Assessing airborne biological hazard from urban wastewater treatment

PII: S0043-1354(99)00264-X

Wat. Res. Vol. 34, No. 4, pp. 1173±1178, 2000 # 2000 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0043-1354/00/$ - see front matter

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ASSESSING AIRBORNE BIOLOGICAL HAZARD FROM URBAN WASTEWATER TREATMENT A. CARDUCCI1*M, E. TOZZI1, E. RUBULOTTA1, B. CASINI1, L. CANTIANI1, E. ROVINI1, M. MUSCILLO2 and R. PACINI3 1 Department of Experimental Pathology, Medical Biotechnologies, Infectious Diseases and Epidemiology, University of Pisa, Via S Zeno 35, I-56127, Pisa, Italy; 2Department of Environmental Hygiene, Instituto Superiore di SanitaÁ, Rome, Italy and 3Leghorn National Health Oce 6, Prevention Department, Biotoxicology Section, Livorno, Italy

(First received 1 August 1998; accepted in revised form 1 May 1999) AbstractÐThe production of microbial aerosols by urban sewage treatment plants may have wide hygienic implications which call for careful evaluation: exposure to such aerosols may in fact represent a health hazard for plant workers and nearby residents alike. This paper describes the results of a study analysing the degree of microbial contamination at di€erent levels of treatment at several plants in the City of Leghorn (Livorno, Italy). Monthly aerosol samples were collected with an agar impact sampler from January to November 1996, from di€erent sites at an activated sludge plant, an anaerobic sludge plant and a wastewater washing station. The total bacterial and coliform counts were determined, and pathogenic enteric bacteria and viruses were determined. These same parameters were also measured in wastewater and sludge samples obtained at the same sites. The results revealed that high-grade airborne contamination existed at several of the studied sites. In particular, pathogenic enteric bacteria (Salmonella enteritidis and S. boydii ) were isolated in 2% of the samples (540 l per sample), reovirus in 46% and enterovirus in 9% (1800 litres per sample in indoor environment and 3000 l outdoor), always in association with the former virus. The biological parameters measured had no evident correlation with meteorological factors such as temperature, relative humidity or wind characteristics. Viral contamination proved to be quite wide-spread and detectable even in the presence of low levels of bacterial contamination. Although virological analysis have been only qualitative, and the di€erent volumes examined for viruses and bacteria cannot allow us to appraise with accuracy the association between these two parameters, the viral presence along with low bacteria contamination suggests more dedicated studies to address with greater accuracy the quantitative aspects of this association. However the monitoring performed allowed for a determination of the areas of greatest potential risk for plant workers, and the preventive measures most suitable to guaranteeing their safety. # 2000 Elsevier Science Ltd. All rights reserved Key wordsÐmicrobial aerosol, viruses, biological hazard, airborne infection

INTRODUCTION

Sewage treatment plants represent an important source of aerosols, particularly those portions containing moving mechanisms and where forced aeration of wastewater is performed (aeration tanks). The aerosols thus formed contain signi®cant quantities of a variety of microorganisms, including, among the predominant saprophytes, pathogens capable of infecting man through inhalation, contact and ingestion. The process of aerosol spread is in¯uenced by a number of atmospheric factors, such as temperature, wind velocity and relative humidity, which interact with each other in various ways depending upon local conditions (Butelli, 1988; Bitton, 1994). The actual risk of infection faced by wastewater *Author to whom all correspondence should be addressed.

treatment plant workers has yet to be properly estimated. A number of authors have reported a particular form of illness, probably viral in origin, that strikes such workers and has accordingly been called ``sewage worker's syndrome'' (Rylander et al., 1976; Clark, 1987; Fannin et al., 1985). It is characterised by general malaise, weakness, acute rhinitis and fever. Other studies have revealed a signi®cant association between exposure to this type of aerosol and the incidence of respiratory and enteric illness, as well as seropositivity in plant workers and residents of neighbouring areas to several viral strains contained in sewage (Clark and Linneman, 1986; Clark, 1987; Proust and Boutin, 1989; Frolich and Zeller, 1993; Heng, 1994). Dispersion of aerosolized enteric viruses and bacteria by sprinkler irrigation with wastewater and possible transmission of infectious agents were also observed (Teltsch et al., 1980; Applebaum et al., 1984; Shuval et al., 1989a,

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1989b). Given the potential health hazard of airborne spread of the products of treatments plants, microbiological studies of such products take on particular signi®cance, especially considering European regulations on safety in the workplace (89/391/CEE; 89/655/CEE; 89/656/CEE; 90/269/ CEE; 90/270/CEE, 90/679/CEE) enacted in the 1994 Italian law 626/94 (Decreto Legislativo). In this perspective, thorough investigation of the risk of infection associated with such plants must include, ®rst a determination of the main sources of aerosol, and second a careful assessment of their potential for spreading disease, both in quantitative and qualitative terms on the basis of the pathogens isolated. To this end, microbial air contamination was monitored in order to obtain measures of both bacteriological (total bacteria count, total coliform and enteric pathogen counts), and virological (cytopathic viruses) parameters for the various components (collection, treatment and disposal) of the sewage treatment system serving the city of Leghorn (Italy), with particular attention to those areas most frequented by plant personnel. MATERIALS AND METHODS

During the course of this one-year study the following facilities were monitored: (1) an activated sludge plant with air supply oxidation; (2) an anaerobic sludge treatment plant; (3) a sewage washing station located along the collecting system. Sampling A ®rst screening was carried out in order to determine the actual sites to be monitored during the study. Thirtyfour di€erent locations were screened by means of sedimentation on open Petri dishes method (30 min exposure at about 50 cm from ground level). This method, although not quantitative and detecting no real air-borne aerosols,

but mainly settleble particles, was used for a ®rst screening and for the choice of the sites to analyse because it was easy to set-up and cost e€ective; it also allowed a high density of sites and simultaneous sampling. The 12 sites yielding the most signi®cant results were chosen, as follows: 1. At the activated sludge plant (all aerosol samples outdoors) 1.1. The primary screen (site 1) at a series of three points separated by a variable distance from the tank: one at 10 m near the entrance gate (site 1A); one at about 20 m, in the vicinity of the Archimedes' screw conveyer (site 1B), and the last at about 40 m, in an elevated position at the entrance to the laboratories (site 1C) 1.2. The secondary screen (site 2) and corresponding area of waste expression and collection (site 2A) 1.3. The grit chamber (site 3) 1.4. The aeration tank (site 4) 2. At the anaerobic sludge treatment plant (all aerosol samples indoors) 2.1. The nearby belt ®lter presses 2.2. The drier outlet 3. The sewage washing station (all aerosol samples indoors). The waste entering the primary screen, and the sludge exiting the belt ®lter presses were analysed at the same time as the aerosol. Aerosol samples were obtained monthly from January until November, 1996, using an SAS agarized terrain impactor samples (Surface Air System Ð PBI). This method is easy and manageable, although the low suction time required for viruses sampling could cause a high risk of drying up. Di€erent air volumes were collected as required by the parameter to be analysed: for bacteria we collected the maximum volume allowing plate count without overcrowding, all counts were normalised to a 1 m3 volume. Then for each sample were collected 180 litres for total bacteria count and 540 l for coliform and pathogenic bacteria counts (Table 1). For viruses we aspired 1800 l in indoor environment and 3000 l outdoor, to take in account the di€erent dilution of viral particles and the results of previous studies (Carducci et al., 1995b).

Table 1. Mean bacterial concentration and presence of pathogenic bacteria and virus at the various sites studied (CFU, colonies forming units; nd, not determined) Samplea

Activated sludge Aerosol: plantb Aerosol: Aerosol: Aerosol: Aerosol: Aerosol: Aerosol: Aerosol: Sludge treatment Aerosol: c plant Aerosol: Washing station Aerosol a

Coliform MPN of Samples Samples Number Bacterial count pathogenic positive count positive of b samples (CFU/mc) (CFU/mc) for pathogenic bacteria for virus, % per litre Mean log Mean log bacteria, %

primary grill

11

563

52

9

10 m from primary grill screw conveyor 40 m from primary grill secondary grill waste expression sand collection tank oxidation tank belt ®lter presses

7 11 7 11 10 10 6 11

48 4303 37 554 38 434 19 88

1 275 1 88 6 46 2 1

± ± ± ± ± 10 ± ±

sludge discharge

10 10

78 2566

1 1168

± ±

17.4  10ÿ3

19.5  10ÿ3

55

MPN of virus per litre 4.51  10ÿ4

nd 45 nd 45 nd 50 nd 45

20.2  10ÿ3

40 60

17.2  10ÿ3 31.6  10ÿ3

1.07  10ÿ4 1.07  10ÿ4 3.92  10ÿ3

Collected air (l): 180 for bacterial count, 540 for coliform and pathogenic bacterial count, for viruses 1800 in indoor environment and 3000 outdoor. b Wastewater primary grill: 11 samples, coliform count (mean log) 279 CFU  105/g, 27% of samples positive for pathogenic bacteria, 55% of samples positive for virus. c Sludge originated by belt ®lter presses: 11 samples, coliform count (mean log) 53 CFU  105/g, 9% of samples positive for pathogenic bacteria, 9% of samples positive for virus.

Assessing airborne biological hazard

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Sewage analysis

Statistical analyses

Bacteriological parameters. Sewage was collected in sterile 400 ml glass bottles and kept refrigerated up to the time of analysis. The total coliform and faecal coliform counts were evaluated by means of the membrane ®lter method utilising, respectively, M-endo agar LES incubated at 378C for 48 h and MFC agar (Difco) incubated at 428C for 48 h. Salmonellae were also cultured by enrichment in Selenite Broth (Difco) and subsequent isolation on XLD agar (Xylose, Lactose Deoxycolate, Difco) at 378C for 24 h. Suspicious colonies were con®rmed through biochemical and serological tests. Virological parameters. Virus isolation was performed by ®rst decontaminating the sewage (50 ml) with chloroform (Carducci et al., 1997) and subsequent seeding onto BGM cells (Bu€alo Green Monkey). A minimum of three subcultures was performed before the presence of viral agents were excluded or con®rmed.

In order to analyse any possible e€ects of weather conditions on the values of the bacteriological parameters detected at the various sampling sites (bacterial and coliform counts), simple linear regression analyses were performed. Any correlations between the presence of enteric virus and bacterial parameters were assessed by means of the Student t test applied to the di€erences in bacterial counts between samples resulting positive for viral presence and those testing negative. As in assessing risk, the concentration of pathogens is more important than the percentages of positive or negative samples, we estimated them according to the Thomas MPN equation (American Public Health Association, 1995) (Table 1).

Sludge analyses

Table 1 presents the mean values of the bacterial and coliform counts and the presence of bacterial pathogens and viruses detected in aerosol samples. At the level of the primary screen, the results con®rm considerable airborne microbial pollution, with bacterial pathogens found in 9% of samples from this site and viruses in 55%. Such values are in good agreement, especially with regard to viral contamination, with those relative to the sewage samples from the same area (27% positive for pathogenic bacteria, 55% for virus). At the site of the Archimedes' screw conveyor (site 1B), high total bacteria and coliform counts were observed, as well as the wide-spread presence of virus (45% of samples), though pathogenic bacteria were absent. In the areas neighbouring site 1 (1A and 1C), on the other hand, the detected level of microbial airborne spread was much lower. The areas of the secondary screen (site 2) and grit chamber (site 3) resulted instead to be highly contaminated with bacteria, and contained elevated levels of virus (45% of samples) and pathogenic bacterial species (10% at site 3). On the other hand, the refuse expression area (site 2A) and the area near the aeration tank (site 4) showed only low-grade microbial dispersion. The sludge treatment plant yielded intermediate levels of airborne bacterial pollution: virus was found in 45% of the aerosols sampled near the belt ®lter presses and 40% of those from the sludge discharge duct, which represents levels higher than those found in the sludge itself (9%). Lastly, the washing station consistently yielded the highest values of both total bacterial and coliform count, as well as airborne virus (60%). Using the Thomas MPN equation, the highest viral concentrations estimated have been observed at the sludge treatment plant and washing station. Despite the signi®cant variability over time of the two bacterial parameters in the various sites, we were unable to determine any clear seasonal trend or correlation to meteorological measures (temperature, relative humidity and wind parameters). Even at the most contaminated points, enteric pathogens

Bacteriological parameters. Stabilised, dried sludge samples (10 g) were collected in sterile plastic bags, then diluted 1:10 in distilled water to prevent clogging the ®lters, and homogenised. Total coliform and faecal coliform concentrations were then determined by means of the membrane ®lter method. Salmonellae were cultured as described previously for sewage. Virological parameters. Sludge samples (50 g) were eluted with 3% beef extract pH 9, and the eluate concentrated by means of tangential ¯ow ultra®ltration (Carducci et al., 1997). Samples were then decontaminated through chloroform treatment and seeded onto BGM cells for at least three culturings. Aerosol analyses Bacteriological parameters. For each sampling site determination was made of the total bacterial count, utilising Rodac plates (International Pool Bioanalysis Italiana SpA, PBI) (Borioli et al., 1988) indicated by the manufacturer as suitable for Surface Air System (SAS, PBI) containing PCA (Plate Count Agar, Difco), and the total coliform count, with Mac Conkey agar plates (Difco). Culturing for enteric bacteria was performed using Rodac plates containing XLD agar. Subcultures of the colonies developing on this medium were then prepared on selective medium in order to characterise the prevailing microbial species and isolate any pathogenic enteric bacteria, speci®cally, shighella and salmonella. Virological parameters. Viral isolation was conducted using Rodac plates containing AT (Agar Tryptose, Difco). After sampling, the agar was minced with the aid of a scalpel and eluted in 3% beef extract at pH 9. In previous experimental trials, the percentage of virus recovery in agarized means was of 40.3% (Carducci et al., 1995b). The eluate was then decontaminated with chloroform and seeded onto BGM cell cultures for at least three culturing to con®rm a cytopathic e€ect. Identi®cation of isolated viral agents Identi®cation of the cytopathic agents isolated was performed utilising the RT-PCR (Reverse TranscriptasePolymerase Chain Reaction) for enterovirus, and the PAGE (Polyacrylamide Gel Electrophoresis) for reoviruses, following the procedure set forth in previous studies (Carducci et al., 1997). Meteorological parameters Weather parameters (wind intensity and direction, temperature and relative humidity) were provided by the Association for Detection of Atmospheric Pollution, the Leghorn±Collesalvetti chapter of an environmental monitoring network.

RESULTS

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A. Carducci et al. Table 2. Pathogen bacteria and viruses isolated by the sludge, mud and aerosol collected from the examined plantsa Mud

Sludge

Months

Primary grill Belt ®lter press

January February March April May June July August September October November

E+R ± ± ± ± E+R R S. blockley R+S. derby ± R+S. derby ± R+S. agona ± R ± ± ± ± ± ± ±

Aerosol Point 1

Aerosol

Point 1B Point 2 Point 3 Belt ®lter press Desiccator barrier Generating station

E+R E+R ± ± R R ± E+R R+S. enteritidis R R ± R ± R R ± ± ± ± ± ±

± ± R R R ± R R ± ± ±

S. boydii ± E+R R R ± R R ± nd ±

E+R R ± ± R R ± R ± ± ±

± E+R ± ± ± R R R ± ± nd

nd E+R ± ± R R R R ± ± R

a

E, enterovirus; R, reovirus; nd, not determined.

were rarely isolated from aerosol samples (only S. enteritidis and S. boydii ), despite their high incidence in the sewage (Table 2). The ®ndings relative to viral parameters were instead much more consistent, especially with regard to the presence of reoviruses, isolated in 46% of samples, while enterovirus was encountered in only 9%, and always in conjunction with the former. No correlation was found between bacterial parameters and the presence of cytopathic virus, in general, or reovirus or enterovirus, in particular. Overall, the microbiological monitoring performed has enabled us to construct a map of the potential biological hazard at each of the studied facilities (Fig. 1) and thereby account for each of the three experimental parameters (total bacterial and coliform counts and presence of pathogenic bacteria and/or virus). DISCUSSION AND CONCLUSIONS

The plants monitored in the present study have proved to be signi®cant sources of aerosol microbes. The highest levels of contamination, in both quantitative and qualitative terms, were found at the washing station. As such facilities have never been investigated in previous studies, they must be regarded as posing a high risk to the personnel responsible for their maintenance and inspection. Moreover, areas in the vicinity of moving mechanical equipment (primary and secondary screen and screw conveyors) or machinery performing wastewater aeration (grit chamber) yielded mid-tohigh concentrations of bacteria, while the areas surrounding the aeration tanks did not appear to be highly contaminated, in contrast to results from previous studies (Delia et al., 1984; Fannin et al., 1985; Butelli, 1988; Colombi et al., 1991a; Carducci et al., 1995a). Such a discrepancy is likely due to the di€erent processes employed for wastewater aeration: in the present study the aeration was performed by air bubbles under pressure, instead of surface aerators.

In the sludge treatment plant the average bacterial and coliform counts revealed in the belt ®lter presses area were lower than those reported in the literature (Colombi et al., 1991a; Garzaroli et al., 1995). However, the high rate of viral isolation in this area demonstrated a considerable level of contamination likely due to the greater resistance of virus to drying as compared to bacteria (Bitton, 1994). Most of the microorganisms isolated from aerosol samples from the various plant sites examined were non-pathogenic intestinal species and saprophytes. Nevertheless, enteric pathogens, such as S. enteritidis and Shigella boydii were not altogether lacking. Although such species are not transmitted to man by air, the potential of aerosol to contaminate the environment cannot be ignored, as it may be deposited on masonry structures, maintenance equipment, balustrades and the like, thus determining a potential risk of exposing personnel to contagion by contact or ingestion. Moreover, the aerosols were frequently found to contain enteric virus. Among these, isolated enteroviruses of human origin represent a signi®cant health hazard, as they can be transmitted by the respiratory route and are known to cause often-serious extra-intestinal pathologies (e.g. Coxsackievirus B, myocarditis or meningitis) (Davis et al., 1993). However, ®ndings of such viruses were rather rare and in all cases linked to the presence of reovirus of human or animal origin, a very wide-spread, resistant specie, which has yet to be conclusively linked to any human pathology other than neonatal biliary atresia (Craven, 1991; Dessanti et al., 1997). The diculties inherent in virological analyses could be overcome, at least in part, by selecting the reoviruses alone as indicators of virological risk, and testing for their presence with appropriate cell cultures and particularly sensitive biomolecular techniques such as PCR (Patti et al., 1997). Although virological analysis have been only qualitative, and the di€erent volumes examined for viruses and bacteria cannot allow us to appraise

Assessing airborne biological hazard

Fig. 1. Map of biological contamination present at each of the studied points.

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with accuracy the association between this two parameters, the viral presence with low bacteria contamination suggests to deepen this aspect with dedicated studies that will address with bigger accuracy the quantitative analysis. The risk of viral infection for employees of sewage treatment plants should not be overlooked, if we should consider that an individual with normal breathing capacity, inhaling 7.5 litres per minutes, in an environment with one single virus per 3 m3 of air could come in contact with two viral particles in 8 h of work (Ward and Akin, 1984) AcknowledgementsÐWe would like to express our gratitude to the Leghorn Azienda Servizi Ambientali (Environmental Services Agency) whose funding and collaboration have made this work possible, and in particular to the agency's Technical Director Eng. Caturegli. We also gratefully acknowledge the invaluable contribution of Dr Freedman to realising this study and Association for Detection of Atmospheric Pollution (Leghorn± Collesalvetti) REFERENCES

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