Food Environ Virol (2013) 5:69–76 DOI 10.1007/s12560-013-9105-5

ORIGINAL PAPER

Effects of Bacterial, Chemical, Physical and Meteorological Variables on Virus Removal by a Wastewater Treatment Plant A. Carducci • M. Verani

Received: 19 July 2012 / Accepted: 31 January 2013 / Published online: 13 February 2013  Springer Science+Business Media New York 2013

Abstract The purpose of wastewater treatment is to minimize chemical and microbial contamination of recipient waters. The present study evaluated the impacts of meteorological variables, such as temperature and rainfall, on the removal of human viruses and indicators by a wastewater treatment plant servicing Pisa, Italy. Data were obtained during four sampling campaigns from 2007 to 2010. Wastewater sewage samples were analyzed for human adenovirus (HAdV) and norovirus using quantitative molecular techniques. In parallel, Escherichia coli, enterococci and somatic coliphages were measured, and meteorological and chemical data were recorded. We detected a continuous presence of HAdV in both influent and effluent samples with an average removal rate of 2.01 log10 Genomic Copies/l. An association between meteorological parameters and viral removal rates was detected only for rainfall and HAdV removal during a specific sampling campaign. No correlation was found between viral data and microbial, chemical and physical ones. Viral removal rates were not strongly influenced by meteorological conditions and were unrelated to other process indicators routinely monitored. Our results suggest that HAdV is a suitable parameter to assess the viral removal efficiency of wastewater treatment plants, particularly in the case of heavy rainfall. Keywords Wastewater treatment  Viral survival  Human adenovirus  Norovirus

A. Carducci (&)  M. Verani Laboratory of Hygiene and Environmental Virology, Department of Biology, University of Pisa, Via S. Zeno 35/39, 56127 Pisa, Italy e-mail: [email protected]

Introduction Wastewater treatment plants (WWTPs) function to reduce the environmental and health impacts of chemicals and biological agents discharged into water. Urban sewage contains high concentrations of enteric bacteria and viruses, i.e. * 106–107 colony forming unit/ml (CFU/ml) of Escherichia coli and 106 genome copies/ml (GC) of adenovirus (AdV) (Carducci et al. 2008, Petrinca et al. 2009). The effluents of WWTPs receiving urban sewage may be discharged into surface or sea waters or may be reclaimed for industrial or agricultural purposes. WWTPs may become a source of contamination if they dysfunction, for instance, by overflowing with partially treated wastewater during heavy rainfall or when an increase in sewage flow is coupled with a reduction in transit time in the aeration tank (Rose et al. 2001). Moreover, temperature influences population dynamics in the aeration tank (Bitton 2011), thereby affecting the activated sludge ecosystem. The microbial removal rate of WWTPs is generally assessed using faecal bacteria (e.g. E. coli and enterococci) as indicators; however, viruses and protozoa are more resistant to removal by sewage treatment (Carducci et al. 2006, 2008; Wen et al. 2009; Petrinca et al. 2009). Numerous types of viruses are transmitted via the faecal– oral route and subsequently can contaminate water (Muscillo et al. 2001). The most actively studied of these are AdV and norovirus (NoV) (Wyn Jones et al. 2011; Rodrı`guez et al. 2012). AdV strains are highly resistant to natural and artificial disinfection processes and procedures (Battistini et al. 2013), can be detected readily by molecular techniques (e.g. polymerase chain reaction- PCR), and are generally cultivable in cell culture. For these reasons, AdV has been proposed as a viral indicator (Sinclair et al. 2009; Silva et al. 2011). NoV is a

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primary causative agent of viral gastroenteritis in people of all age groups. NoV has a positive-sense RNA genome that can be detected in waters by reverse-transcription (RT)–PCR (Bosch et al. 2011). The effects of meteorological events on pathogen reduction by WWTPs have been difficult to evaluate because of the myriad variables involved, including epidemiological dynamics, seasonal cycles of some pathogens, geographic areas, and WWTP technology. Nevertheless, the examination of associations between climatic factors and pathogen removal by WWTPs can be very important in defining the interventions and strategies needed to protect the public health. This is particularly relevant today as climate change increases the frequency of heavy rainfall and hence the frequency of water-borne outbreaks that correlate significantly with heavy precipitation (Curriero et al. 2001). The WWTP servicing the 40,000 inhabitant equivalents of Pisa, Italy, has been monitored regularly since 2006 to evaluate the presence of several human viruses (human AdV—HAdV, hepatitis A virus, human rotavirus, NoV genogroups I and II, and enterovirus) as well as somatic coliphages and indicator bacteria (E. coli and enterococci) (Carducci et al. 2006, 2008, 2009; Verani et al. 2006). In addition to the viral presence, viral load has been measured since 2007 for HAdV and since 2009 for NoV. In parallel, physical and chemical parameters representative of routine functioning of the WWTP have been monitored and recorded as requested by a European Union (EU) Directive (91/271/EEC). In the present study, data from the WWTP were analyzed to detect possible correlations among the various parameters, to determine the relative impact of meteorological variables (temperature and rain), and to assess whether the microbial, physical, and chemical indicators commonly employed were representative of viral removal.

Materials and Methods Sampling Influent and effluent samples were collected from a WWTP servicing Pisa, Italy, where the sewer system is still partially combined. A bypass in case of overflow is included in this system to minimize input flow fluctuations. Sewage is treated by activated sludge, chlorinated, and discharged without reuse into a river that flows into the Mediterranean Sea. The WWTP was monitored routinely for the presence of several human viruses in inflow and outflow waters, primarily for surveillance purposes. Sampling periods were chosen according to previous clinical and environmental data (Carducci et al. 2006). Particular attention was paid to

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winter and spring months, which are characterized by higher circulating gastroenteritis viruses and by greater meteorological variability. Only in the last campaign late summer and autumn months also were considered. We conducted monitoring campaigns from 2007 to 2010, as follows: • • • •

Campaign Campaign Campaign Campaign

1: 2: 3: 4:

March 2007–June 2007 November 2007–April 2008 February 2009–March 2009 September 2009–June 2010

Sample volumes were collected bimonthly at the entry (1 l) and the exit (10 l) of the WWTP and were used to quantify HAdV by quantitative (q) PCR for all the sampling campaigns. NoV GI and GII were detected by RT– PCR during the second campaign and were measured by quantitative (q)RT–PCR during the third and fourth campaigns. The frequency of NoV-positive samples, although not constant, was sufficiently high (88 %) to justify a quantitative analysis to identify correlations with the other considered variables. Parallel samples were analyzed for bacteriological indicators (E. coli and enterococci) and somatic coliphage. On each sampling day, the rain level (mm) and temperature (C) were measured. Data were recorded regarding water inflow to the plant, entry and exit biochemical oxygen demand (BOD), chemical oxygen demand (COD), total nitrogen, total phosphorus and suspended solids. For the third campaign, sampling was conducted such that 3 dry and 3 rainy days could be compared. On each sampling day, six replicate samples were collected both at the entrance and at the exit of the WWTP. Sample Treatment for Virus Detection Samples were concentrated using two-step tangential-flow ultrafiltration. Each sample was applied to a 10 kDa-cutoff polysulphone membrane (Merck Millipore, Italy) and was eluted in 3 % beef extract at pH 9 to yield a 250 ml final sample. Each eluate then was re-concentrated using a MiniUltrasette device (Pall Corporation, Italy) to obtain a concentrated sample of approximately 40 ml. The concentrated samples were adjusted to pH 7, and chloroform was added to eliminate bacteria and enveloped viruses (Carducci et al. 2008). To concentrate viruses further, each sample was precipitated with sterile polyethylene glycol 6000 (Promega Corporation, USA) (Li et al. 1998). Biomolecular Analysis Viral nucleic acids were extracted from concentrated samples using the QIAamp DNA kit (Qiagen, Germany) or the QIAamp RNA mini-kit (Qiagen) according to the manufacturer’s

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protocols. The extracted nucleic acids were assayed with nested (RT)-PCR according to published protocols to reveal the presence of the viral genomes of HAdV and NoV GI and GII (Carducci et al. 2011). The quantitative analysis for HAdV was performed using a previously described qPCR method (Bofill-Mas et al. 2010). A standard curve for HAdV detection was constructed by cloning the entire hexon region of Ad41 into pBR322 (Carducci et al. 2009). NoV quantification was performed using a Norovirus Real Time RT-PCR kit (Andiatec, France), a commercial assay typically used for clinical samples that detects both NoV genogroups. A standard curve for NoV detection was obtained using a specific amplicon from the open reading frame ORF1/ORF2 boundary, which was supplied with the kit (Andiatec). A no-amplification control, a no-template control, and a control for the presence of enzymatic inhibitors (e.g. uracil N-glycosylase) were run in parallel with each reaction. Bacterial Indicators and Somatic Coliphages In addition to the viral detection, samples were analyzed for E. coli, intestinal enterococci and somatic coliphage counts using ISO 7899-1 (1999), ISO 9308-3 (1998) and ISO/DIS 10705-2 (2001), respectively. Physicochemical Analysis According the EU directive 91/271/EEC and a subsequent national regulation (D. Lgs n. 152/1999), the waters at the entrance and exit of the WWTP are monitored routinely for the following parameters using the methods given in parentheses: suspended solids, BOD, COD (APAT-IRSACNR 2003), total nitrogen (ISO 11905-1 1997) and total phosphorus (UNI EN 1189 1999).

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forming units (PFU)/l; BOD = mg/l O2; COD = mg/l O2; total nitrogen = mg/l N; total phosphorus = mg/l P; suspended solids = mg/l. The geometrical mean was used to describe the average microbial concentrations. MedCalc Statistical Software 11.5 was used for the following statistical analyses. •

•

•

Unpaired t tests (different variance) to compare the mean values of entry and exit loads and the PRR for all parameters on dry and rainy days. Linear regression analyses (Pearson’s coefficient) to evaluate correlations between microbial parameters (entry and exit viral and bacterial loads, PRR) and climatic parameters (mm rain and temperature). Multiple regression analyses to (1) assess whether the indicator entry loads and PRR were representative of the corresponding human virus removal parameters and (2) detect potential correlations between the PRR associated with microbial parameters and the PRR associated with physicochemical parameters (inflow rate and PRR for suspended solids, BOD, COD, total nitrogen, and total phosphorus).

For regression analyses, a ‘viral load’ corresponding to half of the detection limit (10 GC/ml) was attributed to samples for which qPCR results were negative (Nordgren et al. 2009).

Results A total of 104 samples were collected (52 entry, 52 exit) for the detection of HAdV and indicators, and 46 samples were collected (23 entry, 23 exit) for NoV detection.

Meterological Monitoring

Microbiological Data

Rainfall (mm) and temperature (C) were monitored daily by management staff at the WWTP.

Figure 1 depicts the trend of indicator loads at the WWTP entrance and exit and the PRR values. The average PRRs and standard deviations (SD) were: 1.59 log10MPN/l (±0.56) for enterococci (Fig. 1a), 1.70 log10MPN/l (±0.65) for E. coli (Fig. 1b), and 2.18 log10PFU/l (±0.43) for somatic coliphages (Fig. 1c). With respect to human viruses (Fig. 2), we detected a virtually constant presence of HAdV (100 % in entry samples, 90 % in exit samples), with a moderate entry virus concentration (8.79 log10 GC/l ± 1.19). At the exit, HAdV concentrations averaged 6.78 log10 GC/l (±1.59), giving an average PRR of 2.01 log10 GC/l (±1.24) (Fig. 2a). NoV, which was assayed during the second sampling campaign, was detected in 72 % of entry samples and 65 % of exit samples. The PRR value for NoV, determined for the third and fourth sampling campaigns, was approximately zero (Fig. 2b).

Statistical Analysis Because we collected samples at the entry and exit of the WWTP on the same days, we could not account for wastewater retention time. Therefore, instead of the plant removal efficiency, we calculated the plant removal rate (PRR) according to the following: PRR ¼ Entry load  Exit load where ‘load’ is the measured amount of the different parameters expressed as follows: NoV = log10GC/l; HAdV = log10GC/l; E. coli = log10 most probable number (MPN)/l; enterococci = log10MPN/l; somatic coliphages = log10 plaque

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Fig. 1 Data of enterococci (a), E. coli (b) and somatic coliphages (c) Entry, exit loads and plant removal rates

Rain Influence No influence of rainfall was found on the entry and exit loads of the considered parameters. Only HAdV was associated with a significantly lower PRR on rainy days

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(Table 1). A linear regression analysis of the PRR on rainy days and mm of rainfall indicated no significant correlation, probably owing to the small sample sizes collected on rainy days. Only in the 2009 campaign, when additional samples were collected specifically to compare 3 rainy and

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Fig. 2 Data of human adenovirus (a) and norovirus GI and GII (b)

3 dry days, the HAdV PRR was significantly negatively correlated with mm of rainfall (Fig. 3). Temperature Influence A linear regression analysis and Pearson’s coefficient (Table 2) indicated a significant positive correlation between temperature and entry load for bacterial indicators and coliphages and a non-significant negative trend between temperature and NoV. No correlations were detected between exit loads and temperature. Temperature significantly influenced the PRR only with respect to E. coli. Utility of Microbial Indicators The values of entry loads were significantly correlated among E. coli, enterococci and coliphages (p \ 0.05). Regarding the PRR, significant correlations were identified only between E. coli and enterococci (p \ 0.05). The NoV entry loads were negatively correlated with HAdV and coliphages (p \ 0.05). No correlations were detected between human viral parameters and bacterial indicator parameters.

Utility of Physicochemical Parameters The PRR of the chemical parameters considered generally were unrelated to the removal rates of microorganisms. Coliphages, which were negatively correlated with COD and positively correlated with total phosphorus (p = 0.02 for both), were the exception.

Discussion The possible role of WWTPs as sources of microbial pathogens in receiving watersheds is an area of great interest owing to water shortages in many parts of the world and a consequent, increasing need for reclaimed waters (Okoh et al. 2007). The activated sludge process is the most widely applied biological wastewater treatment in the world; its primary objective is the removal of soluble biodegradable compounds and suspended solids. For this reason, WWTP efficiency is generally measured on the basis of chemical parameters such as BOD, COD and nutrient removal and its sustainability can be evaluated by

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Table 1 Comparisons (unpaired t test) between the mean values of all the analyzed parameters in samples collected in dry (No 35) and rainy (No 19) days Rainy days Mean ± SD

Dry days Mean ± SD

p

Enterococci (Log10 MPN/) Entry

6.84 ± 0.70

6.82 ± 0.74

0.91

Exit

5.18 ± 0.62

5.23 ± 0.65

0.78

PRR

1.66 ± 0.54

1.59 ± 0.51

0.63

E. coli (Log10 MPN/L) Entry

7.39 ± 0.77

7.48 ± 0.80

0.69

Exit

5.64 ± 0.66

5.80 ± 0.73

0.44

PRR

1.74 ± 0.58

1.67 ± 0.61

0.70

7.22 ± 0.40

0.35

Coliphages (Log10 PFU/L) Entry

7.10 ± 0.40

Exit

4.99 ± 0.53

5.00 ± 0.56

0.93

PRR

2.11 ± 0.40

2.21 ± 0.46

0.45

8.48 ± 1.03

0.30

Human adenovirus (Log10 GC/L) Entry

8.80 ± 1.04

Exit

7.17 ± 1.18

6.13 ± 1.34

\0.001

PRR

1.63 ± 0.88

2.34 ± 1.39

0.05

Norovirus (Log10 GC/L) Entry

5.83 ± 2.87

5.92 ± 2.86

0.93

Exit

5.80 ± 2.75

604 ± 2.94

0.84

PRR

0.02 ± 0.61

-0.11 ± 0.34

0.48

11,548 ± 1,440

11,128 ± 1,001

0.22

Flow (m3/day) Entry BOD (mg/L O2) Entry

185 ± 166

204 ± 109

0.61

Exit

2.47 ± 3.97

4.86 ± 6.43

0.15

PRR

182 ± 165

199 ± 107

0.65

COD (mg/L O2) Entry

867 ± 832

662 ± 404

0.23

Exit

47 ± 26

46 ± 30

0.92

PRR

819 ± 829

615 ± 400

0.23

Total nitrogen (mg/L N2) Entry

47 ± 29

46 ± 18

0.85

Exit

14 ± 5

15 ± 4

0.45

PRR

33 ± 29

31 ± 16

0.72

Total phosphorus (mg/L P) 9±5

0.68

Exit

Entry

9.9 ± 10 2 ± 1.6

2.5 ± 1.3

0.32

PRR

7.9 ± 10

6.5 ± 5.3

0.54

596 ± 1,318

0.86

Suspended solids (mg/L) Entry

538 ± 720

Exit

28.4 ± 20

PRR

509 ± 712

31.16 ± 27 565 ± 1,323

0.71 0.86

Geometric means are considered for microbial parameters. Differences with p \ 0.05 are evidenced in italic

considering various potential effects such as eutrophication or global warming (Rodriguez-Garcia et al. 2011). Many reports have identified variables affecting plant

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Fig. 3 Linear regression between mm of rain and PRR for human adenovirus in the third campaign. filled square PRR Mean values, filled triangle PRR values for each days

performance, demonstrating the influence of treatment process type, retention time, oxygen concentration, pH, temperature and rainfall amount (Doorn et al. 2006). When performing optimally, WWTPs can reduce bacterial and viral pathogens by approximately 2 log units (Carducci et al. 2009, Petrinca et al. 2009; Wen et al. 2009); a disinfection step generally is required to reach higher removal rates. The effectiveness of disinfection depends upon several factors, including the type of disinfectant, its contact time with wastewater, temperature, pH, effluent quality and pathogen type (US EPA 1986). The influence of heavy rainfall on faecal pollution of waters is well-documented, mainly with regard to combined sewer overflows (CSOs) (Rechenburg et al. 2006; Rose et al. 2001; Rodrı`guez et al. 2012). However, some studies have reported lower viral concentrations in water reclamation plant outfalls than in surface waters after rainfalls (Rijal et al. 2009: Rodrı`guez et al. 2012), suggesting that untreated waters released from CSO outfalls impact the microbial quality of waters more than run-off derived from WWTPs. In our study, the average entry and exit loads and removal rates for E. coli and enterococci were consistent with previous surveys (Petrinca et al. 2009; Wen et al. 2009). Precise comparisons are difficult because of differences in sewage concentrations, local climate conditions and plant technologies. Variations in bacterial parameters during the collection periods were not associated with dry versus rainy days or with mm of rainfall. Significant associations were identified between temperature and entry bacterial loads for E. coli and enterococci and between temperature and the PRR for E. coli. Increasing epidemiological interest in enteric viruses as agents of water-borne and food-borne outbreaks (EFSA and ECDC 2011; Brunkard et al. 2011) has motivated studies to assess the removal of viruses by WWTPs

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Table 2 Results of linear regression analysis for temperature effects on entry and exit loads and PRR (p \ 0.05 is evidenced in italic) En-T r

Ex-T p

r

PRR-T p

r

p

Human adenovirus

0.07

0.59

-0.009

0.94

0.08

0.55

Enterocci

0.33

0.01

0.12

0.38

0.26

0.06

E.coli Coliphages

0.42 0.32

0.001 0.001

0.18 0.20

0.19 0.13

0.28 0.04

0.03 0.76

-0.32

0.12

0.04

0.84

Norovirus

-0.32

0.13

BOD

0.16

0.23

0.02

0.85

0.16

0.23

COD

0.04

0.73

-0.19

0.16

0.05

0.68

Total nitrogen

0.11

0.40

0.13

0.36

0.08

0.52

Total phosphorus

0.04

0.76

0.09

0.50

0.02

0.88

Suspended solids

-0.13

0.34

-0.21

0.12

-0.12

0.36

PRR plant removal rate, T temperature, En entry load, Ex exit load, r correlation coefficient, p probability

(Carducci et al. 2009; Kuo et al. 2010; Nordgren et al. 2009; Thompson et al. 2003). The viruses discussed most in these reports are HAdV and NoV. In our study, the average influent and effluent concentrations and the PRR for HAdV were similar to those found in other studies (Hewitt et al. 2011; Katayama et al. 2008). Temperature was not associated with the entry loads, exit loads, or PRR for HAdV, whereas mean HAdV PRR values were significantly higher on dry versus rainy days. This finding was supported by the correlation between PRR values and mm of rain detected in a sampling campaign designed specifically to assess this relationship. In a report describing the impact of CSOs on surface waters (Rodrı`guez et al. 2012), the HAdV concentration in the WWTP outfall increased following rainfall events. The NoV entry and exit presence and loads in our study were consistent with others; reported NoV reduction rates vary from 0.0 to 3.6 log10 (Hewitt et al. 2011; Lodder and de Roda Husman 2005; Nordgren et al. 2009; Ottoson et al. 2006). We observed a PRR close to zero that may be explained by the sporadic occurrence of NoV and by our small sample size. NoV concentrations were not influenced by rain and showed a non-significant tendency towards a negative correlation with temperature, consistent with the greater epidemiological circulation of NoV during the winter season (Katayama et al. 2008). The reduction of enteric microbial parameters was not influenced strongly by meteorological parameters, probably owing to approximations associated with simultaneous sampling at the WWTP entry and exit that did not account for retention time. This approximation could be responsible for the few detectable correlations between PRR and rainfall, a parameter that is known to be related with retention time. Nevertheless, entering flow rates were not

influenced by rain or related to microbial loads, according to the overflow bypass upstream of the WWTP. The present study supports the utility of HAdV as an indicator of viral removal efficiency by a WWTP. Our data identify a potential influence of meteorological events on HAdV abatement when the entire sampling period is considered and when the specific study period of spring 2009 is considered. No physicochemical or bacterial indicators correlated with HAdV loads and reduction, confirming the poor utility of these organisms as indicators of viral removal. Although the effects of meteorological variables on viral contamination of water are well-established, few studies to date have examined the impacts of these variables on viral removal by WWTPs. We assessed monitoring data for the removal of indicators and viruses from the same plant over a long period of time. Moreover, our study evaluates the effects of climatic parameters, namely temperature and rain, on removal rates. Our results underscore the importance of viruses in evaluating the performance of WWTPs. In particular, the constant presence of HAdV in WWTPs and the response of HAdV to rainfall events confirm their usefulness in assessing the virological safety of effluents for reuse or discharge into natural waters. Acknowledgment We wish to thank the native English-speaking experts of ‘‘BioMed Proofreading’’ for the help in editing this article.

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