International Journal of Hygiene and Environmental Health 213 (2010) 210–216

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International Journal of Hygiene and

Environmental Health
journal homepage: www.elsevier.de/ijheh

Chemical and microbiological parameters as possible indicators for human

enteric viruses in surface water
Lars Jurzik a,∗,1 , Ibrahim Ahmed Hamza a,1,2 , Wilfried Puchert b , Klaus Überla c , Michael Wilhelm a
a
Department of Hygiene, Social and Environmental Medicine, Ruhr-University Bochum, Germany
b
Health Department of the State of Mecklenburg-West Pomerania, Schwerin, Germany
c
Department of Molecular and Medical Virology, Ruhr-University Bochum, Germany

a r t i c l e i n f o a b s t r a c t

Article history: There are still conflicting results on the suitability of chemical and microbiological parameters as indica-
Received 19 February 2010 tors for the viral contamination of surface waters. In this study, conducted over 20 months, the abundance
Received in revised form 7 May 2010 of human adenovirus, human polyomavirus, enterovirus, group A rotavirus and norovirus was deter-
Accepted 7 May 2010
mined in Ruhr and Rhine rivers, Germany. Additionally, prevalence of different possible indicators such
as somatic coliphages, E. coli, intestinal enterococci, and total coliforms was also considered. Moreover, the
Keywords:
chemical parameter TCPP (tris-(2-chloro-, 1-methyl-ethyl)-phosphate), characterized by environmental
Surface water
stability and human origin, was included. Furthermore, chemical parameters (fluoride, chloride, nitrate,
Enteric viruses
Somatic coliphages
nitrite, bromide, phosphate, and sulfate) which may influence the stability and subsequently the detec-
TCPP tion rates of viruses in aquatic environment were measured. Quantitative Real-Time (RT-)PCR and double
Indicator agar layer test were used for the quantification of human enteric viruses and somatic coliphages, respec-
tively. The analyses for E. coli, total coliforms, and intestinal enterococci were done with respect to the
standard reference method. The chemical parameters were measured by liquid chromatography of ions
and by gas chromatography-flame photometer detector (GC-FPD), respectively. We demonstrated that
human adenovirus had the highest detection rate (96.3%), followed by somatic coliphages (73.5%), human
polyomavirus (68.6%), and rotavirus (63.5%). However, norovirus GII and enterovirus were found in only
25.7 and 17.8%, respectively. The concentration of the viral genome ranged between 16 and 1.1 × 106 gen.
equ./l (genome equivalents/l) whereas the concentrations for TCPP ranged between 0.01 and 0.9 ␮g/l. The
results of the Pearson correlation showed no association between TCPP and any other microbiological
parameter. None of the other tested chemical parameters correlated negatively, and therefore they do
not influence the stability of enteric viruses. We conclude that neither TCPP nor any other chemical or
microbiological parameter can be used as a reliable indicator for the presence of enteric viruses in river
water.
© 2010 Published by Elsevier GmbH.

Introduction of the drinking water supply, a bypass connection to an irrigation

Several years ago more than 100 species of viruses have been
isolated from sewage water (Cukor and Blacklow, 1984). It is well
known that pathogenic microorganisms may enter surface waters
through discharges of raw and treated sewage, and also manure
runoff from agricultural land (Pusch et al., 2005; van den Berg et
al., 2005). Some enteric viruses have been related to waterborne
outbreaks by a fecal contamination of drinking water. This con-
tamination may be caused by sewage or surface water, breakdown
∗ Corresponding author. Tel.: +49 234 32 28931; fax: +49 234 32 14199.
E-mail address: jurzik@hygiene.rub.de (L. Jurzik).
1 indicator microorganisms.
These authors contributed equally to this work.
2
Permanent address: Environmental Virology Laboratory, Department of Water
Pollution Research, National Research Centre, 12311 Dokki, Cairo, Egypt.
system or leakages in the water distribution system (Hafliger et
al., 2000). Some waterborne outbreaks with enteric viruses have
been described in the last years. One of them took place in southern
Italy with 344 norovirus (NoV) infected people (Boccia et al., 2002).
Additionally, 460 people were infected with enterovirus (EV) dur-
ing an outbreak in Belarus (Amvrosieva et al., 2001). Furthermore, a
recent waterborne norovirus outbreak occurred in Podgorica (Mon-
tenegro) with 1699 people infected due to contaminated municipal
water (Werber et al., 2009).
The main problem with determining enteric viruses in water
directly is that the detection methods like cell culture and
Real-Time PCR, or a combination of both, are expensive, time-
consuming, and labor-intensive. This leads to the concept of
Parameters like somatic coliphages, E. coli, total coliforms, and
intestinal enterococci are often discussed as indicators for enteric

1438-4639/$ – see front matter © 2010 Published by Elsevier GmbH.

doi:10.1016/j.ijheh.2010.05.005
 L. Jurzik et al. / International Journal of Hygiene and Environmental Health 213 (2010) 210–216
Fig. 1. Location of sampling sites, sewage treatment plants and direct discharge. Samples were taken biweekly with a volume of 10 l.
viruses in surface water. Somatic coliphages use E. coli and closely
related species as hosts and hence can be released by these bac-
terial hosts into the feces of humans and other warm-blooded
animals. Coliphages used in water quality assessment are divided
into the major groups of somatic coliphages and F-specific RNA
(F-RNA) phages, which are most frequently studied (Skraber et al.,
2004a). Phages share many properties with human viruses, notably
composition, morphology, and structure. Due to their resistance
against environmental factors, somatic coliphages are more appli-
cable than fecal bacteria for indicating fecal contamination of water
(Contreras-Coll et al., 2002).
In the present study, an additional chemical parameter,
the organophosphate TCPP, Tris (2-chloro-, 1-methyl-ethyl)-
phosphate, has been included to find a suitable indicator for enteric
viruses in surface waters. As could be shown in a human biomoni-
toring study TCPP is absorbed and distributed to the whole body and
the metabolites are excreted via urine (Schindler et al., 2009). Due
to its high environmental stability it passes the sewage treatment
plant without reduction and afterwards enters the surface water
(Andresen et al., 2004; Fries and Puttmann, 2001). Supportive to the
human biomonitoring study a high correlation (R2 = 0.92) between
the TCPP charge of the outlet of sewage treatment plants and the
associated population has been demonstrated (MUNLV, 2008). For
this reasons we tried to evaluate its suitability as indicator for con-
tamination of river water with human enteric viruses.
In addition to the microbiological parameters and TCPP, this
paper also considers those factors which may influence the stability
and the survival of viruses in river water.
Presently, there are no suitable models developed that can reli-
ably predict the presence of enteric viruses in surface water, and
there is less concordance between the published data concerning
the suitability of some viruses/coliphages as indicators for enteric
viruses in water. In our recent study we detected at the Ruhr and
Rhine rivers (water supply of about 5.3 million people) nucleic
acids of several human pathogenic viruses during winter 2007/08
(Hamza et al., 2009). In this study water samples have been tested
211
over a period of 20 months to determine the relation between
human viruses and some chemical and microbiological parameters
in surface waters in Germany.
Methods
Study area
The water samples were collected from five sampling sites
located along the river Ruhr, which meets the Rhine at Duisburg
(North Rhine-Westphalia, NRW), Germany. In detail, water sam-
ples were collected near to Hagen, Bochum, Essen, Muelheim (all
situated along the river Ruhr in the given order), and Duesseldorf
(river Rhine, 30 km upstream of Duisburg). The distances to the
next upstream sewage treatment plant is 1.5–10 km. The location
of sampling sites is shown in Fig. 1.
Sampling and virus concentration
Water samples (10 l each) were collected from February 2008 to
September 2009, in a depth of minimum 0.3 m, to avoid disinfectant
effect of the UV light (Hijnen et al., 2006). Water temperature and
the pH value were measured in situ.
The samples were transported within the next 4 h to the labora-
tory and were subsequently analyzed. The concentration of viruses
and coliphages by filtration procedure (VIRADEL) has already been
described before, followed by PEG-6000 precipitation as a re-
concentration step (Hamza et al., 2009).
In brief MgCl2 was added to the sample with a final concentra-
tion of 0.05 M. Additionally, the pH was adjusted at 3.5 with HCl
(APHA, 1998). A negatively charged membrane with a pore size
of 0.45 ␮m (Millipore, Bedford, USA) and a diameter of 142 mm
was used. After filtration the membrane was rinsed with 200 ml of
0.5 mM H2 SO4 with a pH of 3.5. The viruses were eluted with 70 ml
of a buffer containing 0.05 M KH2 PO4 , 1 M NaCl, 0.1% Triton X-100
with a pH of 9.2. The eluate was neutralized using 1N NaOH. After-
212 L. Jurzik et al. / International Journal of Hygiene and Environmental Health 213 (2010) 210–216

wards it was reconcentrated by adding 12.5% polyethylene glycol Table 1

Detection rate of microbiological parameters measured during our study. For HAdV,
(PEG-6000, Merck, Darmstadt, Germany) and 2.5% NaCl. The eluate
E. coli, total coliforms and intestinal enterococci more than 90% of all collected
was stirred at 4 ◦ C for 4 h, centrifuged at 10,000 × g for 30 min and samples were positive.
pellet was suspended in 3 ml PBS.
Parameter N No. (%) of No. (%) of
positive negative
Quantitative Real-Time PCR samples samples

HAdV 190 183 (96.3) 7 (3.7)

Viral nucleic acids were extracted from 200 ␮l of the con- HPyV 188 129 (68.6) 59 (31.4)
centrated virus suspension using the QIAamp DNA Blood Mini NoV GII 187 48 (25.7) 139 (74.3)
EV 174 31 (17.8) 143 (82.2)
Kit (Qiagen, Hilden, Germany) according to the manufacturer’s
RoV 181 115 (63.5) 66 (36.5)
instructions. The Real-Time (RT-) PCR was performed as previously Somatic coliphages 185 136 (73.5) 49 (26.5)
described (Hamza et al., 2009), except Taqman probe was used E. coli 182 150 (82.4) 32 (17.6)
instead of sybr green protocol for human adenovirus (HAdV) qPCR Total coliforms 185 179 (96.8) 6 (3.2)
i enterococci 192 175 (91.1) 21 (10.9)
to increase the specificity to human adenovirus strains (Heim et
al., 2003). In brief, the PCR was carried out using Quantitect probe HAdV – adenovirus; EV – enterovirus; NoV GII – norovirus GII; RoV – rotavirus;
RT-PCR kit (Qiagen, Hilden, Germany). The amplification conditions HPyV – human polyomavirus; i enterococci – intestinal enterococci.

were optimized for each virus group and were as follows: (i) HAdV;
95 ◦ C for 15 min; 45 cycles of 95 ◦ C for 15 s, 60 ◦ C for 60 s, (ii) RoV;
50 ◦ C for 30 min, 95 ◦ C for 15 min, 45 cycles of 95 ◦ C for 15 s, 56 ◦ C for
30 s, 72 ◦ C for 1 min. (iii) NoV GII; 50 ◦ C for 30 min, 95 ◦ C for 15 min,
10 cycles of 95 ◦ C for 15 s, 56 ◦ C for 1 min, 35 cycles of 95 ◦ C for 15 s,
63 ◦ C for 1 min. (iv) HPyV; 95 ◦ C for 15 min, 50 cycles of 95 ◦ C for
15 s, 60 ◦ C for 1 min. (v) EV; 50 ◦ C for 30 min, 95 ◦ C for 15 min, 45
cycles of 95 ◦ C for 15 s, 60 ◦ C for 1 min. The specificity of the PCR
assay was tested previously by sequence analysis (Hamza et al.,
2009).
Detection limit of the assay and PCR inhibition control
was tested as has been published before by Hamza et al.
(2009).
Quantification of somatic coliphages
Somatic coliphages were quantified using the double layer
plaque assay according to the method of the International Orga-
nization for Standardization (ISO, 2002). For surface water the E.
coli strain DSM 13127 was used.
Bacteriological analysis
The bacteriological analyses were done according to the method
of the International Organization for Standardization (ISO, 2001).
For E. coli, total coliforms, and enterococci detection: 100 ml of
water was passed through 0.45 ␮m membrane filters (47 mm
diameter, Millipore, Bedford, USA), which were subsequently
placed onto different culture media (Oxoid, Cambridge, UK). E. coli
and total coliforms: lactose utilization on lactose-TTC agar, then
oxidase- and indol-testing. Intestinal enterococci: a two step selec-
tive agar method with Slanetz-Bartley- and Aesculin agar (ISO,
2000).
Chemical analysis
The chemical analysis of dissolved bromate and chloride,
fluoride, nitrate, nitrite, phosphate and sulfate was conducted
according to the method of the International Organization for
Standardization (DIN, 2001, 2008) and was done by ICS-90 liq-
uid chromatography of ions (Dionex, USA). TCPP in surface water
was analyzed by gas chromatography-flame photometer detector
(GC-FPD) after solid phase extraction (SPE). The method has been
described previously by Prösch et al. (2002).
The detection limits for the chemical parameters were: 0.1 mg/l
for fluoride, 0.002 mg/l for bromate, 1.0 mg/l for chloride, 0.02 mg/l
for nitrite, 1.0 mg/l for nitrate, 0.1 mg/l for phosphate, 1.0 mg/l for
sulfate, and 0.02 ␮g/l for TCPP.
Statistical analysis
Statistical analysis was performed to investigate the association
between microbiological and viral load in surface waters. For the
chemical parameters the data below the detection limit were set
to 1/2 of the detection limit. Statistica software (9.0) was used to
calculate the Pearsons correlation. To evaluate the concentration of
the detected parameters at the different sampling site ANOVA test
was performed.
Results
Detection of viral nucleic acids and bacteria
Water samples were taken at five different sampling sites along
the rivers Ruhr and Rhine over a period of 20 months and were
concentrated using the VIRADEL method. For each parameter a
statistical test was performed to clarify whether there are signifi-
cant differences for the concentration of the detected parameters
between the five sampling sites. Only for somatic coliphages a sig-
nificant difference was found (data not shown). Table 1 represents
detection rate of microbiological parameters measured during the
study, whereas the descriptive statistic for the viral parameters is
presented in Table 2. HAdV (human adenovirus, n = 190) showed
the highest detection rate (96.3%) with a median concentration of
2.9 × 103 gen. equ./l and a maximum of 7.3 × 105 gen. equ./l. While
the detection rates of HPyV (human polyomavirus, n = 188), somatic
coliphages (n = 185), and RoV (rotavirus, n = 181) were 68.6, 73.5,
and 63.5%, respectively. NoV GII (norovirus GII, n = 187) and EV
(enterovirus, n = 174) have been detected in only 25.7 and 17.8%
of the examined samples. All samples were negative for norovirus
GI.
Due to the fact that temperature is one of the main factors
influencing the stability of enteric viruses in surface water the
correlation with other microbiological parameters has been con-
ducted with different temperatures (i) ≥10 ◦ C and (ii) <10 ◦ C. The
temperature profile of water samples can be seen in Fig. 2.
Physic-chemical parameters
The median pH value was 7.6 (5.9–9.2) and the water temper-
ature ranged between 1.1 and 25.5 ◦ C. TCPP was measured in 50
samples, whereas the other parameters (fluoride, chloride, nitrite,
nitrate, bromide, phosphate and sulfate) were measured in 69
water samples. The median concentrations (range) were as follows:
TCPP 0.06 ␮g/l (0.01–0.9 ␮g/l), fluoride 0.1 mg/l (0.05–0.3 mg/l);
chloride 34.0 mg/l (13.0–5.7 × 102 mg/l); nitrite 0.073 mg/l
(0.02–0.6 mg/l); bromide 0.03 mg/l (0.001–1.0 mg/l); nitrate
 L. Jurzik et al. / International Journal of Hygiene and Environmental Health 213 (2010) 210–216 213

Table 2
Descriptive statistics of the physic-chemical- and microbiological parameters for the Ruhr- and Rhine river. The surface water samples have been collected from February
2008 to September 2009. For the calculation of median, min–max and percentile only the positive samples have been taken.

N Median Range Percentile 10–90%

HAdV (gen. equ./l) 183 2.9 × 103 5.7 × 101 to 7.3 × 105 1.2 × 102 to 1.8 × 104
EV (gen. equ./l) 31 7.8 × 103 1.0 × 102 to 1.1 × 106 6.3 × 102 to 5.8 × 104
NoV GII (gen. equ./l) 48 1.0 × 103 3.1 × 101 to 6.4 × 104 1.0 × 102 to 1.9 × 104
HPyV (gen. equ./l) 129 1.4 × 103 3.7 × 101 to 5.2 × 105 1.1 × 102 to 2.1 × 104
RoV (gen. equ./l) 115 3.5 × 103 1.6 × 101 to 3.8 × 105 1.1 × 102 to 4.5 × 104
Somatic coliphages (pfu/l) 136 1.4 × 102 3.0–8.1 × 104 1.6 × 102 to 4.0 × 104
E. coli (cfu/100 ml) 150 1.1 × 103 1.0 × 101 to 4.1 × 104 1.6 × 102 to 9.0 × 103
Total coliforms (cfu/100 ml) 179 1.4 × 103 1.0 × 101 to 4.6 × 104 2.0 × 102 to 1.1 × 104
i enterococci (cfu/100 ml) 175 6.2 × 101 1.0–1.7 × 104 8.5–1.1 × 103
Fluoride (mg/l) 69 0.1 0.05–0.3 0.05–0.2
Chloride (mg/l) 69 34.0 13.0–5.7 × 102 21.1–2.8 × 102
Nitrite (mg/l) 69 0.073 0.02–0.6 0.02–0.2
Bromide (mg/l) 69 0.03 0.001–1.0 0.001–0.1
Nitrate (mg/l) 69 15.1 9.0–23.0 12.0–18.2
Phosphate (mg/l) 69 0.05 0.05–0.15 0.05–0.1
Sulfate (mg/l) 69 40.3 22.0–1.2 × 102 31.0–85.9
TCPP (␮g/l) 50 0.06 0.01–0.9 0.01–0.3

cfu/100 ml – colony forming unit per 100 ml; gen. equ./l – genome equivalent per liter; pfu/1 l – plaque forming unit per liter; HAdV – adenovirus; EV – enterovirus; NoV GII
– norovirus GII; RoV – rotavirus; HPyV – human polyomavirus; i enterococci – intestinal enterococci.

15.1 mg/l (9.0–23.0 mg/l); phosphate 0.05 mg/l (0.05–0.15 mg/l);
sulfate 40.3 mg/l (22.0–1.2 × 102 mg/l) (Table 1).
Correlation among measured parameters
With respect to the water temperature three correlation anal-
yses have been performed (Table 3). At temperatures ≥10 ◦ C:
somatic coliphages, E. coli, total coliforms, and intestinal ente-
rococci showed significant correlation with HPyV. Whereas at
temperatures <10 ◦ C: E. coli and total coliforms showed signifi-
cant association with RoV. Taken together the two temperature
profiles a slight change for association between E. coli and total
coliforms with RoV was observed. The relation between E. coli, and
total coliforms were stable throughout the year, while the corre-
lation between enterococci and E. coli or coliforms showed high
variation for the two temperature profiles. No correlation could be
detected for HAdV and other tested parameters. Due to the fact that
the detection rate of NoV GII and EV is lower than 50% we felt that
it is not advisable to perform a correlation analysis.
In general the correlation of chemical parameters was weak or
moderate (r = 0.24–0.35) with bacteria and viruses (Table 4). For
TCPP no correlation with any microbiological parameter could be
detected. Nitrate and phosphates shows a moderate correlation
with intestinal enterococci (r = 0.48). This was also the case for
phosphate and somatic coliphages (r = 0.54). But none of the viral
parameters showed significant association with chemical parame-
ters.
Discussion
This 20-month study was designed to determine the rela-
tionship between enteric viruses, bacteria, and TCPP. The other
physic-chemical parameters along the Ruhr and Rhine river were
measured to find out if they correlate with the detection rate or
concentrations of enteric viruses or not. High dissemination rate of
enteric viruses and bacterial fecal indicators was found in Ruhr and
Rhine rivers, 96.3% of the samples were positive for HAdV, 96.8%
for total coliforms, 91.1% for intestinal enterococci, 68.6% for HPyV,

Table 3
Pearson correlation between measured parameters. The data for the correlation analysis were divided into two groups: (i) temperatures ≥10 ◦ C and (ii) temperatures <10 ◦ C.
The results are shown in this table. The level of significance is p < 0.05 (written in bold). Due to the low detection rate of enterovirus (EV) and norovirus GII (NoV GII) (<50%)
no correlation has been conducted with these parameters. Missing data have been casewise deleted and positive and negative results have been included. HAdV – human
adenovirus; RoV – rotavirus; HPyV – human polyomavirus; i enterococci – intestinal enterococci.

Parameter Parameter (r)

Temperature ≥10◦ C; N = 63 Temperature <10 ◦ C; N = 63 Both temperatures; N = 144

HAdV HPyV (−0.08) HPyV (0.00) HPyV (0.01)

RoV (−0.03) RoV (−0.03) RoV (0.02)
Somatic coliphages (−0.07) Somatic coliphages (0.27) Somatic coliphages (0.12)
E. coli (−0.01) E. coli (−0.11) E. coli (−0.09)
Total coliforms (−0.03) Total coliforms (−0.13) Total coliforms (−0.10)
i enterococci (0.04) i enterococci (−0.09) i enterococci (−0.06)

HPyV RoV (0.09) RoV (0.15) RoV (0.17)

Somatic coliphages (0.41) Somatic coliphages (−0.07) Somatic coliphages (−0.03)
E. coli (0.49) E. coli (−0.10) E. coli (−0.04)
Total coliforms (0.67) Total coliforms (−0.12) Total coliforms (−0.06)
i enterococci (0.41) i enterococci (−0.08) i enterococci (−0.03)

RoV Somatic coliphages (−0.07) Somatic coliphages (−0.04) Somatic coliphages (−0.05)
E. coli (−0.02) E. coli (0.46) E. coli (0.31)
Total coliforms (−0.04) Total coliforms (0.46) Total coliforms (0.29)
i enterococci (−0.06) i enterococci (0.08) i enterococci (0.09)

E. coli Total coliforms (0.88) Total coliforms (0.97) Total coliforms (0.95)
i enterococci (0.80) i enterococci (0.44) i enterococci (0.47)

Total coliforms i enterococci (0.74) i enterococci (0.42) i enterococci (0.44)

214 L. Jurzik et al. / International Journal of Hygiene and Environmental Health 213 (2010) 210–216

Table 4
Pearson correlation of physic-chemical parameters and bacteria/viruses, respectively. In the grey fields the correlated parameters with a level of statistical significance
(p < 0.05) are listed. No correlation of TCPP with a microbiological parameter could be detected. Missing data have been casewise deleted.

Fluoride (r/p/N) Chloride (r/p/N) Nitrite (r/p/N) Bromide (r/p/N) Nitrate (r/p/N) Phosphate (r/p/N) Sulfate (r/p/N) TCPP (r/p/N)

E. coli −0.01 −0.15 0.17 0.12 0.41 0.26 −0.23 −0.01

0.95 0.23 0.18 0.32 0.00 0.04 0.07 0.96
65 65 65 65 65 65 65 49
Total coliforms −0.09 −0.15 0.26 0.24 0.43 0.38 −0.24 0.07
0.48 0.23 0.04 0.04 0.00 0.00 0.06 0.65
65 65 65 65 65 65 65 49
i enterococci −0.19 −0.25 0.08 0.38 0.48 0.47 −0.35 −0.13
0.12 0.04 0.50 0.00 0.00 0.00 0.00 0.36
69 69 69 69 69 69 69 50
HAdV −0.22 −0.21 −0.05 0.24 0.10 0.32 −0.32 −0.02
0.08 0.09 0.71 0.05 0.43 0.01 0.01 0.90
67 67 67 67 67 67 67 50
HPyV −0.10 −0.24 0.04 0.21 0.24 0.12 0.21 −0.01
0.46 0.04 0.73 0.08 0.04 0.35 0.08 0.95
67 67 67 67 67 67 67 49
RoV −0.25 −0.06 0.04 0.00 0.21 −0.13 −0.08 0.08
0.04 0.65 0.75 0.97 0.09 0.31 0.54 0.60
67 67 67 67 67 67 67 46
Somatic coliphages 0.02 −0.04 0.15 0.41 0.40 0.54 −0.16 −0.04
0.87 0.77 0.23 0.00 0.00 0.00 0.19 0.81
67 67 67 67 67 67 67 50

82.4% for E. coli, 73.5% for coliphages, 63.5% for RoV, 25.7% for NoV
GII, and 17.8% for EV. The high detection rate obtained for HAdV
and HPyV could be a result of their stability in surface and sewage
water. On the other hand, it is known that HPyV and HAdV are
excreted regularly in urine because they produce latent infections
in healthy individuals (Contreras-Coll et al., 2002; de Bruyn and
Limaye, 2004; Westrell et al., 2006). Besides, the high efficiency of
the virus concentration step might be the reason for the high detec-
tion rate of these viruses (Hamza et al., 2009). In contrast to HPyV
and HAdV other viruses like RoV are commonly detected during
winter time because the maximum of infection, and therefore the
maximum of excretion, is from February to April (Petrinca et al.,
2009). This varying seasonality might be the explanation for the
different correlations at temperatures <10 and ≥10 ◦ C. Although
the water temperature is a main factor influencing the persistence
of viruses, other parameters like hardness, turbidity, and UV light
might affect the stability of enteric viruses and bacteria (John and
Rose, 2005).
In our study it was clear that the correlation between differ-
ent microbial parameters could be changed throughout the year
Fig. 2. Median value of the water temperature at 2–4 sampling sites during the study
period from February 2008 to September 2009. The samples have been grouped into
two seasons: (i) ≥10 ◦ C and (ii) <10 ◦ C.
depending on the water temperature. We found that at tempera-
tures ≥10 ◦ C some parameters correlate with HPyV. Similarly, RoV
showed significant association with E. coli and total coliforms at
temperatures <10 ◦ C, while it was not the case at higher tempera-
tures.
The detection rates for HAdV was very high compared to other
studies; in South Africa 22.2% (10/45) and in California 52% (11/21)
of the samples were positive for HAdV. Moreover Genthe et al.
detected HAdV in 49–88% (29–53/60) of the surface water samples
(Genthe et al., 1995; Jiang and Chu, 2004; van Heerden et al., 2005).
Due to the high detection rates obtained for the nucleic acids of
HAdV (96.3%), it could be suggested as an indicator for the sewage
contamination of river water; the same finding has been observed
before (Albinana-Gimenez et al., 2006; Pina et al., 1998).
Somatic coliphages were detected in 73.5% of the examined
samples (185). The concentration of somatic coliphages ranged
between 3.0 and 8.1 × 104 pfu/l. Compared to another study in
France, the concentration ranges between 4 × 102 and 6 × 105 pfu/l,
we found lower levels of somatic coliphages in river water (Hot
et al., 2003). There are still conflicting results due to the question
whether somatic coliphages can reliable predict the viral con-
tamination of surface waters. There are some publications that
considered coliphages as good indicators of enteric viral pollution
because they have been detected in wastewater and other fecal con-
taminated waters in numbers at least equal to the enteric viruses
(Fannin et al., 1977; Kott, 1977; Kott et al., 1974). Hot et al. reported
that somatic coliphages are not suitable parameters for predicting
the presence of waterborne viruses and the results of our study
supports this finding (Hot et al., 2003). In our study, even the use of
three cut-off levels (>25, >50, and >100 pfu/l) did not show an asso-
ciation between somatic coliphages and human enteric viruses in
river water. Only at temperatures ≥10 ◦ C a correlation with HPyV
could be detected.
The lack of correlation of somatic coliphages with other viruses
might be explained with some limitations. At first they might infect
some closely related member of the Enterobacteriaceae so that they
even can be detected in surface water without fresh fecal contam-
ination (Hayes, 1968). Furthermore, some hosts may multiply or
metabolize in water environments to the extent that they sup-
port the replication of the phages (Grabow, 2001). Additionally,
there is a significant reproduction of bacteriophages, if the bacterial
concentration is higher than 104 cfu/ml (Wiggins and Alexander,
1985).
 L. Jurzik et al. / International Journal of Hygiene and Environmental Health 213 (2010) 210–216
As an alternative, F-RNA phages could be analyzed as possi-
ble indicators for enteric viruses. However, somatic coliphages
are detectable by relatively simple and inexpensive plaque assays,
which yield results within 24 h, plaque assays for F-RNA coliphages
are not quite as simple, because the culture of host bacteria has to
be in the logarithmic growth phase at a temperature above 30 ◦ C
to ensure that F-fimbriae are present (WHO, 2008). And this aspect
does not agree with the concept of indicator microorganisms.
Traditionally, bacterial indicators have been used to assess the
concentration of enteric viruses in waters. But the concentration
of fecal bacteria can provide some level of indication of enteric
viruses when the contamination originates from human sources.
This relationship may not exist when the source of pollution is of
animal origin (Payment et al., 2000). Additionally, the concentra-
tion of microorganisms in surface water fluctuates strongly and
this might affect the correlation between viruses and bacteria neg-
atively. Therefore some other indicator organisms are needed.
With this study we can support the findings of other working
groups who concluded that E. coli, total coliforms, and intestinal
enterococci cannot be applied as indicators for viral particles in
surface water (Skraber et al., 2004a,b).
In the present work, we studied the correlation between TCPP, a
flame retardant, and enteric viruses in river water. TCPP is absorbed
throughout the whole body, excreted via urine and TCPP is not regu-
larly measured but due to its stability and human origin it seemed to
be a promising tool to predict the concentration of enteric viruses in
surface water. In a study from 2004 the concentration of TCPP in the
Ruhr river ranged between 0.020 and 0.2 ␮g/l and the sewage treat-
ment plant effluents exhibit concentrations up to 0.4 ␮g/l. With a
median concentration of 0.06 ␮g/l (0.0.1–0.9 ␮g/l) our study is in
the line with these findings. We found no correlation with viruses
or bacteria. This lack of association might be due to the low number
of samples (n = 46–50). Even with a cut-off level of 0.05 ␮g/l no cor-
relation with any microbiological parameter could be detected. We
suggest that TCPP does not seem to be a good indicator for human
enteric viruses in river water. Parameters like nitrate, nitrite, phos-
phate, and ammonia may influence the stability of viruses. No
negative correlation between chemical and microbiological param-
eters was observed. The explanation might be that chemical and
microbiological parameters have different transformation or decay
rates, and therefore the elimination of some of them does not imply
the removal of others.
The data obtained by Real-Time PCR cannot discriminate
between infectious viral particles, non-infectious viral particles
and free nuclide acids. For the statistical analysis different recov-
ery rates of the viral parameters can lead to different detection
rates/concentration (e.g. for somatic coliphages and for HAdV), then
might influence the correlation coefficient. But when we tried to
find a correlation between the presence/absence of viruses without
considering the concentration we have nearly the same association
(r value).
Conclusion
Taken together, our findings indicate that none of the tested
parameters seems to be a good indicator for viral contamination
of river water. In addition, TCPP could not be a good indicator for
human enteric viruses in river water. Therefore, the ideal indication
is provided by the viral pathogen itself.
Acknowledgements
Ibrahim A. Hamza received a PhD fellowship from the Academy
of Scientific Research and Technology (ASRT) of Egypt. The authors
would like to thank Ulrike Bandow for her technical assistance, Elke
215
Uhlenbrock, Sabine Wagener and Gregor Perna for help in collecting
the samples.
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