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Chemical and microbiological parameters as possible indicators for human enteric viruses in surface water
International Journal of Hygiene and Environmental Health 213 (2010) 210–216
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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 b c
Department of Hygiene, Social and Environmental Medicine, Ruhr-University Bochum, Germany Health Department of the State of Mecklenburg-West Pomerania, Schwerin, Germany Department of Molecular and Medical Virology, Ruhr-University Bochum, Germany
a r t i c l e
i n f o
Article history: Received 19 February 2010 Received in revised form 7 May 2010 Accepted 7 May 2010 Keywords: Surface water Enteric viruses Somatic coliphages TCPP Indicator
a b s t r a c t There are still conflicting results on the suitability of chemical and microbiological parameters as indicators for the viral contamination of surface waters. In this study, conducted over 20 months, the abundance of human adenovirus, human polyomavirus, enterovirus, group A rotavirus and norovirus was determined 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 chemical parameter TCPP (tris-(2-chloro-, 1-methyl-ethyl)-phosphate), characterized by environmental stability and human origin, was included. Furthermore, chemical parameters (fluoride, chloride, nitrate, nitrite, bromide, phosphate, and sulfate) which may influence the stability and subsequently the detection rates of viruses in aquatic environment were measured. Quantitative Real-Time (RT-)PCR and double agar layer test were used for the quantification of human enteric viruses and somatic coliphages, respectively. 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 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 contamination may be caused by sewage or surface water, breakdown
∗ Corresponding author. Tel.: +49 234 32 28931; fax: +49 234 32 14199. E-mail address: [email protected] (L. Jurzik). 1 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. 1438-4639/$ – see front matter © 2010 Published by Elsevier GmbH. doi:10.1016/j.ijheh.2010.05.005
of the drinking water supply, a bypass connection to an irrigation 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) during an outbreak in Belarus (Amvrosieva et al., 2001). Furthermore, a recent waterborne norovirus outbreak occurred in Podgorica (Montenegro) 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, timeconsuming, and labor-intensive. This leads to the concept of indicator microorganisms. Parameters like somatic coliphages, E. coli, total coliforms, and intestinal enterococci are often discussed as indicators for enteric
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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 bacterial 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 applicable 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 biomonitoring 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 contamination 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 reliably 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
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 samples 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 laboratory 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 reconcentration step (Hamza et al., 2009). In brief MgCl2 was added to the sample with a final concentration 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-
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wards it was reconcentrated by adding 12.5% polyethylene glycol (PEG-6000, Merck, Darmstadt, Germany) and 2.5% NaCl. The eluate was stirred at 4 ◦ C for 4 h, centrifuged at 10,000 × g for 30 min and pellet was suspended in 3 ml PBS.
Table 1 Detection rate of microbiological parameters measured during our study. For HAdV, E. coli, total coliforms and intestinal enterococci more than 90% of all collected samples were positive. Parameter
N
No. (%) of positive samples
No. (%) of negative samples
HAdV HPyV NoV GII EV RoV Somatic coliphages E. coli Total coliforms i enterococci
190 188 187 174 181 185 182 185 192
183 (96.3) 129 (68.6) 48 (25.7) 31 (17.8) 115 (63.5) 136 (73.5) 150 (82.4) 179 (96.8) 175 (91.1)
7 (3.7) 59 (31.4) 139 (74.3) 143 (82.2) 66 (36.5) 49 (26.5) 32 (17.6) 6 (3.2) 21 (10.9)
Quantitative Real-Time PCR Viral nucleic acids were extracted from 200 l of the concentrated virus suspension using the QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The Real-Time (RT-) PCR was performed as previously described (Hamza et al., 2009), except Taqman probe was used instead of sybr green protocol for human adenovirus (HAdV) qPCR to increase the specificity to human adenovirus strains (Heim et al., 2003). In brief, the PCR was carried out using Quantitect probe RT-PCR kit (Qiagen, Hilden, Germany). The amplification conditions 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).
HAdV – adenovirus; EV – enterovirus; NoV GII – norovirus GII; RoV – rotavirus; HPyV – human polyomavirus; i enterococci – intestinal enterococci.
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
Quantification of somatic coliphages Somatic coliphages were quantified using the double layer plaque assay according to the method of the International Organization 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 selective 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 liquid 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.
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 significant differences for the concentration of the detected parameters between the five sampling sites. Only for somatic coliphages a significant 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 conducted 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 temperature 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
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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.
HAdV (gen. equ./l) EV (gen. equ./l) NoV GII (gen. equ./l) HPyV (gen. equ./l) RoV (gen. equ./l) Somatic coliphages (pfu/l) E. coli (cfu/100 ml) Total coliforms (cfu/100 ml) i enterococci (cfu/100 ml) Fluoride (mg/l) Chloride (mg/l) Nitrite (mg/l) Bromide (mg/l) Nitrate (mg/l) Phosphate (mg/l) Sulfate (mg/l) TCPP (g/l)
N
Median
Range
Percentile 10–90%
183 31 48 129 115 136 150 179 175 69 69 69 69 69 69 69 50
2.9 × 103 7.8 × 103 1.0 × 103 1.4 × 103 3.5 × 103 1.4 × 102 1.1 × 103 1.4 × 103 6.2 × 101 0.1 34.0 0.073 0.03 15.1 0.05 40.3 0.06
5.7 × 101 to 7.3 × 105 1.0 × 102 to 1.1 × 106 3.1 × 101 to 6.4 × 104 3.7 × 101 to 5.2 × 105 1.6 × 101 to 3.8 × 105 3.0–8.1 × 104 1.0 × 101 to 4.1 × 104 1.0 × 101 to 4.6 × 104 1.0–1.7 × 104 0.05–0.3 13.0–5.7 × 102 0.02–0.6 0.001–1.0 9.0–23.0 0.05–0.15 22.0–1.2 × 102 0.01–0.9
1.2 × 102 to 1.8 × 104 6.3 × 102 to 5.8 × 104 1.0 × 102 to 1.9 × 104 1.1 × 102 to 2.1 × 104 1.1 × 102 to 4.5 × 104 1.6 × 102 to 4.0 × 104 1.6 × 102 to 9.0 × 103 2.0 × 102 to 1.1 × 104 8.5–1.1 × 103 0.05–0.2 21.1–2.8 × 102 0.02–0.2 0.001–0.1 12.0–18.2 0.05–0.1 31.0–85.9 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 analyses have been performed (Table 3). At temperatures ≥10 ◦ C: somatic coliphages, E. coli, total coliforms, and intestinal enterococci showed significant correlation with HPyV. Whereas at temperatures <10 ◦ C: E. coli and total coliforms showed significant 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 correlation 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 parameters. Discussion This 20-month study was designed to determine the relationship 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) RoV (−0.03) Somatic coliphages (−0.07) E. coli (−0.01) Total coliforms (−0.03) i enterococci (0.04)
HPyV (0.00) RoV (−0.03) Somatic coliphages (0.27) E. coli (−0.11) Total coliforms (−0.13) i enterococci (−0.09)
HPyV (0.01) RoV (0.02) Somatic coliphages (0.12) E. coli (−0.09) Total coliforms (−0.10) i enterococci (−0.06)
HPyV
RoV (0.09) Somatic coliphages (0.41) E. coli (0.49) Total coliforms (0.67) i enterococci (0.41)
RoV (0.15) Somatic coliphages (−0.07) E. coli (−0.10) Total coliforms (−0.12) i enterococci (−0.08)
RoV (0.17) Somatic coliphages (−0.03) E. coli (−0.04) Total coliforms (−0.06) i enterococci (−0.03)
RoV
Somatic coliphages (−0.07) E. coli (−0.02) Total coliforms (−0.04) i enterococci (−0.06)
Somatic coliphages (−0.04) E. coli (0.46) Total coliforms (0.46) i enterococci (0.08)
Somatic coliphages (−0.05) E. coli (0.31) Total coliforms (0.29) i enterococci (0.09)
E. coli
Total coliforms (0.88) i enterococci (0.80)
Total coliforms (0.97) i enterococci (0.44)
Total coliforms (0.95) i enterococci (0.47)
Total coliforms
i enterococci (0.74)
i enterococci (0.42)
i enterococci (0.44)
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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.
E. coli
Total coliforms
i enterococci
HAdV
HPyV
RoV
Somatic coliphages
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)
−0.01 0.95 65 −0.09 0.48 65 −0.19 0.12 69 −0.22 0.08 67 −0.10 0.46 67 −0.25 0.04 67 0.02 0.87 67
−0.15 0.23 65 −0.15 0.23 65 −0.25 0.04 69 −0.21 0.09 67 −0.24 0.04 67 −0.06 0.65 67 −0.04 0.77 67
0.17 0.18 65 0.26 0.04 65 0.08 0.50 69 −0.05 0.71 67 0.04 0.73 67 0.04 0.75 67 0.15 0.23 67
0.12 0.32 65 0.24 0.04 65 0.38 0.00 69 0.24 0.05 67 0.21 0.08 67 0.00 0.97 67 0.41 0.00 67
0.41 0.00 65 0.43 0.00 65 0.48 0.00 69 0.10 0.43 67 0.24 0.04 67 0.21 0.09 67 0.40 0.00 67
0.26 0.04 65 0.38 0.00 65 0.47 0.00 69 0.32 0.01 67 0.12 0.35 67 −0.13 0.31 67 0.54 0.00 67
−0.23 0.07 65 −0.24 0.06 65 −0.35 0.00 69 −0.32 0.01 67 0.21 0.08 67 −0.08 0.54 67 −0.16 0.19 67
−0.01 0.96 49 0.07 0.65 49 −0.13 0.36 50 −0.02 0.90 50 −0.01 0.95 49 0.08 0.60 46 −0.04 0.81 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 detection 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 different 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 temperatures ≥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 temperatures. 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 contamination 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 contaminated 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 association 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 contamination (Hayes, 1968). Furthermore, some hosts may multiply or metabolize in water environments to the extent that they support 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).
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As an alternative, F-RNA phages could be analyzed as possible 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 concentration of microorganisms in surface water fluctuates strongly and this might affect the correlation between viruses and bacteria negatively. 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 regularly 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 treatment 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 correlation 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, phosphate, and ammonia may influence the stability of viruses. No negative correlation between chemical and microbiological parameters 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 recovery 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
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Uhlenbrock, Sabine Wagener and Gregor Perna for help in collecting the samples. References Albinana-Gimenez, N., Clemente-Casares, P., Bofill-Mas, S., Hundesa, A., Ribas, F., Girones, R., 2006. Distribution of human polyomaviruses, adenoviruses, and hepatitis E virus in the environment and in a drinking-water treatment plant. Environ. Sci. Technol. 40, 7416–7422. Amvrosieva, T.V., Titov, L.P., Mulders, M., Hovi, T., Dyakonova, O.V., Votyakov, V.I., Kvacheva, Z.B., Eremin, V.F., Sharko, R.M., Orlova, S.V., et al., 2001. Viral water contamination as the cause of aseptic meningitis outbreak in Belarus. Cent. Eur. J. Public Health 9, 154–157. Andresen, J.A., Grundmann, A., Bester, K., 2004. Organophosphorus flame retardants and plasticisers in surface waters. Sci. Total Environ. 332, 155–166. APHA, American Public Health Association, 1998. Standard Methods for the Examination of Water and Wastewater. American Public Health Association, Washington, D.C. 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Contents lists available at ScienceDirect
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 b c
Department of Hygiene, Social and Environmental Medicine, Ruhr-University Bochum, Germany Health Department of the State of Mecklenburg-West Pomerania, Schwerin, Germany Department of Molecular and Medical Virology, Ruhr-University Bochum, Germany
a r t i c l e
i n f o
Article history: Received 19 February 2010 Received in revised form 7 May 2010 Accepted 7 May 2010 Keywords: Surface water Enteric viruses Somatic coliphages TCPP Indicator
a b s t r a c t There are still conflicting results on the suitability of chemical and microbiological parameters as indicators for the viral contamination of surface waters. In this study, conducted over 20 months, the abundance of human adenovirus, human polyomavirus, enterovirus, group A rotavirus and norovirus was determined 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 chemical parameter TCPP (tris-(2-chloro-, 1-methyl-ethyl)-phosphate), characterized by environmental stability and human origin, was included. Furthermore, chemical parameters (fluoride, chloride, nitrate, nitrite, bromide, phosphate, and sulfate) which may influence the stability and subsequently the detection rates of viruses in aquatic environment were measured. Quantitative Real-Time (RT-)PCR and double agar layer test were used for the quantification of human enteric viruses and somatic coliphages, respectively. 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 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 contamination may be caused by sewage or surface water, breakdown
∗ Corresponding author. Tel.: +49 234 32 28931; fax: +49 234 32 14199. E-mail address: [email protected] (L. Jurzik). 1 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. 1438-4639/$ – see front matter © 2010 Published by Elsevier GmbH. doi:10.1016/j.ijheh.2010.05.005
of the drinking water supply, a bypass connection to an irrigation 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) during an outbreak in Belarus (Amvrosieva et al., 2001). Furthermore, a recent waterborne norovirus outbreak occurred in Podgorica (Montenegro) 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, timeconsuming, and labor-intensive. This leads to the concept of indicator microorganisms. Parameters like somatic coliphages, E. coli, total coliforms, and intestinal enterococci are often discussed as indicators for enteric
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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 bacterial 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 applicable 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 biomonitoring 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 contamination 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 reliably 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
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 samples 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 laboratory 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 reconcentration step (Hamza et al., 2009). In brief MgCl2 was added to the sample with a final concentration 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-
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wards it was reconcentrated by adding 12.5% polyethylene glycol (PEG-6000, Merck, Darmstadt, Germany) and 2.5% NaCl. The eluate was stirred at 4 ◦ C for 4 h, centrifuged at 10,000 × g for 30 min and pellet was suspended in 3 ml PBS.
Table 1 Detection rate of microbiological parameters measured during our study. For HAdV, E. coli, total coliforms and intestinal enterococci more than 90% of all collected samples were positive. Parameter
N
No. (%) of positive samples
No. (%) of negative samples
HAdV HPyV NoV GII EV RoV Somatic coliphages E. coli Total coliforms i enterococci
190 188 187 174 181 185 182 185 192
183 (96.3) 129 (68.6) 48 (25.7) 31 (17.8) 115 (63.5) 136 (73.5) 150 (82.4) 179 (96.8) 175 (91.1)
7 (3.7) 59 (31.4) 139 (74.3) 143 (82.2) 66 (36.5) 49 (26.5) 32 (17.6) 6 (3.2) 21 (10.9)
Quantitative Real-Time PCR Viral nucleic acids were extracted from 200 l of the concentrated virus suspension using the QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The Real-Time (RT-) PCR was performed as previously described (Hamza et al., 2009), except Taqman probe was used instead of sybr green protocol for human adenovirus (HAdV) qPCR to increase the specificity to human adenovirus strains (Heim et al., 2003). In brief, the PCR was carried out using Quantitect probe RT-PCR kit (Qiagen, Hilden, Germany). The amplification conditions 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).
HAdV – adenovirus; EV – enterovirus; NoV GII – norovirus GII; RoV – rotavirus; HPyV – human polyomavirus; i enterococci – intestinal enterococci.
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
Quantification of somatic coliphages Somatic coliphages were quantified using the double layer plaque assay according to the method of the International Organization 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 selective 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 liquid 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.
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 significant differences for the concentration of the detected parameters between the five sampling sites. Only for somatic coliphages a significant 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 conducted 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 temperature 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
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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.
HAdV (gen. equ./l) EV (gen. equ./l) NoV GII (gen. equ./l) HPyV (gen. equ./l) RoV (gen. equ./l) Somatic coliphages (pfu/l) E. coli (cfu/100 ml) Total coliforms (cfu/100 ml) i enterococci (cfu/100 ml) Fluoride (mg/l) Chloride (mg/l) Nitrite (mg/l) Bromide (mg/l) Nitrate (mg/l) Phosphate (mg/l) Sulfate (mg/l) TCPP (g/l)
N
Median
Range
Percentile 10–90%
183 31 48 129 115 136 150 179 175 69 69 69 69 69 69 69 50
2.9 × 103 7.8 × 103 1.0 × 103 1.4 × 103 3.5 × 103 1.4 × 102 1.1 × 103 1.4 × 103 6.2 × 101 0.1 34.0 0.073 0.03 15.1 0.05 40.3 0.06
5.7 × 101 to 7.3 × 105 1.0 × 102 to 1.1 × 106 3.1 × 101 to 6.4 × 104 3.7 × 101 to 5.2 × 105 1.6 × 101 to 3.8 × 105 3.0–8.1 × 104 1.0 × 101 to 4.1 × 104 1.0 × 101 to 4.6 × 104 1.0–1.7 × 104 0.05–0.3 13.0–5.7 × 102 0.02–0.6 0.001–1.0 9.0–23.0 0.05–0.15 22.0–1.2 × 102 0.01–0.9
1.2 × 102 to 1.8 × 104 6.3 × 102 to 5.8 × 104 1.0 × 102 to 1.9 × 104 1.1 × 102 to 2.1 × 104 1.1 × 102 to 4.5 × 104 1.6 × 102 to 4.0 × 104 1.6 × 102 to 9.0 × 103 2.0 × 102 to 1.1 × 104 8.5–1.1 × 103 0.05–0.2 21.1–2.8 × 102 0.02–0.2 0.001–0.1 12.0–18.2 0.05–0.1 31.0–85.9 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 analyses have been performed (Table 3). At temperatures ≥10 ◦ C: somatic coliphages, E. coli, total coliforms, and intestinal enterococci showed significant correlation with HPyV. Whereas at temperatures <10 ◦ C: E. coli and total coliforms showed significant 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 correlation 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 parameters. Discussion This 20-month study was designed to determine the relationship 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) RoV (−0.03) Somatic coliphages (−0.07) E. coli (−0.01) Total coliforms (−0.03) i enterococci (0.04)
HPyV (0.00) RoV (−0.03) Somatic coliphages (0.27) E. coli (−0.11) Total coliforms (−0.13) i enterococci (−0.09)
HPyV (0.01) RoV (0.02) Somatic coliphages (0.12) E. coli (−0.09) Total coliforms (−0.10) i enterococci (−0.06)
HPyV
RoV (0.09) Somatic coliphages (0.41) E. coli (0.49) Total coliforms (0.67) i enterococci (0.41)
RoV (0.15) Somatic coliphages (−0.07) E. coli (−0.10) Total coliforms (−0.12) i enterococci (−0.08)
RoV (0.17) Somatic coliphages (−0.03) E. coli (−0.04) Total coliforms (−0.06) i enterococci (−0.03)
RoV
Somatic coliphages (−0.07) E. coli (−0.02) Total coliforms (−0.04) i enterococci (−0.06)
Somatic coliphages (−0.04) E. coli (0.46) Total coliforms (0.46) i enterococci (0.08)
Somatic coliphages (−0.05) E. coli (0.31) Total coliforms (0.29) i enterococci (0.09)
E. coli
Total coliforms (0.88) i enterococci (0.80)
Total coliforms (0.97) i enterococci (0.44)
Total coliforms (0.95) i enterococci (0.47)
Total coliforms
i enterococci (0.74)
i enterococci (0.42)
i enterococci (0.44)
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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.
E. coli
Total coliforms
i enterococci
HAdV
HPyV
RoV
Somatic coliphages
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)
−0.01 0.95 65 −0.09 0.48 65 −0.19 0.12 69 −0.22 0.08 67 −0.10 0.46 67 −0.25 0.04 67 0.02 0.87 67
−0.15 0.23 65 −0.15 0.23 65 −0.25 0.04 69 −0.21 0.09 67 −0.24 0.04 67 −0.06 0.65 67 −0.04 0.77 67
0.17 0.18 65 0.26 0.04 65 0.08 0.50 69 −0.05 0.71 67 0.04 0.73 67 0.04 0.75 67 0.15 0.23 67
0.12 0.32 65 0.24 0.04 65 0.38 0.00 69 0.24 0.05 67 0.21 0.08 67 0.00 0.97 67 0.41 0.00 67
0.41 0.00 65 0.43 0.00 65 0.48 0.00 69 0.10 0.43 67 0.24 0.04 67 0.21 0.09 67 0.40 0.00 67
0.26 0.04 65 0.38 0.00 65 0.47 0.00 69 0.32 0.01 67 0.12 0.35 67 −0.13 0.31 67 0.54 0.00 67
−0.23 0.07 65 −0.24 0.06 65 −0.35 0.00 69 −0.32 0.01 67 0.21 0.08 67 −0.08 0.54 67 −0.16 0.19 67
−0.01 0.96 49 0.07 0.65 49 −0.13 0.36 50 −0.02 0.90 50 −0.01 0.95 49 0.08 0.60 46 −0.04 0.81 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 detection 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 different 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 temperatures ≥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 temperatures. 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 contamination 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 contaminated 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 association 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 contamination (Hayes, 1968). Furthermore, some hosts may multiply or metabolize in water environments to the extent that they support 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).
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As an alternative, F-RNA phages could be analyzed as possible 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 concentration of microorganisms in surface water fluctuates strongly and this might affect the correlation between viruses and bacteria negatively. 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 regularly 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 treatment 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 correlation 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, phosphate, and ammonia may influence the stability of viruses. No negative correlation between chemical and microbiological parameters 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 recovery 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
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