. Microbiol. 31,354-358.
11{)43-135480/0201-0169502.00/0
METHODS FOR DETECTION OF VIRUS IN WASTEWATER, APPLIED TO SAMPLES FROM SMALL SCALE TREATMENT SYSTEMS B. LYDHOLM and A. L. NIELSEN Department of Veterinary Virology and Immunology, The Royal Veterinary and Agricultural University of Copenhagen, Denmark. (Receipted No~emher 19781
Abstract--Two small scale treatment systems were investigated to evaluate their effect on indigenous viruses in wastewater. In the effluent from an emscher tank system, treating a mixture of outlet from toilet, bath and kitchen, virus was demonstrated rather constantly. Samples were taken from a pasteurization plant where outlet from toilets was treated by addition of formaldehyde and exposure to 76°C for 30 min. A few samples from influent were positive, but no virus was found in the effluent. A comparison was made of four methods to detect viruses in wastewater. Alum precipitation followed by tris elution, acidic precipitation followed by elution with beef extract and two-phase concentration all made it possible to isolate indigenous viruses from wastewater and gave comparable results. No virus was demonstrated by direct inoculation. This corresponds well with the findings that the contents of virus was so low as to require concentration to be demonstrable.
INTRODUCTION
Treatment of wastewater can be carried out in several ways. The system chosen will to a large extent depend on the socioeconomic level and the size of the community. For buildings outside urban districts septic and emscher tank systems can be applied. In Denmark 15-20~ of the population use this type of treatment, which has been practised since the beginning of this century. The effluent from such systems is carried off to a seepage area or to a stream. The main purpose of this mechanical treatment is to get a reduction in organic matter, whereas less attention has been given to the content of pathogens. Such considerations are taken in newer developed systems for small communities, especially in systems employing pasteurization. The usefulness of this type of system is however somewhat limited because of high operating expenses. I.t is to be expected, that the reduction of the virus concentration of wastewater in septic tank or emscher tank systems is small, whereas a good reduction presumably will be obtained in a well functioning pasteurization plant. Examination of the effect of these systems on the virus content is difficult because the available methods are not sufficiently quantitative and equally reliable under different conditions. Several methods have been attempted to recover viruses from wastewater. A number of review articles have appeared of these, e.g. Sobsey (1976). Acidic precipitation of the proteins in the wastewater might also trap the viruses and possibly be used as a method of concentration (Lund, 1977). Katzenelson et al. (1976) used this in a secondary concentration after membrane filtration of tapwater. It was found by Lund (1973) that virus might be bound to particles and therefore removal of any part of the sample before a quantitative assay should be
avoided. This was supported by Wellings et al. (1976), who considered the indigenous virus to be both adsorbed to and embedded in the solids. These works throw new light upon earlier methods for virus recovery and show that seeding experiments should be estimated with care. The purpose of this work was to evaluate the efficiency of two small scale treatment systems, a pasteurization and an emscher tank system. Simultaneously a comparison was made of four different methods for the detection of indigenous viruses from wastewater. The methods chosen were direct inoculation, acidic precipitation followed by elution with beef extract, alum precipitation followed by tris-buffer elution, and two-phase separation.
MATERIAL
AND
METHODS
Stud), site
At the Tvind Schools located in the country outside Ulfborg in Jutland wastewater from the 500 inhabitants is treated in two separate small scale systems, each receiving about half of the wastewater. When the schools started in 1972 a traditional emscher tank system (Trix, Nordisk Triclair) was set up to treat the mixed outlet from bath, kitchen and toilet. This system is composed of two separate tanks, both with sedimentation of particles from the sewage in the upper part and a digestion chamber at the bottom, partly separated from the stream of water above. This system is designed to give a 30~o reduction in the B O D before the water is discharged into a brook. In 1975, when the schools were extended, an alternative form of treatment was chosen to purify sewage from the new houses. The toilet outlets from these houses are treated separately. By use of vacuum it is sufficient to use 1 1 of water per flushing. This sewage with high 169
170
B. LYDHOLMand A. L. NIELSEN
amount of total suspended solids is mixed with formaldehyde to give a concentration of 170-200mg/l and is immediately afterwards pasteurized at 76°C for 30 min. The effluent from this system is kept in open basins until land application. Outlet from bathrooms and kitchens is conducted to a seepage area. Rainwater is collected and discharged into the same brook as the emscher tank effluent.
Sample collection Samples of influent and effluent from both the emscher tank system and the pasteurization plant were collected. Samples were also taken from the influent to the seepage area. Samples of raw sewage were collected from the treatment plant at Damhusaaen where part of the domestic and industrial wastewater from Copenhagen is treated. In all cases the volume was I I. Transport of the samples to the laboratory was carried out within 4 h. The samples were left overnight at 4°C and processed the following day.
Virus assay Tube cultures of HeLa cells were grown in Eagles medium with 3~o calf serum, 200 IU benzyl-penicillin per ml and 260 #g streptomycinsulfate per ml. Each tube contained 2 ml of medium, which was renewed twice during the incubation period of 7 days at 37°C. Just before inoculation mycostatin was added to the sample to give a final concentration of 1000 IU per ml. The inoculum was 0.1 ml and each sample was inoculated into four tubes. Three hours after inoculation of the two-phase concentrates and of the direct samples the medium was renewed to avoid cell degeneration because of the harmful effect of these materials. Blind passages were carried out twice on negative cultures (Lund & HedstriSm, 1969). If three or four of the tubes were positive at the primary inoculation serial ten-fold dilutions were carried out in Hanks' balanced salt solution, and each dilution inoculated into three tubes. Virus concentration was calculated according to the 50~o-endpoint method of Reed & Muench (1938). The isolated virus strains were typed by use of neutralization test.
Methods used to isolate virus from wastewater Alum precipitation. 75 mg A!2(SO4)3"18 H20 (Merck) was added to 100 ml of the sample. After shaking for 2 h the mixture was centrifuged at 4080 g for 15 min at 4°C. The precipitate was eluted with tris buffer at pH 9.0 (Lal &Lund, 1975), in the proportion 1:4. After shaking for 2 h the sample was recentrifuged and the eluate neutralized. Acidic precipitation (Lund, 1977). 1 N HCI was added to 200 ml sewage until pH 3.5 was obtained. The sample was mixed by shaking for 1 h and centrifuged at 16,000 g for 30 min at 4°C. The precipitate was mixed with four volumes of a 10~o solution of beef extract (Difco) at pH 7. After mechanical stirring for 2 h centrifugation was repeated and the eluate neutralized. Two-phase concentration (Lund & Hedstri~m, 1966). 20 g of 5 M NaCl, 58 g of 30~ polyethylene glycol (Carbowax 6000), 2.7 g of sodium dextran sulfate (2000 DS, Pharmica) were added to 200 ml sample and pH adjusted to 7.2. After thorough shaking for t h the sample was left overnight at 4°C in a separatory funnel. A bottom-phase and an interphase were collected each giving 2 ml. Direct inoculation. 2 ml wastewater was adjusted to pH 7.2.
RESULTS
In Table 1 a comparison is made between the different methods used to detect viruses in wastewater. The results shown for the two-phase concentration are the values from the bottom-phase isolations as only a few inter-phase samples turned out positive and then in a lower concentration than in the corresponding bottom-phase samples, The two-phase separation then gave a 100-fold concentration based on volume reduction. This factor was used to estimate the virus concentration in the total sample, and cons~uently the lowest detectable virus concentration with this method was 0.05 TCIDso per mi wastewater. Lowest detectable amount per ml for direct inoculation was 4.6 TCIDso. In the two precipitation methods the sensibility of virus detection varies because the quantity of the eluent was dependent on the amount of precipitate. The average amount of total suspended solids in the sewage was 0.2~o for the raw sewage, for the sewage from the vacuum toilets 4Yo and for the emscher tank effluent 0.1~. When direct inoculation was used 33Yo of the samples were harmful to the cell cultures, none of these or any of the other direct inoculated samples gave a positive result in the blind passages. With both acidic Decontamination precipitation and two-phase concentration 5 out of 17 The eluates, the concentrates from the two-phase samples were positive, and with alum precipitation separation and the samples to be inoculated directly virus was detectable in 6 out of 18 samples. A comwere decontaminated by addition of ether in the pro- parison between the methods did not reveal any difportion 1:I (normally 2ml each). After thorough ference in the estimated concentrations of virus. shaking the mixture was allowed to stand for at least Before the examination of the wastewater from the 3 h before most of the ether was pipetted off and the small community at the Tvind Schools two samples of rest was removed by bubbling air through the sample raw wastewater from a municipal treatment plant in for 20 min or more. Copenhagen were tested by means of the different iso-
171
Detection of virus in wastewater Table 1. Comparison of different methods for the isolation of viruses from sewage
Indigenous virus recovered (TCIDs0/ml wastewater) Direct Alum Acidic Two-phase ~ inoculation precipitation precipitation concentration
Type of sample
Date
Raw sewageb Average pH 7.5
6/7/77 7/7/77
0~ 0
0.9 0
0.8 0.8
0 0.3
Influent to the pasteurization plant (before addition of formaldehyde)J Average pH 9.0
28/7/77 19/8/77 31/8/77 14/9/77 20/9/77 27/9/77 4/10/77 31/10/77
0 0 tox t 0 0 0 0 tox
0 0 0 0 0 0 1.3 0
ND ~ 0 0 0 0 0 0 0
1.8 ND 0 0 0 0 0.1 0
Effluent from the emscher tank system~ Average pH 7.5
19/8/77 31/8/77 14/9/77 20/9/77 27/9/77 4/10/77 11/10/77 31/7/77
tox tox 0 tox 0 tox 0 0 0/18
0 0 0.3* 0.6 0.7 0 0 0.2* 6/18
0 0 0 0 0.1 * 0.1 * 0 0.2 5/17
0 0 0 1.8 0 0 0 0.05* 5/17
Samples positive/samples tested
a The value of TCIDs0 per ml sample is calculated on the basis of virus detected in the bottom-phase. b Sewage collected from the influent at the treatment plant at Damhusaaen, located in Copenhagen. c "O" means that no viruses were isolated from the sample by the applied method. For sensibility of detection see the text. d Sewage from two small scale treatment systems at the Tvind Schools. The mean temperature was 13°C in the period of sample collection. ' Not done. f "tox" means that the sample inoculated had a toxic effect on the cell cultures. * Only one out of four inoculated tubes was positive. lation methods. The sewage was expected to contain virus because of the size of the population and the time of the year (July). All the methods except the direct inoculation revealed viruses. The highest concentration detected was 0.9 TCIDs0 per ml. At the Tvind Schools virus was found in 2 out of 8 samples in the influent to the pasteurization plant before the formaldehyde was added. The highest concentration was 1.8 TCIDs0 per ml, estimated from two-phase concentration. Twice it was possible to get samples after addition of formaldehyde, but before pasteurization (4 and 11 October). N o n e of these samples turned out positive, although the corresponding sample without formaldehyde taken on 4 October was positive. On 11 October no sample without formaldehyde was tested. Virus was never detected in the effluent from the pasteurization plant in the examination period (19 August-31 October 1977). In the same way no virus was demonstrated in the influent to the seepage area and to the emscher tank. In the effluent from the emscher tank system virus was isolated 5 out of 8 times in a concentration up to !.8 TCIDs0 per ml. In Table 1 only the wastewater types from which virus was detected are shown. In Table 2 the virus types isolated are given. In the raw municipal sewage only coxsackievirus B5 was
found. This virus was also demonstrated in both systems at the Tvind Schools. Adenovirus 2 was isolated in one sample. This was also the case with echovirus 13, which was found by use of the two-phase concentration method, but only in the interphase. Polioviruses were detected several times after the 20 September in the emscher tank system. DISCUSSION The results of the present report show that the twophase concentration, alum precipitation and acidic precipitation all were applicable for the isolation of viruses from waste-water. The two-phase concentration method has been used in several works, giving good results both on natural and on artificially contaminated samples (Lund & Hcdstr6m, 1966; Shuval et at., 1967; Lal & Lund, 1975). In the present work the method was usable even for the sewage from the pasteurization plant, where the amount of total suspended solids was high. The viruses embedded in the wastewater particles may not have been detected when the two-phase concentration was used, as this method 'does not employ an elution step. Lal & Lund (1975) t6sted the alum precipitation method on artificially contaminated sludge. They obtained a good recovery and a high increase in virus
172
B. LYDHOLMand A. L. NIELSEN Table 2. Virus types isolated from the sewage samples mentioned in Table 1
Type of sample Raw sewage lnfluent to the pasteurization plant (before addition of formaldehyde)
Effluent from the emscher tank system
Date
Direct inoculation
6/7/'77 7/7/77 28/7/77 19/8/77 31/8/77 14/9/77 20/9/77 27/9/77 4/10/77 31i10/77 19/8/77 31/8/77 14/9/77 20/9/77 27/9/77 4/10/77 11/10/77 31/10/77
Alum precipitation
Acidic precipitation
Two-phase concentration
Coxs B5
Coxs B5 Coxs B5
Coxs B5 Coxs B5
~"Echo 13" L Adeno 2
Adeno 2
Coxs B5 Polio 3 Polio 3 Polio 3
Polio 3 Polio 2 Polio 3 Polio 1 Polio 3
Polio 2
" Only isolated from the inter-phase. Coxs = Coxsackievirus concentration and consequently a high degree in sensibility. Although they worked with seeded sewage, their results may be transferred to natural conditions as the viruses embedded in wastewater particles probably will be loosened during several hours' shaking in eluent. In the present work the vigorous stirring for 2 h presumably also had such a loosening effect. The acidic precipitation gave a good recovery of viruses although the concentration might be lower than in the two other concentration methods. Perhaps some inactivation of the viruses occur during the period with low pH. In a single seeding experiment with coxsackievirus B3 added to sewage a 100% recovery was obtained by this method, but the effect of acidification may be different for other virus types. Therefore a shortening of the period with low pH may be recommended. The concentrations estimated by two-phase separation and the precipitation methods were between 0 and 1.8 TCIDs0 per ml wastewater. These values are smaller than the lowest detectable amount for direct inoculation, which is 4.6 TCIDs0 per ml for the used assay system, 0.1 ml inoculated into four tubes per sample. Buras (1974) obtained good results by use of the direct inoculation method on sewage from Israel, which had a higher content of virus (average 1069 PFU per 100 ml) than the Danish sewage tested in the present report. Six samples out of 18 tested were harmful to the cell cultures so even if the concentration in the sewage had been higher this might have interfered with the results. Maybe the viruses embedded in solids were not detected in the direct inoculated samples, as in the case with the two-phase concentration. There seems to be no difference in the virus types
isolated by the different methods except the isolation of echovirus 13 from the inter-phase. The virus assay system used, HeLa cells in tube cultures with one week of incubation and two blind passages, gives a limitation to the types that can be detected. It is possible to isolate adeno-, polio-, echo-, coxsackie B- and a few coxsackie A viruses, but probably not reoviruses. For most coxsackie A types inoculation in new-born mice would be required and the viruses of infectious hepatitis and non-bacterial gastroenteritis cannot be cultivated from wastewater samples. The adenoviruses were detected in blind passages, and would not have appeared if these had not been carried out (Lund & Hedstr6m, 1969). The poliovirus isolated from the emscher tank effluent probably resulted from vaccination of a group of people in the beginning of September. The group belonged to the part of the school connected to the emscher tank system. The treatment system employing pasteurization and addition of formaldehyde is difficult to evaluate because of the few positive samples in the influent. Laboratory studies with artificially contaminated sewage show that the addition of 100mg formaldehyde per 1 at pH from 8 to 10.5 gives at least a 2.4 logt0 reduction in the content of poliovirus type 1 (Sobsey et al., 1974). Foliguet & Doncoeur (1972) obtained a 5 loglo reduction in poliovirus type 1 during heating of raw sludge to 80°C in 10 min. These results indicate that a considerable reduction in the virus concentration will be achieved by addition of 170-200 mg formaldehyde per litre sewage at pH 9 and heating to 76°C for 30 min as used at the Tvind Schools. In the.period of sample collection no positive samples were found in the effluent from the system. The Salmonella content of the wastewater in the open
Detection of virus in wastewater basins after the pasteurization plant has been measured regularly by the municipal laboratory, but has only been demonstrated twice; once because of human errors and once because of overloading. The types isolated were S. indiana, S. montevideo and S. typhimurittm (Faulenborg, 1978). The samples of wastewater from bathroom and kitchen did not contain virus in detectable amounts. Bacteriological examinations have shown that 20-40% of the total amount of fecal colibacteria can be found in wastewater from bath and kitchen (Tullander, 1967). The results of the present work may be due to the low concentration of virus in the sewage. No virus was detected in the emscher tank influent during the sampling period probably because of the heterogenity of the flow and of the composition of the water. The planned time of sedimentation in the tanks was 2 h during which sedimentation took place. Presumably also a homogenization happened from which a liberation of the viruses embedded in fecal matter results, leading to a constant flow of virus from the system. In this water Salmonella was found in three out of five samples; the types isolated were S. heidelberg and S. singapore (Faulenborg, 1978). This way of discharging the effluent must be considered inappropriate especially because of the small flow in the brook. If circumstances (soil structure, groundwater level, available area) allow, it must be recommended to lead the water to a seepage area. Apart from the initial difficulties in the operation of the pasteurization plant the treatment in this system seems to be satisfactory from a virological point of view. As none of the methods used gave 9 positive sampies, which were the total positive number detected, the effectivity of each method was less than 100~o. From the results obtained in the present study none of the concentration methods can be preferred to the others. It cannot be excluded that the same samples tested a number of times employing just one method might have given similar results. Acknowledgements--This work was carried out at the Royal Veterinary and Agricultural University of Copen-
173
hagen, where we were permitted to work during our graduate study of biology at the University of Copenhagen. We wish to express our gratitude to Professor Ebba Lund and her staff at the Department of Veterinary Virology and Immunology for their generous assistance. We also want to thank the Tvind Schools for their kind co-operation.
REFERENCES
Buras N. (1974) Recovery of viruses from wastewater and effluent by direct inoculation method. Water Res. 8, 19-22. Faulenborg G. (1978) Personal communication. Foliguet J. M. & F. Doncoeur. (1972) Inactivation assays of enteroviruses and salmonella in fresh and digested wastewater sludges by pasteurization. Water Res. 6, 1399-1407. Katzenelson E., B. Fattal and T. Hostovesky. (1976) Organic flocculation: an efficient second-step concentration method for the detection of viruses in Tap Water. Applied and Em'ironmental Microbioloqy 32, 638-639. Lal S. M. and E. Lund (1975) Recovery of virus by chemical precipitation followed by elution. Pro#. in Water Technol. 7, 687-693. Lund E. and C. E. Hedstr6m (1966) The use of an aqueous polymer phase system for enterovirus isolations from sewage. Am. J. Epidemiology 84, 287-291. Lund E. and C. E. Hedstr6m. (1969) A study on sampling and isolation methods for detection of virus in sewage. Water Res, 3, 823-832. Lund E. (1973) The effect of pretreatments on the virus contents of sewage samples. Water Res. 7, 873-879. Lund E. (1977) Personal communication. Reed L. J. and H. Muench. (1938) A simple method of estimating fifty percent endpoints. Am. J. Hygiene 27, 493-497. Shuval H. I., S. Cymbalista, B. Fattal and N. Goldblum. (1967) Concentration of enteric viruses in water by hydroextraction and two-phase separation. Transmission of t,irus by the water route. G. Berg (ed.) 45-55. Sobsey M. D., C. Wallis and J. Melnick. (1974) Chemical disinfection of holding-tank sewage. Appl. Microbiol. 28, 861-866. Sobsey M. D. (1976) Methods for detecting enteric viruses in water and wastewater. Viruses in Water. G. Berg (ed.) 89-127. Tullander V. (1967) Hushb.llsavloppsvatten 5. Bakteriologisk Sammens~ittning. Byggforskningen, Informationsblad no. 23. Wellings F. M., A. L. Lewis and C. W. Mountain. (1976) Demonstration of solids-associated virus in wastewater and sludge. Appl. Env. Microbiol. 31,354-358.