ENTERIC VIRUSES IN A WASTEWATER TREATMENT PLANT IN ROME E A. AULICINO, A. MASTRANTONIO, E ORSINI, C. BELLUCCI, M. MUSCILLO and G. LAROSA Istituto Superiore di Sanit& Roma, Laboratorio di Igiene Ambientale (Received 13 July, 1994; accepted 13 July, 1995)

Abstract, Over the course of a year a series of samples were taken from a wastewater treatment plant handling domestic sewage at each stages of the process, to detect the presence of enteric viral and classical bacterial indicators, and physicochemical parameters. The viruses were isolated on BGM cell cultures and counted according to the Most Probable Number method. The values of enteric viruses varied from 102 to 104/L in raw sewage and from 10° to 103 in final effluent. The efficiency of the plant at each stages during processing was evaluated. The parameters analysed show a systematic reduction of values between input and output, with average bacteriological reductions of 88% (fecal streptococci), 93% (fecal coliforms) and 94% (total coliforms), viral load reduced by 0-99%. COD and suspended solids showed a reduction of 61% and 71% respectively. The 40% of isolated viruses were submitted to identification procedures using molecular techniques and pools of antisera. The viral types identified were enteroviruses (poliovirus and coxsackievirus B) and reoviruses. Viruses appear less easily removed than classical bacterial indicators. Reoviruses were removed less efficiently than enteroviruses.

Key words: enteric viruses, domestic sewages, enteroviruses, reoviruses, waste water treatment, raw and treated sewages

1. Introduction Domestic sewage contains an unhealthy mix of both harmless and infectious microorganisms such as enteric viruses, and represents the major pollution source of rivers, lakes, seas, soils, etc. The presence of enteric viruses in surface and ground waters and in soils contaminated by effluents may be considered a potential public health hazard since the viruses are a source of contamination for human beings. The study of the enterovirus load in biological treatment plants can be important in evaluating the size of the viral presence in effluents and consequently in establishing sanitary impact on receiving water bodies with a view to improving the their quality. In recent several studies on surface water virological quality have been carried out on the river and coastal waters of Italy. The results showed widespread virological pollution in the coastal areas of the Tyrrhenian and Adriatic seas, with isolation of enteroviruses and reoviruses, and in the Tiber fiver waters, where Hepatitis A Water, Air, and Soil Pollution 91: 327-334, 1996. (E) 1996 Kluwer Academic Publishers. Printed in the Netherlands.

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virus was also isolated (Aulicino et al., 1992; Morace et al., 1993; Muscillo et al., 1994; Patti et al. in press). Very few investigations on the quality of wastewater in terms of its enteric virus content have been carried out in Italy, whereas there have been several studies from other countries providing such information (Buras, 1974; Irving et al., 1981; Dahling et al, 1989; Morris et al., 1984 a, b; Payment et al., 1986; Schwartzbroad et al., 1984; Smith et al., 1982; Krikelis et al., 1985). In some studies virus levels in raw sewages and in effluents have been reported as high as 105 particles/L and 104/L respectively. This study was carried out: 1) to evaluate the extent of viral pollution in wastewater and in final effluent of a wastewater treatment plant that collects parts of sewage of a big city, 2) to evaluate the trend of bacteriological, virological and physicochemical parameters at different stages of treatment. 2. Materials and Methods 2.1.

SAMPLES AND SAMPLING PROCEDURE

The study was carried out from March 1992 to February 1993 in a activated sludge treatment plant serving a population of 400,000 in the east of Rome. This plant receives about 2.57 m3/s of sewage from two incoming flows and the estimated average transit time is 9 h. Processing includes a pretreatment (screening and grit separation), a primary treatment (4 settling tanks of 3,780 m 3 each one) and a secondary treatment by activated sludge (4 aeration-activated sludge basins). All raw sewage in this plant undergoes primary treatment (2.57 m3/s), but, because the aeration-activated basins can only process primary sewage up to 1,5 m3/s, secondary treatment is applied only to this fraction of the primary sewage. Consequently final effluent consists of about 40% raw sewage processed by only primary treatment (physicochemical process) and 60% sewage processed also by secondary treatment (activated sludge treatment). From June to September, terminal chlorination with sodium hypochlorite was carried out on effluent. After 35 rain of contact, chlorine levels in final effluent had usually fallen to 0-0.22 mg/L. The final effluent is discharged to a fiver that flows into the Tiber. Samples (from 0.1 to 10 L) of raw sewage, primary effluent, secondary effluent and final effluent were collected every month, taken from a 24 hour composite sample, made up of individual samples collected every 3 h. Sodium thiosulphate solution (10%) was added to chlorinated samples (0.1/100 mL of sample). All samples were transported immediately to the laboratory, stored at 4-8°C and submitted to analysis within 24 hours of collection.

ENTERIC VIRUSES IN A WASTEWATER TREATMENT PLANT IN ROME

2.2.

329

VIRAL, BACTERIAL AND PHYSICOCHEMICAL ANALYSES

Total and fecal coliforms, fecal streptococci, COD, suspended solids and residual chlorine analyses were performed according to the Standard Methods (APHA, 1992). Enteric viruses were concentrated by the adsorption-elution technique. From 100 mL to 10 L of samples at pH 6.5-7.5 were filtered across 2 electropositive membranes (Virosorb 1 MDS-AMF Comp., Cuno Division, Meriden, U.S.A.) (Chang et al, 1981; Dahling et al., 1989). 1 and 10 L samples were filtered accross membranes of 142 mm, whereas 47 mm diameter membranes were used to concentrate the 100 mL samples (APHA, 1992). The 142 and 47 mm filters were eluted with 70-80 mL and 10-15 mL respectively of 3% beef extract solution at pH 9 (Lab-Lemco powder L29, Oxoid). The elution step was carried out three times, very slowly. Because the filters occlusion sample volumes over 1 L or samples with high levels of turbidity were clarified by centrifugation at 6,000 rpm for 30'. The pellet was magnetically stirred for 30' in 3% beef extract solution at pH 9 (1:4) to elute virus particles, and submitted to centrifugation at 6,000 rpm for 30'. The supematant was neutralized with a solution ofHC1 (1 mol/L) and added to the clarified sample, which was filtered as described above. Decontamination was performed by shaking eluates for 30' with 3/10 of volume of sample of chloroform, removing the chloroform phase, adding 1/10 of volume of sample of an antibiotics and fungicide mix, and incubating samples for two hours at 37°C (APHA, 1992). An additional treatment with chloroform (10%) was necessary because of the persistent presence of contaminants (overall protozoa). The enteric viruses were enumerated following the method of Most Probable Number of Cytopathogenic Units (MPNCU) through inoculation onto BGM (Buffalo Green Monkey) cell monolayers, adsorption for 2 hours at 37°C, removal of the samples and overlaying with MEM (Minimum Esssential Medium) Earle's salts with 2% fetal calf serum (Gibco). 75 cm 2 and 25 cm 2 plastic flasks were inoculated with 10, 1 and 0.1 mL and, if necessary, 0.01 and 0.001 mL of eluates (Lucena E et al., 1985). Cell monolayers were examined every three days for 28 days for appearance of cytopathic effect. Three successive passages were performed. The MPNCU L-1 was calculated using Standard Method MPN tables (APHA, 1992). The presumptive diagnosis of isolated strains was made on the basis of the appearance of cytopathic effect and on the time necessary to destroy cell monolayers. About 40% of the isolated strains were submitted to identification tests. Enteroviruses were typed by serum neutralization tests using pools of antisera obtained from the Dutch National Institute of Public Health and Environmental Protection (Bilthoven, The Netherlands). Reovirus diagnosis was confirmed submitting dsRNA extracted from cell lysates to polyacrilamide gel electrophoresis (PAGE), using the methods set out in Sambrook et al. (1989) with some modifica-

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Table I Average values for selected microbiological and physicochemical water characteristics at each stage

of treatment Parameters

Total coliforms lgl0/100 mL Fecal coliforms lglo/100 mL Fecal streptococci lgt0/100 mL COD

mg/L Total solids mg/t,

Raw

Primary sewage

mean

% mean removal

Secondary sewage

mean

% removal

Final effluenta

mean

% removal

7.2

6.8

63

5.8

96

6.0

94

6.7

6.6

35

5.04

98

5.7

93

6.1

6.1

-8

5.04

92

5.1

88

252

152

42

36

88

104

61

199

89

58

18

91

62

71

a The effluent consists of 60% secondary sewage and 40% primary sewage

tions (Muscillo et al., 1994). After the electrophoresis, segments of dsRNA were made visible through silver staining (Dolan et al., 1985).

3. Results Table I shows the average values for bacteriological and physicochemical parameters after different stages of treatment. Table II shows the results referred to viruses. Because raw and treated sewages samples n ° 6, 7 and 8 were assayed in quantity of 0.1 L the results referred to these samples are reported as MPN/0.1 L. The results in table I show that this plant produced an overall bacterial reduction between 88% and 94%. The chemical oxygen demand and the total suspended solids were reduced by 61% and 71%, respectively. The primary sedimentation process registered average percentages of reduction of 35% and 63% for total coliforms and fecal coliforms respectively. Fecal streptococci increased by 8%. The biological oxidation treatment process (secondary sedimentation) was the most effective of the treatments. At the end of this step, percentages of removal for bacteriological parameters were from 92 to 98%. COD and total solids showed removal percentages of 88% and 91% respectively. The final effluent removal percentages were lower than the ones for secondary effluent, as a consequence of the fraction of primary sewage not treated by the secondary process, carried out to the outlet. There were no substantial differences in bacteria reduction in chlorinated and non-chlorinated effluent, as a result of the low level of chlorine and the high level

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F. A. AULICINOET AL. Table III Percentages of positive samples for enteroviruses and reoviruses % positive samples

Sample

Enterovimses Reoviruses

Raw sewage Primary sewage Secondary sewage Final effluent

83 (10/12) 75 (6/8) 27 (3/11) 54 (6/11)

100 (12/12) 100 (8/8) 100 (11/11) 100 (11/11)

In parentheses are reported the numbers of the positive samples for enteroviruses and reoviruses out of the number of total positive samples.

of bacteria. Viruses generally seemed to decrease in chlorinated effluent in respect with non-chlorinated ones (Table II). Viral density in raw sewage entering the plant ranged from 1 x 102 to 1 x 104 MPNCU/L and all the samples contained viruses (Tables II and III). After primary settling and secondary treatment viral titres were reduced to 1 x 10 - 1 x 103 and 1 x 10 ° - 1 x 102 MPNCU/L respectively, and all the samples still contained viruses. In final effluent viruses were reduced by 30-99,5%. Viral titres in these samples generally increased with respect to those of the secondary effluent (Table II). Viral absence was registered in one chlorinated sample out of 12 total samples. It is not sure that this sample is really free of viruses, because may be that insufficient sample quantity has been assayed. A total of 290 viral isolations were made during this study: 75 enteroviruses and 215 reoviruses. The detection of reoviruses was dependent on the observation of cell cultures over a long period. Enterovimses showed cytopathic effect, with 80% of the cell layer destroyed in 4-8 days after inoculation, while most reoviruses were detected after 10 to 25 days of incubation. Identification tests, carried out on about 50% of presumed enteroviruses (39/75 strains) and on 40% of presumed reoviruses (80/215 strains), confirmed the diagnosis for all strains of reovirus and for most of the strains of enteroviruses. Poliovirus types 1,2,3 and coxsackievirus B made up 20.5% (8/39) and 58.9% (23/39), respectively, of the presumptive enteroviruses isolated. 13% of the isolated strains showed the presence of both polio and coxsackievirus (5/39), while 7.6% remained untyped (3/39 strains). Echoviruses were not isolated, but BGM is, in any case, not generally considered a reliable host system for echovirus detection in environmental samples (Schmidt et al., 1978). Reoviruses were isolated in 100% of positive samples (Table III). The percentage of enteroviruses positive samples was 83% in raw sewage and fell to 54% in final

ENTERICVIRUSESINA WASTEWATERTREATMENTPLANTIN ROME

333

effluent. Enteroviruses were isolated with lower frequency than reoviruses at all steps of the treatment, but mainly at secondary treatment (Table III).

4. Discussion

This treatment plant showed an overall performance lower than the performance of the activated sludges secondary process, both in terms of microbiological parameters and of physicochemical ones. The quality of final effluent worsened with respect to that of the secondary sewage since the final effluent was composed of sewage treated only by the primary process (40%) and, cleaner, secondary sewage (60%). The percentage of overall bacterial reduction was high, but the quantity of these indicators of fecal contamination was also high in the final effluent (Table I). The primary treatment showed the lowest removal efficiency for all the parameters considered. During this study, in September and in October fecal streptococci titres increased after primary sedimentation. We also found increases in fecal coliforms and in enteric viruses at this stage (in enteric viruses also at the final stage) during the sampling in December (sample No. 10 - Table II). Primary treatment is not designed to improve microbiological quality but is mainly for the removal of settleable suspended solids. An increased number of bacteria can be a consequence of the disagregation of fecal clumps or of a regrowth. In a previous study in the same plant increments of coliforms and fecal streptococci were observed at this step of treatment (Volterra et al., 1983). The increase of viral particles, which should be removed with solids, is surprising. They may be eluated and freed from solids as a consequence of particular conditions such as pH variations or the breakup of solids. The viral levels found at each step of treatment and in the final effluent are similar to values reported by some authors (Buras, 1974; Dahiling et al., 1989; Krikelis et al., 1985, Irving et al., 1981; Morris, a-b 1984), but higher than those reported by others (Payment et al., 1986; Schwartzbroad et al., 1985; Smith et al., 1982). Most samples treated by centrifugation+filtration showed lower levels of viral particles than samples concentrated by filtration alone (Table II). The centrifugation pretreatment could cause viral losses, because many viral particles could have been not detached from suspended solids or could have been inactivated by the additional pH change. Viruses appear less easily removed than coliforms and fecal streptococci. Bacteria as cellular organisms are more subject to environmental stresses and can decay more rapidly than acellular organisms such as viruses. Reoviruses were less efficiently removed than enteroviruses (Table III). The different processes of the treatment plant, include sedimentation, are probably

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less effective in removing reoviruses, because these viruses could be less easily adsorbed onto solids than are enteroviruses. Reoviruses were found to be more numerous than enteroviruses, nevertheless they are understimated. Enteroviruses showed cytopathic effect in flasks with higher quantities of samples, reoviruses in ones with lower quantifies, when the two strains of virus were both present in the same sample. We assume that the rapidly cytopathic enteroviruses destroyed the cell monolayer so early that the cells were not been available for reovirus isolation. Despite these disadvantages, we think that the method followed in this study was useful, because using a simple virus concentration technique, only one cell line, the liquid overlay assay and observations of cell cultures for a long period we detected both enteroviruses and slowly growing reoviruses.

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