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Influence of wastewater treatment process and the populationsize on human virus profiles in wastewater

Joanne Hewitt a,c,*, Margaret Leonard b,1, Gail E. Greening a, Gillian D. Lewis c

a Institute of Environmental Science & Research Ltd, Kenepuru Science Centre, PO Box 50-348, Porirua, New Zealandb Institute of Environmental Science & Research Ltd, Christchurch Science Centre, PO Box 29-181, Christchurch, New ZealandcSchool of Biological Sciences, University of Auckland, Auckland, New Zealand

a r t i c l e i n f o

Article history:

Received 1 May 2011

Received in revised form

27 August 2011

Accepted 13 September 2011

Available online 19 September 2011

Keywords:

Virus removal

Wastewater

Norovirus

Adenovirus

Enterovirus

* Corresponding author. Institute of EnvironZealand. Tel.: þ64 4 914 0690; fax: þ64 4 914

E-mail address: joanne.hewitt@esr.cri.nz1 Present address. Christchurch Polytechn

0043-1354/$ e see front matter ª 2011 Elsevdoi:10.1016/j.watres.2011.09.029

a b s t r a c t

Human adenovirus (AdV and AdV species F), enterovirus (EV) and norovirus (NoV)

concentrations entering wastewater treatment plants (WWTP) serving different-sized

communities, and effectiveness of different treatment processes in reducing concentra-

tions were established. Data was combined to create a characteristic and unique descriptor

of the individual viral composition and termed as the sample virus profile.

Virus profiles were generally independent of population size and treatment process

(moving bed biofilm reactors, activated sludge, waste stabilisation ponds). AdV and EV

concentrations in wastewater were more variable in small (<4000) and medium-sized

(10,000e64,000) WWTP than in large-sized (>130,000 inhabitants) plants. AdV and EV

concentrations were detected in influent of most WWTP (AdV range 1.00e4.08 log10infectious units (IU)/L, 3.25e8.62 log10 genome copies/L; EV range 0.7e3.52 log10 plaque

forming units (PFU)/L; 2.84e6.67 log10 genome copies/L) with a reduced median concen-

tration in effluent (AdV range 0.70e3.26 log10 IU/L, 2.97e6.95 log10 genome copies/L; EV

range 0.7e2.15 log10PFU/L, 1.54e5.28 log10 genome copies/L). Highest culturable AdV and EV

concentrations in effluent were from a medium-sized WWTP. NoV was sporadic in all

WWTP with GI and GII concentrations being similar in influent (range 2.11e4.64 and

2.19e5.46 log10 genome copies/L) as in effluent (range 2.18e5.06 and 2.88e5.46 log10 genome

copies/L).

Effective management of WWTP requires recognition that virus concentration in

influent will vary e particularly in small and medium plants. Irrespective of treatment

type, culturable viruses and NoV are likely to be present in non-disinfected effluent, with

associated human health risks dependent on concentration and receiving water usage.

ª 2011 Elsevier Ltd. All rights reserved.

1. Introduction wastewater (Farthing, 1989; Wadell, 1984). Effective treatment

Enteric viruses transmitted by the faecaleoral route are

excreted by infected people in high concentrations in faeces,

and are potentially present in large numbers in domestic

mental Science & Resea0770.(J. Hewitt).ic, PO Box 540, Christchurier Ltd. All rights reserved

of such wastewater to reduce the infectious virus load is

important to minimise public health risks in receiving water

(Hafliger et al., 2000; Hewitt et al., 2007; Le Guyader et al., 2006).

The potential risk to human health depends on the types of

rch Ltd, Kenepuru Science Centre, PO Box 50-348, Porirua, New

ch 8140, New Zealand..

wat e r r e s e a r c h 4 5 ( 2 0 1 1 ) 6 2 6 7e6 2 7 66268

viruses present, the types of viruses that survive treatment,

their viability and concentration and the use of the receiving

water, such as for drinking water, recreational activity or

other use.

Human adenoviruses (AdV), enteroviruses (EV) and nor-

oviruses (NoV) are commonly found in influent and effluent

wastewater (da Silva et al., 2007; Haramoto et al., 2007; Irving

and Smith, 1981; Krikelis et al., 1985; Laverick et al., 2004;

Metcalf et al., 1995; Nordgren et al., 2009) and are important

etiological agents of many human illnesses (Green, 2007;

Pallansch and Roos, 2007; Wold and Horwitz, 2007). Human

AdV and EV outbreaks have occurred because of recreational

water becoming contaminated with human faecal waste

(Sinclair et al., 2009). Outbreaks of NoV have been frequently

associated with contact with either contaminated water or

shellfish (Hewitt et al., 2007; Le Guyader et al., 2006; Lees, 2000;

Simmons et al., 2007; Sinclair et al., 2009).

Most data on virus occurrence and concentration in

wastewater and removal efficiency are sourced from WWTP

that generally serve cities or large communities with pop-

ulations greater than 100,000 inhabitants. There is little pub-

lished data on the removal of enteric viruses by waste

stabilisation ponds (WSP) that serve small populations (da

Silva et al., 2007; Lewis et al., 1986; Nordgren et al., 2009)

mainly due to the expense and availability of virusmonitoring

data in wastewater or treated effluents.

In this study, the presence and quantity of three human

enteric viruses, AdV (including specifically human AdV

species F (AdV-F), types 40 and 41), EV and NoV genogroups I

and II (GI and GII) were determined in influent and effluent

wastewater from tenNewZealandWWTP. TheWWTP studied

represented those serving different-sized communities,

geographical locations and a range of wastewater treatment

processes. Real-time (reverse transcription, RT)-quantitative

PCR (qPCR) and cell culture assays, where applicable, were

used for virus detection and quantitation. The data from each

virus analysis was combined for each sample to create

a characteristic and unique descriptor of the individual viral

composition and was termed the ‘virus profile’. Each virus

profile included the occurrence and concentration for each

Table 1 e Wastewater treatment plants (WWTP) location, treapopulation size.

WWTP Treatment processa

A Moving bed biofilm reactor

B Trickling filter, activated sludge,

waste stabilisation pond

C Activated sludge, nitrogen removal

D Activated sludge, nitrogen removal

E Activated sludge

F Moving bed biofilm reactor

G Waste stabilisation pond, aerator

H Waste stabilisation pond

I Waste stabilisation pond

J Waste stabilisation pond, wetland

a WWTP A, C, E and F also include UV as part of the treatment. Samples

b Population range served by the wastewater treatment plants; large 130

virus type tested and for each method used for the analysis of

that virus. Virus profiles were compared and similarities

visualised between samples using non-metric multi-dimen-

sional scaling (MDS) and analysis of similarity (ANOSIM). The

aim of the study was to obtain an understanding of virus

prevalence in the influent of WWTP serving various pop-

ulation sizes, the persistence of enteric viruses through

treatment and the influence of treatment type on the virus

profiles of the effluent.

2. Materials and methods

2.1. WWTP and samples

Primary screened influent and treated effluent wastewater

samples (1 L) were collected from ten community WWTP

(AeJ). Table 1 shows the WWTP treatment process, waste-

water source and the population size served by each plant.

The selectedWWTP served large (130,000e1,000,000), medium

(10,000e64,000) and small (<1100e4000) communities (L-

WWTP, M-WWTP, S-WWTP respectively) and were located

across New Zealand. The wastewater treatment processes

included moving bed biofilm reactor (MBBR) and activated

sludge (AS) plants that served large populations, to WSP that

served small populations. WWTP D, F, H, I and J processed

mainly domestic wastewater whilst WWTP A, B, C, E and G

processed domestic wastewater and significant amounts of

industrial influent, including animal abattoir wastes. Three

samples were taken from each WWTP, one on each of three

sampling windows during the New Zealand summer (24 Nov

to 12 Dec 2003, 19 Jan to 14 Feb 2004, and 8 Mar 04 to 2 Apr

2004). The timing of sampling was defined by criteria for

a wider study investigating a range of analytes in wastewater.

Influent and effluent wastewater samples were temporally

separate, even when collected on the same day, owing to the

time the wastewater took to move through the WWTP.

Effluent samples were collected prior to any mechanical or

chemical disinfection processes.

tment process, predominate wastewater source and

Wastewater source Approximatepopulation sizeb

Domestic and significant industrial Large

Domestic and significant industrial Large

Domestic and significant industrial Large

Mainly domestic Medium

Domestic and significant industrial Medium

Mainly domestic Medium

Domestic and significant industrial Medium

Mainly domestic Small

Mainly domestic Small

Mainly domestic Small

for this study were taken prior to UV disinfection.

,000e1,000,000; medium 10,000e64,000; small <1100e4000 people.

wat e r r e s e a r c h 4 5 ( 2 0 1 1 ) 6 2 6 7e6 2 7 6 6269

2.2. Virus controls

Human AdV type 2 (American Type Tissue Culture Collection

(ATTC) #VR-846, Manassas, VA), human AdV type 41 (ATCC #

VR-930), and Sabin poliovirus type 2 (PV2) LSc ab (monovalent

Pfizer vaccine strain) were used as positive controls for the

AdV qPCR and EV RT-qPCR assay respectively. PV2 was also

used for seeding experiments and as a positive control for the

EV culture assay. For NoV GI and GII RT-qPCR positive

controls, 10% (wt/vol) faecal suspensions from NoV positive

sampleswere prepared, clarified by the addition of chloroform

(10%, wt/vol) and then centrifuged at 13,000� g for 10 min.

2.3. Sample processing

Within 24 h of collection, viruses from1 Lwastewater samples

were concentrated to 10 mL (modified from Green and Lewis,

1995; Greening et al., 2002; Lewis and Metcalf, 1988; Wait

and Sobsey, 1983). Samples (1 L) were first centrifuged at

2400� g for 20 min at 4 �C and the supernatant held at 4 �C for

later use. Sodium nitrate (2 M) in 3% (v/w) beef extract (pH 5.5)

was added to the resulting pellet and pH adjusted to 5.5.

Viruses were eluted for 1 h at 4 �C with continuous mixing on

a horizontal shaker at 180 rpm, and centrifuged at 10,000� g

for 20 min. The resulting supernatant was added to the first

supernatant and pH adjusted to 7e7.2. Viruses were then

concentrated by adding polyethylene glycol (PEG) 6000 and

NaCl to give final concentrations of 10% (w/v) and 2% (w/v)

respectively. Samples were mixed at room temperature to

dissolve the components, then placed on a horizontal shaker

(120 rpm) for a minimum of 2 h at 4 �C and then centrifuged at

10,000� g for 25 min at 4 �C. The supernatant was discarded

and the pellet resuspended in 10 mL phosphate buffered

saline (pH 7.4), so that 1 mL concentrate was equivalent to

0.1 L wastewater. The pH was adjusted to 8.0, sonicated in an

Ultrasonic Cleaner (Model FX10, Unisonics Pty Ltd., Sydney,

Australia) for 2 min and viruses left to elute from the pellet for

1 h at room temperature with occasional vortexing. Samples

were sonicated for another 2 min and centrifuged at 10,000� g

for 20 min. Chloroform (10 mL) was added, tubes inverted

every 5 min for 15 min and then centrifuged at 1200� g for

5 min. The chloroform phase was discarded and 10 mL chlo-

roform added to the aqueous and interfacial phases, mixed

and then centrifuged at 1200� g for 5 min. Pen-

icillinestreptomycin (P/S) (Gibco, Invitrogen Corporation,

Carlsbad, CA) and amphotericin B (Gibco) were added.

Samples were stored at �80 �C, if not analysed within 24 h.

Viral nucleic acidwas extracted from the concentrates (200 mL)

using the High Pure Viral Nucleic Acid kit (Roche Molecular

Biochemicals Ltd, Mannheim, Germany) as per manufac-

turer’s instructions and eluted in 50 mL elution buffer.

Poliovirus-negative wastewater was seeded with 2.0, 3.0 and

4.0 log10 plaque forming units (PFU)/L PV2 in triplicate and

recovered using L20B cells that are very selective for PV

growth.

2.4. Virus culture assays

Cells were propagated in medium 199 (Gibco) (A549 and BGM)

or Dulbecco’s Modified Eagle Medium High Glucose (DMEM)

(293) (Gibco) supplemented with 10% (v/v) foetal calf serum

(FCS), and P/S (Gibco). Human AdV type 2, human AdV type 41

and PV2 stocks were prepared from infected A549, 293 and

BGM cell monolayers respectively by freezeethaw lysis fol-

lowed by sonication for 3 min in an Ultrasonic Cleaner (Uni-

sonics Pty Ltd). Lysates were extracted by the addition of an

equal volume of chloroform and clarified by centrifugation at

400� g for 5 min. Preparations were aliquoted and stored at

�80 �C. Nucleic acid extracts from each virus stock were used

to generate DNA plasmids.

Quantitation of culturable AdV was determined by

observing cytopathic effect (CPE) in A549 cells following

sample inoculation. A maximum of 2 mL concentrate (equiv-

alent to 0.2 L sample) was inoculated by adding 10 mL per well

in 96 well plates and applying the most probable number

(MPN) method after 28 day incubation. Guanidine hydrochlo-

ride (100 mg/mL) was used to suppress EV growth in the AdV

assay as previously described (Hurst et al., 1988). Confirmation

and species typing of AdV was done by extracting DNA from

the selected cell lysates of each sample, followed by AdV

specific PCR (Allard et al., 2001) and DNA sequencing. Human

AdV type 2 was used as both extraction and PCR-positive

control. Quantitation of culturable EV was determined by the

agar cell suspension assay using BGM cells (Greening et al.,

2002) with a maximum of 2 mL concentrate, equivalent to

0.2 L wastewater, inoculated (0.2 mL� 10 plates). PV2 (10 PFU)

was used as a positive control in the EV culture assay. Titres

were expressed as log10 MPN infectious units (IU)/L for AdV

and log10 PFU/L for EV. The theoretical limit of detection per

sample based on the amount of sample tested was therefore

0.7 log10 IU/L for culturable AdV and 0.7 log10 PFU/L for cul-

turable EV.

2.5. RT-qPCR and qPCR

For the generic human AdV and specific human AdV-F qPCR

assays, the PCR was carried out using qPCR Supermix-UDG

(Invitrogen) with either 0.6 mM (generic AdV) or 0.2 mM

(human AdV-F) of each primer and 0.25 mM (generic HAdV) or

0.2 mM (HAdV-F) of each probe (Heim et al., 2003; Wolf et al.,

2010). For EV RT-qPCR, the Platinum Quantitative RT-PCR

ThermoScript� One-Step System (Invitrogen), 0.6 mM

primers, 0.25 mM probe and PCR conditions were used as

previously described (Donaldson et al., 2002). For NoV, the RT

step was carried out using SuperScript� III-First Strand

Synthesis System for RT-PCR (Invitrogen) with 0.1 mM of each

reverse primer (Wolf et al., 2010), followed by qPCR assay

performed as a multiplex assay using qPCR Supermix-UDG

(Invitrogen), with 0.4 mM of each primer and 0.2 mM of each

probe (modified from Wolf et al., 2010). Primers and probes

were verified in our laboratory for the detection of the selected

viruses in wastewater (data not shown) and were considered

appropriate for the purposes of this study. Armored RNA�-

Norwalk Virus GI was used to monitor potential RT-qPCR

inhibition as previously described (Hewitt et al., 2007).

Where (RT)-qPCR inhibition was suspected, the nucleic acid

was diluted 1/5 and the sample retested. All qPCR assays were

carried out using either a Rotor-Gene� 3000 (generic AdV, EV)

or 6000 (NoV GI and GII, human AdV-F) real-time rotary

analyzer (Corbett Life Science, Sydney, Australia). All samples

wat e r r e s e a r c h 4 5 ( 2 0 1 1 ) 6 2 6 7e6 2 7 66270

were analysed at least in duplicate. Quantification of viruses

(genome copies) was determined by comparing the cycle

threshold (Ct) value against the appropriate standard curve

generated from a dilution series ranging between 107 and 10

genome copies per reaction of the appropriate DNA plasmid

prepared from PCR products as previously described (Wolf

et al., 2010). Titres were expressed as log10 genome copies/L

wastewater. Using the standard curve, the lowest concentra-

tion detected was 1.5 log10 genome copies/L in the PCR assays.

In each (RT)-qPCR, virus positive extraction controls, DNA

plasmid controls, and nuclease-free water controls were used.

2.6. Data analysis

Virus quantities as determined by culture and (RT)-qPCR were

log10 transformed prior to statistical analysis. For S-WWTP, M-

WWTP and L-WWTP, box plots were used to show the median

concentrations (log10/L) with the 25the75th percentile values.

Thewhiskers extended to themost extremedata point nomore

than 1.5 times the interquartile (25the75th) range from the box.

For representation on the box plots, sampleswith log10/L values

greater than 1.5 times the interquartile range fromthe boxwere

shown as individual points (stars). For data sets where all

samples were positive, in addition to the median, mean

concentrations with a standard deviation were also calculated.

To analyse themultivariate data and summarise (ordinate)

patterns of virus presence and concentration between

samples, MDS and ANOSIM were used to assess similarities

and dissimilarities according to WWTP size, wastewater type

(influent or effluent) and treatment process. MDS was chosen

as it creates a spatial representation of multivariate sample

data which does not assume linear relationships between

variables (concentrations), and hence is robust and suitable

for comparison of data descriptions comprised of multiple

components of variable magnitude. Log10 transformed culture

and PCR concentration data for each samplewere placed in an

Excel spreadsheet (Microsoft, Redmond, WA) to give a matrix

consisting of one row per dataset (AdV PCR, AdV culture, EV

PCR, EV culture, NoV GI and GII) with one column per sample

(n¼ 60). The data was then exported into PRIMER v6 software

(Primer-E Ltd., Plymouth, United Kingdom) and the

Manhattan distance between samples calculated for each

dataset. MDS plots were created from each resemblance

matrix, and were presented as two-dimensional figures to

display distances between different virus profiles. Plots were

generated to represent the relationship between influent and

Table 2 e Prevalence of culturable adenovirus and enterovirus

Virus Wastewatertype

No

WWTPA, B, C(large)

Adenovirus Influent 9/9, 100%

Effluent 3/9, 33%

Enterovirus Influent 8/9, 89%

Effluent 7/9, 78%

effluent wastewater samples, population size and between

the three categorised groups of WWTP treatment types e

MBBR, AS and WSP (Table 1). One way ANOSIM (Clarke and

Gorley, 2006) was performed on the resemblance data to

determine the statistical significances. Using PRIMER, a test

statistic Rwas generated that ranged from�1 toþ1 depending

on the similarity between samples within a group compared

to the similarity between samples from a different group.

The relationship between AdV and human AdV-F concen-

trations determined by PCR was also determined for 30

influent samples using Microsoft Excel and the correlation

coefficient (r) calculated to determine thedegreeof correlation.

3. Results

3.1. Culturable viruses

Prevalence (% samples positive) and concentration (log10/L) of

AdV and EV detected by cell culture are summarised in Table 2

and Fig. 1 respectively. Seeding and recovery experiments

showed at least 2 log10/L could be recovered by culture

following the virus concentration method, which equated to

a recovery efficiency of at least 10%.

Most influent samples (26/30, 87%) contained either cul-

turable AdV and/or EV with concentrations ranging from 1.00

to 4.08 log10 IU/L and 0.7e3.52 log10 PFU/L respectively. M-

WWTP G and S-WWTP H and I were negative for both cul-

turable AdV and/or EV. The highest median (Fig. 1a, b), and

mean culturable AdV and EV concentrations in influent were

observed in the three L-WWTP (AdV,mean 2.61� 0.45 log10 IU/

L; EV, mean 2.06� 1.05 log10 PFU/L). However, virus concen-

trations for individual WWTP were not necessarily dependent

on the population size that the WWTP served. Culturable AdV

and EV concentrations in influent from S-WWTP and M-

WWTP could be as high as in L-WWTP influent, but with

a lower detection frequency (Table 2, Fig. 1). For example, the

highest concentration of culturable AdV and EV detected in

a single influent sample occurred in S-WWTP J (3.92 log10 PFU/

L) and M-WWTP D (3.52 log10 PFU/L) respectively.

In effluent, culturable AdV and/or EV were detected in 19/

30 (63%) samples with the highest concentrations from M-

WWTP F (AdV, 3.26 log10 IU/L; EV, 2.15 log10 PFU/L). The

median concentration, as grouped by population size, in

effluent samples was consistently lower (AdV range

0.70e3.26 log10 IU/L; EV range 0.7e2.15 log10 PFU/L) than in the

in wastewater treatment plants (WWTP) AeJ.

. positive/total samples, % positive samples

WWTPD, E, F, G(medium)

WWTPH, I, J(small)

Total

10/12, 83% 5/9, 55% 24/30, 80%

6/12, 50% 3/9, 33% 12/30, 40%

7/12, 58% 3/9, 33% 18/30, 60%

4/12, 33% 1/9, 11% 12/30, 40%

Fig. 1 e Box plots showing quantitation (log10/L) of (a) adenovirus (AdV) by culture, (b) enterovirus (EV) by culture, (c) AdV by

PCR, (d) EV by PCR, (e) norovirus (NoV) GI by PCR, and (f) NoV GII by PCR, in influent (Inf) and effluent (Eff) samples from

wastewater treatment plants serving small, medium and large populations. Box plots showmedian concentrations (log10/L)

with the 25the75th percentile values. The whiskers extend to the most extreme data point no more than 1.5 times the

interquartile (25the75th) range from the box.

wat e r r e s e a r c h 4 5 ( 2 0 1 1 ) 6 2 6 7e6 2 7 6 6271

influent samples, with the L-WWTP showing a larger median

decrease than the smaller WWTP (Fig. 1a and b). The 11

effluent samples negative for both AdV and EV were collected

fromM-WWTPD, E and G and S-WWTPH, I and J, but only two

WWTP (M-WWTP G and S-WWTP H) were negative on all

three sampling occasions.

Further studies showed that culturable AdV detected using

A549 cells in both influent and effluent was mainly from

human AdV species AeE (data not shown).

3.2. Virus detection and quantitation by PCR

Prevalence (% samples positive) and concentration (log10genome copies/L) of AdV, EV, NoV GI and NoV GII detected by

real-time qPCR are summarised in Table 3 and Fig. 1

respectively.

In influent, most samples contained AdV and EV, with AdV

present in higher concentrations (range 3.25e8.62, mean

6.28� 0.34 log10 genome copies/L) than EV (range 2.84e6.67,

mean 5.30� 0.60 log10 genome copies/L) (Fig. 1c and d). PCR

quantitation showed that total AdV and human AdV-F

concentrations were similar, indicating that the majority of

AdV were AdV-F. Fig. 2 shows the total human AdV and AdV-

F concentrations (genome copies/L) and correlation between

them (R2¼ 0.9001) in influent samples. Whilst the highest

AdV concentration (8.62 log10 genome copies/L) in influent

detected by PCR was in S-WWTP H, the mean AdV concen-

tration by PCR in positive influent samples was significantly

Table 3 e Prevalence of adenovirus, enterovirus, and norovirus GI and GII, as determined by real-time qPCR for wastewatertreatment plants (WWTP) serving large, medium and small populations.

Virus Wastewater type No. positive/total samples, % positive samples

WWTPA, B, C(large)

WWTPD, E, F, G(medium)

WWTPH, I, J(small)

Total

Adenovirusa Influent 9/9, 100% 10/12, 83% 8/9, 89% 27/30, 90%

Effluent 9/9, 100% 11/12a, 92% 7/9a, 89% 27/30, 90%

Enterovirus Influent 9/9, 100% 12/12, 100% 9/9 100% 30/30, 100%

Effluent 7/9, 78% 7/12, 58% 5/9, 55% 19/30, 63%

Norovirus GI Influent 5/9, 56% 7/12, 58% 3/9, 33% 15/30, 50%

Effluent 6/9, 67% 5/12, 42% 6/9, 67% 17/30, 57%

Norovirus GII Influent 6/9, 67% 5/12, 42% 2/9, 22% 13/30, 43%

Effluent 6/9, 67% 6/12, 50% 5/9, 55% 17/30, 57%

a Three samples negative by generic adenovirus PCR assay (Heim et al., 2003) were positive by the human adenovirus species F PCR assay (Wolf

et al., 2010).

wat e r r e s e a r c h 4 5 ( 2 0 1 1 ) 6 2 6 7e6 2 7 66272

higher in L-WWTP than in M-WWTP and S-WWTP ( p< 0.05).

The lowest variation in AdV and EV concentrations as

determined by comparison of the range of PCR concentra-

tions was observed in L-WWTP (Fig. 1a and b). The frequency

of NoV GI and GII in influent was less, particularly in the S-

WWTP, than AdV and EV, with generally lower concentra-

tions (NoV GI and GII range 2.11e4.64 and 2.19e5.46 log10genome copies/L respectively (Fig. 1e and f)). The highest

median NoV concentrations in influent were from samples

collected from L-WWTP. In influent, 10/30 (33%) samples were

positive for both NoV GI and GII, with 4/30 (13%) samples

positive for NoV GI only and 5/30 (17%) samples positive for

NoV GII only. Eleven influent samples (37%) were negative for

both NoV GI and GII.

In effluent, median AdV and EV concentrations (2.97e6.95

and 1.54e5.28 log10 genome copies/L respectively) of each

sizedWWTPwere lower than the influent samples (Fig. 1c and

d). For all WWTP sizes, median AdV concentrations in effluent

weregreater thanEV.HighAdVconcentrations (up to 6.95 log10genome copies/L) were frequently detected including from S-

WWTP. In effluent, AdVwasmore commonly detected than EV

and NoV. Effluents from all WWTP were positive by either the

y = 1.0801x - 0.1593R2 = 0.9001

0

2

4

6

8

10

0 2 4 6 8 10

all human AdV species (log10 genome copies /L)

hu

man

A

dV

-F

(lo

g10

gen

om

e co

pies/L

)

Fig. 2 e Concentration (log10 genome copies/L) and

correlation of adenovirus (AdV) and human AdV species F

(AdV-F) in influent wastewater samples (n[ 30).

generic AdV or human AdV-F PCR assay (data not shown). In

contrast toAdVandEV, theprevalence andmedianNoVGI and

GII concentrations in PCR-positive effluent samples (range

2.18e5.06 and 2.88e5.46 log10 genome copies/L respectively),

were not generally lower than in influent samples (Table 3,

Fig. 1e and f). Effluent from M-WWTP E was negative for both

NoV GI and GII on all three sampling occasions. More samples

were positive for both NoVGI andGII in effluent (16/30) than in

influent (10/30)with fewer effluent samples positive forGI only

(1/30) and GII only (1/30) than the influent. Twelve effluent

samples (40%) were negative for both NoV GI and GII.

As expected, overall virus prevalence and concentrations

detected by PCRwere greater than by culture. Themean (� SD)

AdV concentration by PCRwas 3.6� 1.2 log10 and 3.9� 0.9 log10greater than culture for influent and effluent samples respec-

tively. For EV, the valueswere 3.0� 0.9 log10 and 1.4� 1.4 log10.

It should be noted that some RT-PCR inhibition was observed

in the one-step EV RT-qPCR assay and so RNA from all EV PCR

negative sampleswas diluted (1/5) to reduce inhibition effects.

An additional six samples were identified as EV positive using

dilutedRNA; four fromeffluent and two from influent samples.

For AdV and EV, there were three influent and two effluent

samples respectively that were positive by culture, but

repeatedly negative by PCR despite diluting the sample.

3.3. Virus profiles

Figs. 3e5 show MDS plots of virus profiles generated from all

PCR and culture quantitative data from the influent and

effluent wastewater samples. Virus profiles with the highest

similarity are shown by ordinate points plotted nearest to

each other, whilst those far apart represent virus profiles that

are less similar. Statistical analysis (ANOSIM) of influent and

effluent sample groups showed that virus profiles of influent

samples were more similar to each other (i.e. within the

group) than to the effluent samples (i.e. between the groups)

(ANOSIM R¼ 0.238, where 0 is highly similar (no difference)

and�1 is highly dissimilar). The similarities between the virus

profiles of influent and effluent are also observed in Fig. 3 as

two clusters showing a degree of separation. This dissimilarity

Fig. 3 e Non-metric multi-dimensional scaling plot

showing virus profile comparison of influent (solid

symbols) and effluent (non-solid symbols) wastewater

samples from each wastewater treatment plant on three

occasions. Samples grouping closer together show a more

similar virus profile than those that are further apart.

Fig. 5 e Non-metric multi-dimensional scaling plot

showing virus profile of effluent samples collected from

moving bed biofilm reactor (MBBR D), activated sludge (AS

3) and waste stabilisation pond (WSP C) wastewater

treatment plants. Virus profiles show no clear similarity to

each other.

wat e r r e s e a r c h 4 5 ( 2 0 1 1 ) 6 2 6 7e6 2 7 6 6273

between the influent and effluent sample groups ismost likely

due to the overall reductions in median AdV and EV concen-

trations from all WWTP (Fig. 1).

MDS analysis showed no strong differentiation within

influent or effluent virus profiles of the different-sizedWWTP,

with virus profiles of influent samples appearing independent

of the population size that theWWTP served (Fig. 4). However,

virus profiles of L-WWTP influent samples showed greater

similarity to each other, as shown by a clustering in the centre

of the plot, than the virus profiles of M-WWTP and S-WWTP

that are further apart (Fig. 4). This is a possible reflection of the

smaller variation between AdV and EV concentrations in the

samples taken from the L-WWTP. MDS analysis of the virus

Fig. 4 e Non-metric multi-dimensional scaling plot

showing virus profile comparison of influent samples from

wastewater treatment plants serving large (:), medium

(D) and small (,) populations. Samples grouping closer

together (:) show a more similar virus profile than those

that are further apart.

profiles of the effluent showed no strong differentiation

between the three groups of treatment processes (MMBR, AS

andWSP) as shown by the dispersal of virus profiles of effluent

samples (albeit distributed in two main groups with no iden-

tified commonality) (Fig. 5).

4. Discussion

An important function of wastewater treatment is to remove

pathogens efficiently from wastewater before discharge or

disposal to minimise public health risks associated with

contact of infectious pathogens via contaminated water,

shellfish or aerosols.

In this study, we showed that AdV, EV and NoV were

present in wastewater from large (>100,000), medium

(10,000e64,000), and small (<4000) populations in New Zea-

land. We demonstrated that although wastewater treatment

processes removed EV and AdV, prevalence of NoV was

similar in both influent and effluent.

Both culturableAdVandEVwereoften found inwastewater

effluent at concentrations similar to those reported elsewhere

(Haramoto et al., 2007; Krikelis et al., 1985; Lodder and de Roda

Husman, 2005). The range of AdV and EV concentrations by

PCR in influent (3.25e8.62 and 2.84e6.67 log10 genome copies/L

respectively) was also similar to other studies (Bofill-Mas et al.,

2006; Katayama et al., 2008; Schvoerer et al., 2001). The high

incidence and concentration ofAdVby PCR in effluent samples

demonstrated in this and other studies (Haramoto et al., 2007;

Pina et al., 1998) may be due either to the high stability of the

dominant human AdV species F (type 40 and 41) through the

various WWTP processes, or the environmental resistance of

AdV (Nwachuku et al., 2005).

Occasionally, samples were positive by culture but nega-

tive by PCR. This discrepancy is probably due to a much

smaller quantity of material tested in the PCR than in the

wat e r r e s e a r c h 4 5 ( 2 0 1 1 ) 6 2 6 7e6 2 7 66274

culture assays. In the culture assay, the equivalent of 200 mL

sample is assayed per virus compared with an equivalent of

1 mL per PCR. Following a 1/5 dilution, even less sample was

tested resulting in a potential further reduction in sensitivity.

Undetected (RT)-qPCR inhibition may also cause false nega-

tive results.

Demonstration of NoV infectivity is not yet possible

because of NoV culture limitations (Duizer et al., 2004). From

this study, assuming that NoV has similar survival properties

and abundance as AdV and EV, we deduce that a proportion

of NoV detected in effluents could be infectious due to the

concurrent presence of culturable enteric viruses, and the

ratio between culturable enteric viruses and PCR concentra-

tions (1.4e3.9 log10 difference). Overall occurrence of NoV GI

and GII was sporadic in both influent and effluent samples

from all WWTP. In effluent, although variable, NoV concen-

trations were in the same range as previous reports (Laverick

et al., 2004; Sima et al., 2011) but concentrations in influent

were generally lower than the 6e8 log10 genome copies/L re-

ported in overseas studies (Laverick et al., 2004; Lodder and de

Roda Husman, 2005; Nordgren et al., 2009; Pusch et al., 2005;

Sima et al., 2011; van den Berg et al., 2005). One possible

reason why NoV concentrations were frequently low and

sporadic in influent wastewater may be due to lower NoV

prevalence in the community than AdV and EV during the

study period. Generally NoV infection is associated with

peaks in winter (Mounts et al., 2000), but there is no clear

seasonal peak for NoV infection in New Zealand. A similar

number of outbreaks are reported by the Norovirus Reference

Laboratory in the summer months as during other seasons of

the year (ESR, 2009). Therefore, we would not expect that the

timing of the study would result in a low NoV prevalence.

Whilst NoV surveillance data system does not capture all

outbreaks and cases in the community, during Januar-

yeMarch 2004, only 12 NoV outbreaks were recorded by the

New Zealand Public Health Services, which was the same

number of outbreaks as reported in the previous quarter.

Most outbreaks were caused by NoV GII (ESR, 2004a,b). Similar

to our finding for NoV, da Silva et al. (2007) reported that NoV

(GI) occurrence in four WWTP studied was ‘erratic’. We found

that NoV GI and GII prevalence were similar whereas most

studies report that either NoV GII is more prevalent than NoV

GI (da Silva et al., 2007; Katayama et al., 2008; Lodder and de

Roda Husman, 2005) or vice versa (Nordgren et al., 2009).

Data from other studies conducted in our laboratory have

showed that no seasonal variation in the prevalence or

quantitation of EV or AdV in influent wastewater is observed

(data not shown). As the overall incidence and concentration

of NoV in influent and effluent were similar, it may also

suggest that NoV was more stable than AdV and EV through

the various WWTP processes. As reported elsewhere (Sima

et al., 2011), the NoV concentration in effluent was seem-

ingly often unrelated to the influent, and so the efficiency of

removal could not be determined or compared directly to AdV

or EV.

The influent virus profiles were generally independent of

the WWTP size, indicating a similar epidemiological distri-

bution of viruses in NZ. The virus profiles in effluent samples

were independent of the treatment process. da Silva et al.

(2007) found NoV concentrations were similar in the WWTP

serving different-sized populations. In contrast, our data

showed less variance in virus prevalence and concentrations

in large WWTP due to the population size. Large communities

are more likely to have virus infections occurring almost

continuously in the community, whereas in small commu-

nities, infection is likely to be sporadic and may affect a larger

proportion of the community. The virus prevalence in large

communities may also be more consistent due to compara-

tive homogeneity of the wastewater from extended infra-

structure, and better comparative mixing as wastewater

travels long distances into the WWTP. The individual virus

concentrations were unrelated to the size of population

contributing to the treatment plant. For example, an

extremely high AdV concentration was observed on one

occasion in the influent of a small WWTP (8.6 log10 genome

copies/L, WWTP H). This may be due to the occurrence of

a possible family/small community outbreak prior to

sampling, resulting in up to 1011AdV/gram faeces being

excreted in the wastewater (Wadell, 1984). Such an unpre-

dictable increase in virus concentrations would be highly

significant in a small WWTP.

Human AdV species AeE, mainly responsible for non-

enteric illnesses, were predominately isolated using A549

cells, that are not optimal for growing human AdV-F strains.

However, PCR data indicated that AdV-F was the predominant

human AdV species present in influent and effluent samples

at concentrations up to 8.6 log10 genome copies/L. The prev-

alence of human AdV-F (a common cause of gastroenteritis in

children) (Uhnoo et al., 1986) in water has been previously

observed (Dong et al., 2010; Haramoto et al., 2007). Further

work using 293 cells showed that culturable human AdV-F

was present in several influent and effluent samples (unpub-

lished data). It is likely that culturable AdV concentrations

were underestimated in this study, due to the use of A549

cells, which would influence the accuracy of gastro-enteric

health risk assessments. If AdV-F survives the WWTP

process, then viable viruses are likely to persist in fresh and

seawaters (Enriquez et al., 1995) with the potential for trans-

mission by the waterborne route.

The estimated log10 reduction of viruses through WWTP

can be theoretically determined by comparing overall virus

presence in pre and post treated wastewater. For accuracy,

sampling should ensure that samples are as representative as

possible and also account for the time required for waste-

water to pass through the plant. These issues can be prob-

lematic, as it is difficult to determine the actual time

wastewater spends in a WWTP especially in a WSP treatment

plant (Berg, 1973). Although wastewater is theoretically

retained for weeks under normal circumstances, this may be

only days if short circuiting occurs where flow bypasses most

of the pond. Use of tracer dyes is the only practical method to

determine the hydraulic retention time but this was outside

the scope of the project. For these reasons, in this study, the

median influent concentration for each population category

was calculated and compared with the median effluent

concentration for each population size. Although we

demonstrated that the median AdV and EV concentrations

were reduced through the treatment processes independent

of treatment types, median NoV GI and GII concentrations

(0e4 log10/L) in the effluent were often greater than those in

wat e r r e s e a r c h 4 5 ( 2 0 1 1 ) 6 2 6 7e6 2 7 6 6275

influent (0e3 log10/L). Other studies have reported NoV

reductions ranging from 0.0 to 3.6 log10 (Kuo et al., 2010;

Lodder and de Roda Husman, 2005; Nordgren et al., 2009;

Ottoson et al., 2006; Pusch et al., 2005; van den Berg et al.,

2005). One probable reason for the lack of reduction

observed for NoV may be due to its sporadic occurrence. Even

over a 24 h period, this could result in variable NoV concen-

trations entering a WWTP, as has been previously described

for other enteric viruses (Berg, 1973).

In summary, this study presents virus profiles for different

WWTP serving a range of populations and using different

treatment processes. While culturable AdV and EV were

detected frequently in effluent samples from several WWTP,

the concentrations were generally lower than the influent

samples. Typical influent concentrations of culturable viruses

were approximately 2e3 log10/L compared to 1e2 log10/L in

the effluent. The frequent presence of NoV in the effluent

along with that of culturable enteric viruses is of particular

concern because of the low infectious dose needed to cause

NoV disease, although it is not possible to determine the

infectivity of NoV. Over the three-month study period, we

showed that virus presence and concentration were generally

independent of the size of population served by the WWTP

and, for effluent, the type of WWTP process. However, a nar-

rower range of AdV and EV concentrations in both influent

and effluent samples was observed in the larger plants

compared with the smaller plants. Smaller plants were

characterised by generally variable virus concentrations with

sporadic spikes in both the influent and effluent. These spikes

in virus concentration need to be taken into account when

assessing the treatment requirements for small communities.

The potential health effects of these discharges on small

receiving waterways used for recreation and food gathering

could be significant.

5. Conclusions

� Effective management of wastewater treatment plants

requires recognition that human virus concentrations in

both influent and effluent wastewater are generally inde-

pendent of the WWTP size and treatment process, and that

virus concentrations in influent will vary e particularly in

small and medium treatment plants.

� Irrespective of treatment type, human viruses are likely to

be present in non-disinfected effluent, with associated

human health risks from virus-contaminated water. The

extent of the risk may be dependent on viability, concen-

tration, and the potential virus transmission routes of the

receiving water usage.

Acknowledgements

This work was funded by the New Zealand Ministry for the

Environment. We appreciate the assistance of Beverly Horn

and Kelly Roberts, and thank Andrew Ball, Richard Lardner,

Jeremie Langlet and Stephen On for their critical review of the

manuscript.

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