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Bacteriological and virological analysis of water from four fresh water beaches
Water Res. Vol. 16, pp. 939 to 943, 1982 Printed in Great Britain
0043-1354,82 060939-05103.00/0 Pergamon Press Ltd
BACTERIOLOGICAL AND VIROLOGICAL ANALYSIS OF WATER FROM FOUR FRESH WATER BEACHES PIERRE
PAYMENT,MIREILLE LEMtEUX andMICHELTRUDEL
Centre de Recherche en Virologic, InstitutArmand-Frappier, Universit8 du Quebec, C.P. I00, Laval-des-Rapides, Quebec. Canada H 7 N 4Z3
(ReceivedSeptember1981) AMtraet--Water samples from four beaches were analysed for the presence of total coliforms, fecal coliforms, fecal streptococci, salmonellae and enteric viruses. Analysis of the data did not reveal any correlation between the presence of viruses in 20 I. of water and the presence of the enteric bacteria. There was also no correlation between the presence of salmonellae and the presence of the other bacterial indicators. A positive correlation between virus isolation and water turbidity was demonstrated when turbidity was greater than l0 N.T.U.
INTRODUCTION The fact that sewage discharged into rivers is rarely adequately treated presents a major health hazard, that is amplified by the fact that viruses survive in significant numbers even after wastewater treatment (Gerba et al., 1975). The bacteria and viruses which are present in sewage represent a risk to swimmers who use contaminated waters for recreational purposes. Cabelli et al. (1979) have epidemiologically demonstrated that swimmers show a greater incidence of gastrointestinal troubles when swimming in polluted waters, than non-swimmers. Dutka 0973) stated
that coliforms are an inadequate index of water quality and Gerba (1979) have reported that enteroviruses were detected 44% of the time in recreational waters which were considered acceptable as judged by coliform and fecal coliform standards. It is therefore questionable whether bacterial standards are adequate to insure virologically safe recreational waters. The present study was undertaken to evaluate the extent of viral pollution in recreational waters, to study the efficiency of two viral concentration methods and to evaluate whether bacterial standards are adequate to monitor for viral pollution.
et al.
Instruments, Model D R T 16) were recorded. Samples were brought to the laboratory within 2h and were concentrated upon arrival. Virus concentration
One 20-I. sample was acidified to pH 3.5 with 1.2 M hydrochloric acid and adjusted to a final concentration of 1.5 m M aluminium chloride. The water was then filtered on a stack of 142mm fiberglass filtersdiscs: an AP-25 Millipore prefilter,a 0.45 and 0.25/~m Duo-Fine Filters (Filterite Corporation, Timonium, MD). Viruses were elutedfrom the filtersusing 100 ml of a 2~ lysine solution, pH 9.5, and the eluate was immediately neutralized using 1.2 M hydrochloric acid. Further concentration to 5 ml was obtained by hydroextraction against polyethylene glycol 6000 (PEG-6000)(Union Carbide, Montreal),
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Water samples were obtained 3 times weekly from 4 beaches located on Deux-Montagnes Lake, (Fig. I) during
July samples andwere August collected 1979. in FOrplastic virological containers analysis, 30cm twobelow 20-1. the surface at a water depth of one meter. For bacteriological analysis two samples were collected (using the same sampling method): one sample (200ml) was used to estimate total coliforms, fecal coliforms and fecal streptococci and a second one (4 I.) was used to detect salmonellae. For each sample, water temperature, pH and turbidity (HF
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Fig. 1: Area map indicating the geographical situation of the 4 beaches and direction of water flow.
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The second 20-I. sample v,as adjusted to pH 6 and adjusted to a final concentration of 10ram magnesium chloride and treated as described above.
dt d [
RESt l.r~, Bacteriological and virological results fTom the
Tissue culture Vero cells/a continuous cell line of African green monkey' kidney cells) were used in this study'. The cells ',*.,ere grown in equal parts of medium 199 with Hanks balanced salt solution and Eagle's minimum essential medium {MEM) {Earle's base), with 5?; fetal calf serum (FCS) and antibiotics {penicillin, 100 U ml- ~; streptomycin, 100 ~g ml-~l. MEM medium with _~o'~°.FCS and antibiotics was used as the maintenance medium,
water of the 4 beaches are illustrated in Figs 2-5. I-he first beach, Plage Paul Sauv~. is located in the Oka Provincial Park and is the only beach organized to receive several hundred bathers. Bacteriological analysis (Fig. 2) of its water revealed slightly elevated bacterial counts but within legal limits: less than 10130 total coliforms and less than 200 fecal coliforms. Exceptionalty high coliform counts were observed on 7,
Virus isolation Plaque assay on Vero cells was performed using a 1 ml aliquot of the concentrate. The remainder of the concentrate (4 ml) was used to detect viruses by cytopathic effect on Veto cells (CPE). Briefly, 0.5 ml of the concentrate was inoculated in a 25 cm-' flask and allowed to adsorb to the flask for 2 h before completing with maintenance medium and incubating at 37'C. Cell cultures were examined daily for CPE and if negative after 7 days, were frozen, thawed, and reinoculated. All cultures showing specific cytopathic effect were subcultured and then frozen at -70°C until identification was performed,
13 and 14 August, but viruses were isolated only from the 14 August sample. During the month of July, 8 out of 10 samples yielded viruses but most had a tow bacterial content. Salmonellae were isolated from 7/10 samples, the most frequent isolate being Salmonellae
Virus identification Poliovirus isolates were identified using an indirect enzyme-immunoassay (EIA) test (Payment et al., 1982). Non-reactive isolates in the EIA were classified as nonpolioviruses and by electron-microscopy were all picornavirus-like, Bacteriolo~Ty
limits and lower than at the first beach (Fig. 2). All 9 samples submitted for salmonella analysis were negative. Out of 18 samples 8 were positive for virus and
typhimurium. Turbidity ranged from 0.8 to 26 N T U (Nephelometric Turbidity Units) with an average value of 12 N T U (Table 1). Figure 3 reports the findings at Plag¢ Roger, a small private beach also opened for recreational bathing. Bacterial counts were usually within legal
on several occasions when bacterial counts were very low or negative (5, 16, 19, 30, 31 July and 16 August). Turbidity ranged from 0.9 to 28 N T U with an average of 11 N T U (Table 1). Figure 4 represents samples taken from Plage El Paraiso, a beach which was closed due to very high bacterial counts detected during a period of several years and which has been transformed into a yacht
The membrane filtration procedures described in Standard Methods (APHA, 1975) were used to enumerate the bacteria. Total coliforms were measured on M-endo medium at 35°C, fecal coliforms on MFC-medium at 45°C and fecal streptococci on M-Enteroccus agar. Salmonellae were detected by filtering 41. of water through a layer of Celite and a Millipore HC membrane before enrichment and growth as described in Standard Methods.
club. Most of our bacteriological samples revealed low bacterial counts, except on August 14, when high bacterial counts were detected and viruses isolated. Six of the 18 samples were positive for virus, five dur-
Statistical analysis The statistical evaluation of the relationships between the presence or absence of virus (in the water) and the bacterial parameters was performed using a corrected chisquare analysis.
ing July, one of which (July, 16) was positive for salmonellae and viruses. Turbidity ranged from 0.8 to I00 N T U with an average value of 18 N T U (Table 1).
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Fig. 5. Figs 2-5. B~eriological and virological data from the water of 4 l~actms. The presence of a circle at a given date indicates that a sample was taken for virus or salmonella isolation. An open circle indicates a negative result while a solid circle indicates a positive result. The asterisks on 4, 5, 9 and 10 July indicate that the samples were not analysed for the presence of fecal streptococci.
'~42
PT~RRE P~,'~'MI:Nrct ,L Table I, Results o( the ',iroIog~cal anal2sls of the ~ater from the 4 beaches
Date
Paul-Sau',e Turb.* A1Cl3t MgCl_,~ Turb.
04.07.79 ND 05,07,79 0.8 -09.07.79 1 4 . 0 +(P3~ 10.07.79 II.0 -12.07.79 8.5 16.07.79 24.5 +(NP) 17.07.79 1 8 . 0 +(P3) 19.07.79 1 5 . 0 +(PI) 30.07.79 II.0 +(NP) 31.07.79 1 3 . 0 +(PI) 02.08.79 6.4 06.08.79 11.0 07.08.79 8.2 09.08.79 6.2 13.08.79 11.0 14.08.79 26.0 +(P3) 16.08.79 8.2 -21.08.79 16.0 12.0 7/18 (mean)
-(NP)§ --, (P3) --
ND 0.9 19.5 8.7 13.0 +(NP) 16.0 +(P3} 12.0 +(P1) 24.0 11.0 +(PI) 10.0 5.8 -8.0 12.0 4.8 -4g -28.0 6.0 7.0 6/18 11.0 tmean~
Roger AICI 3
MgCI:
Turb.
-tP3~ t(P3) ...... . . . . . . . . . . +(PI) +(PI) +(NPJ +(NP) +(NPI +(PI) +(PI) -----+(P3) --
ND 0.8 19.5
-+(P3) 7/18
5'18
6.2
14.0 52.0 18.0 26.0 6.8 22.0 5.4 5.0 8.0 6.4 5.3 100.0 11.0 10.0 18.0 (meanl
El Paralso AtCI 3 MgCI,
Deux-Montagncs Turb. AICI3 MgCI,
ND ND ND 2.7 ---IP3) ~IP3) 37.0 ........ 12.0 . . . . . 52,0 +(NP) +(NP) 3 0 . 0 +{NP) +(NP) +{NP) +(NP} 12.0 +(NP} +(NP) +(NP) 22.0 a-INP) --6.4 -+(NP) +(PI) +(P1) 29.0 +(Pt) +{PI) 104.0 . . . . -18.0 -12.0 . . . . 22.0 ...... 13.0 +(P3) 104.0 +{P3) ND ND ND ND ND ND 5/'18 4/17 32.0 6, 15 6/15 rmeanl . . . . .-{P3) ....
* Turbidity in nephelo metric turbidity units INTU}. t Virus concentrated by adsorption at pH 3.5 in the presence of 1.5 mM aluminium chloride (AICI3). ++Virus concentrated by adsorption at pH 6.0 in the presence of 10 mM magnesium chloride (MgClz). §PI: Polio I; P3: Polio 3; NP: non-Polio: ND: not done. Figure 5 represents samples taken from Camping Deux-Montagnes a private camping ground where the beach was closed for bathing. It has very turbid waters and bacterial counts were more erratm than at the other beaches with occasionally very high values, During our study 8/15 samples were positive for viruses and 1/8 positive for salmonellae. This last sample was also positive for viruses and had high bacterial counts. Again most samples from which viruses were isolated were collected during July. Turbidity ranged from 2.7 to 104 N T U with an average value of 32 N T U (Table 1). Detailed results of the virological analysis are reported in Table 1. As reported, the a l u m i n u m chloride method permitted the detection of 1 or 2 more positive samples than did the magnesium chloride method. A few isolates were detected by only one of the methods but not at a slgnificatively higher rate. Thirteen isolates were identified as poliovirus type 1. Table 2. Correlation between turbidity and the presence of viruses Virus isolation Turbidity (NTU) >5 > I0 > 15 > 20 > 30
Negative (n = 34)
Positive (n = 32)
Chi-square analysist
32 17 7 4 I
29 25 17 12 a
0.941 (NS) 0.033(S) 0.012(S) 0.030 (S) 0.319 (NS)
* Nephelometric turbidity units. ~"Corrected chi-square analysis value; S: significative, NS: non-significative,
14 as poliovirus type 3 and 19 were classified as nonpolioviruses. In order to d e m o n s t r a t e a relationship between the presence of viruses a n d the bacterial indicators a cotrected chi-Square analysis was performed. There was no correlation between the presence of viruses and the presence of bacteria at any level of contamination. The same analysis was performed on the data related to the isolation of salmonellae and viruses, again without correlation. There was also no correlation between the presence of viruses and the temperature or pH of the water. A correlation was found between increased t u r b i d i t y a n d t h e p r e s e n c e o f v i r u s e s ( T a b l e 2 ) . Turbidity in excess of 10 N T U correlated with the isolation of viruses in the sample.
DISCUSSION Water quality in C a n a d a as well as in most countries is m o n i t o r e d by bacterial indicators, usually the coliforms a n d the fecal streptococci. These indicators have been adequate to insure the disappearance of most water-related diseases from the countries that have adopted these standards. Because viruses were not easy to detect in surface waters and because they were usually p r ¢ ~ n t in low numbers most felt that they were not a public health problem, Recent epidemiologic studies have shown that bathing in seven slightly polluted waters resulted in a higher incidence of gastroenteritis (Cabelli e¢ a l . , 1979). O t h e r studies have s h o w n that viruses were frequently isolated from swimming pool or wading pool water meeting all bacteriological water quality criteria and containing
Bacteriological and virological analysis of water residual chlorine (Keswick et al., abstract Q-43, 81st Annual Meeting of the ASM, Dallas, 1981). The results of our study clearly indicate that none of the standard bacteriological indicators and even the salmonellae, are sui~cient to assess the virological quality of fresh surface water. Because viruses can surrive for longer periods of time than any of the indicator bacteria, any human fecal pollution can be the source of viral pathogens that will be present for some time after the bacterial indicators have been inactivated (Gerba et at., 1979). The correlation between turbidity and the presence of viruses in the water was expected as it is known that sediments can contain viable microorganisms and are easily put back in suspension by any water movement (Gerba et al., 1979). The beaches studied are known to be contaminated by untreated sewage effluents which have not been located or from leaking septic tanks. Because of these ponctual contaminations and because turbidity cannot be normally correlated with pathogens if the water is not contaminated by fecal material, the correlation found with turbidity appears to be fortuitous, Similar studies in marine waters have also failed to demonstrate a correlation between viral and bacterial indicators (Hugues et al., 1979; Marzook et al., 1980; Gerba et al., 1980). Because of the accumulated data on the presence of viruses in water that are considered safe for bathing and because of the incidence of gastroenteritis in swimmers, further studies are needed to determine if these gastroenteritis are of viral origin
943
and if so what is the correlation between viral concentration and disease incidence. Acknowledgements--We thank Robert Plante and the technical staff of the laboratory of the Quebec Ministry of Environment for performing all bacteriological analysis. This work was supported by grant EQ-1422 of the Quebec Ministry of Education (FCAC program).
REFERENCES APHA (1975) Standard Methods for the Examination of Water and Wastewater, 14th Edition. American Public Health Association. Washington, DC. CabeUi V. J., Dufour A. P., Levin M. A., McCabe L. J. & Haberman P. W. (1979) Relationship of microbial indicators to health effects at marine beaches. Am. J. publ. Hlth 69, 690--696. Dutka B. J. (1973) Coliforms are an inadequate indicator of water quality. J. environ. Hlth 36, 39-46. Gerba C. P., Goyal S. M.. Labelle R. L., Cech I. & Bodgam C. F. (1979) Failure of indicator bacteria to reflect the occurrence of enteroviruses in marine waters. Am. J. Publ. HIth 69, 1116-1119. Gerba C. P., Wallis C. & Melnick J. L. (1975) Viruses in water: the problem, some solutions. Environ. Sci. Technol. 9, 1122-1126. Hugues B., Plissier M., Mothon F. & Bocquet J. P. (1979) Recherche virale darts les eaux r~cr~atives. Rev. franc. Sant6 Publ. 7, 53-61. Marzook Y., Goyal S. M. & Gerba C. P. (1980). Relationship of viruses and indicator bacteria in water and wastewater of Israel. Water Res. 14, 1585-1590. Payment P., Tremblay C. & Trudel M. (1982). Rapid identification and serotyping of poliovirus isolates by an immunoassay. J. virolog. Meth. In press.
0043-1354,82 060939-05103.00/0 Pergamon Press Ltd
BACTERIOLOGICAL AND VIROLOGICAL ANALYSIS OF WATER FROM FOUR FRESH WATER BEACHES PIERRE
PAYMENT,MIREILLE LEMtEUX andMICHELTRUDEL
Centre de Recherche en Virologic, InstitutArmand-Frappier, Universit8 du Quebec, C.P. I00, Laval-des-Rapides, Quebec. Canada H 7 N 4Z3
(ReceivedSeptember1981) AMtraet--Water samples from four beaches were analysed for the presence of total coliforms, fecal coliforms, fecal streptococci, salmonellae and enteric viruses. Analysis of the data did not reveal any correlation between the presence of viruses in 20 I. of water and the presence of the enteric bacteria. There was also no correlation between the presence of salmonellae and the presence of the other bacterial indicators. A positive correlation between virus isolation and water turbidity was demonstrated when turbidity was greater than l0 N.T.U.
INTRODUCTION The fact that sewage discharged into rivers is rarely adequately treated presents a major health hazard, that is amplified by the fact that viruses survive in significant numbers even after wastewater treatment (Gerba et al., 1975). The bacteria and viruses which are present in sewage represent a risk to swimmers who use contaminated waters for recreational purposes. Cabelli et al. (1979) have epidemiologically demonstrated that swimmers show a greater incidence of gastrointestinal troubles when swimming in polluted waters, than non-swimmers. Dutka 0973) stated
that coliforms are an inadequate index of water quality and Gerba (1979) have reported that enteroviruses were detected 44% of the time in recreational waters which were considered acceptable as judged by coliform and fecal coliform standards. It is therefore questionable whether bacterial standards are adequate to insure virologically safe recreational waters. The present study was undertaken to evaluate the extent of viral pollution in recreational waters, to study the efficiency of two viral concentration methods and to evaluate whether bacterial standards are adequate to monitor for viral pollution.
et al.
Instruments, Model D R T 16) were recorded. Samples were brought to the laboratory within 2h and were concentrated upon arrival. Virus concentration
One 20-I. sample was acidified to pH 3.5 with 1.2 M hydrochloric acid and adjusted to a final concentration of 1.5 m M aluminium chloride. The water was then filtered on a stack of 142mm fiberglass filtersdiscs: an AP-25 Millipore prefilter,a 0.45 and 0.25/~m Duo-Fine Filters (Filterite Corporation, Timonium, MD). Viruses were elutedfrom the filtersusing 100 ml of a 2~ lysine solution, pH 9.5, and the eluate was immediately neutralized using 1.2 M hydrochloric acid. Further concentration to 5 ml was obtained by hydroextraction against polyethylene glycol 6000 (PEG-6000)(Union Carbide, Montreal),
A N
*~ n_~ ~'L"~t'*'~ j ~ ~ ~ " " ~',,!~i ,~:~ ,=
I
MATERIALS AND METHODS Collection
of samples
939
ff_~. s , ~ ,~
: .~~/"~
1--~"~~~ i
Water samples were obtained 3 times weekly from 4 beaches located on Deux-Montagnes Lake, (Fig. I) during
July samples andwere August collected 1979. in FOrplastic virological containers analysis, 30cm twobelow 20-1. the surface at a water depth of one meter. For bacteriological analysis two samples were collected (using the same sampling method): one sample (200ml) was used to estimate total coliforms, fecal coliforms and fecal streptococci and a second one (4 I.) was used to detect salmonellae. For each sample, water temperature, pH and turbidity (HF
:::~
;i
ILE ~m~oT
/
~
,
,~ /
j
~
mm '
'
Fig. 1: Area map indicating the geographical situation of the 4 beaches and direction of water flow.
'4£I)
PIERRI-
P~.~MENF
The second 20-I. sample v,as adjusted to pH 6 and adjusted to a final concentration of 10ram magnesium chloride and treated as described above.
dt d [
RESt l.r~, Bacteriological and virological results fTom the
Tissue culture Vero cells/a continuous cell line of African green monkey' kidney cells) were used in this study'. The cells ',*.,ere grown in equal parts of medium 199 with Hanks balanced salt solution and Eagle's minimum essential medium {MEM) {Earle's base), with 5?; fetal calf serum (FCS) and antibiotics {penicillin, 100 U ml- ~; streptomycin, 100 ~g ml-~l. MEM medium with _~o'~°.FCS and antibiotics was used as the maintenance medium,
water of the 4 beaches are illustrated in Figs 2-5. I-he first beach, Plage Paul Sauv~. is located in the Oka Provincial Park and is the only beach organized to receive several hundred bathers. Bacteriological analysis (Fig. 2) of its water revealed slightly elevated bacterial counts but within legal limits: less than 10130 total coliforms and less than 200 fecal coliforms. Exceptionalty high coliform counts were observed on 7,
Virus isolation Plaque assay on Vero cells was performed using a 1 ml aliquot of the concentrate. The remainder of the concentrate (4 ml) was used to detect viruses by cytopathic effect on Veto cells (CPE). Briefly, 0.5 ml of the concentrate was inoculated in a 25 cm-' flask and allowed to adsorb to the flask for 2 h before completing with maintenance medium and incubating at 37'C. Cell cultures were examined daily for CPE and if negative after 7 days, were frozen, thawed, and reinoculated. All cultures showing specific cytopathic effect were subcultured and then frozen at -70°C until identification was performed,
13 and 14 August, but viruses were isolated only from the 14 August sample. During the month of July, 8 out of 10 samples yielded viruses but most had a tow bacterial content. Salmonellae were isolated from 7/10 samples, the most frequent isolate being Salmonellae
Virus identification Poliovirus isolates were identified using an indirect enzyme-immunoassay (EIA) test (Payment et al., 1982). Non-reactive isolates in the EIA were classified as nonpolioviruses and by electron-microscopy were all picornavirus-like, Bacteriolo~Ty
limits and lower than at the first beach (Fig. 2). All 9 samples submitted for salmonella analysis were negative. Out of 18 samples 8 were positive for virus and
typhimurium. Turbidity ranged from 0.8 to 26 N T U (Nephelometric Turbidity Units) with an average value of 12 N T U (Table 1). Figure 3 reports the findings at Plag¢ Roger, a small private beach also opened for recreational bathing. Bacterial counts were usually within legal
on several occasions when bacterial counts were very low or negative (5, 16, 19, 30, 31 July and 16 August). Turbidity ranged from 0.9 to 28 N T U with an average of 11 N T U (Table 1). Figure 4 represents samples taken from Plage El Paraiso, a beach which was closed due to very high bacterial counts detected during a period of several years and which has been transformed into a yacht
The membrane filtration procedures described in Standard Methods (APHA, 1975) were used to enumerate the bacteria. Total coliforms were measured on M-endo medium at 35°C, fecal coliforms on MFC-medium at 45°C and fecal streptococci on M-Enteroccus agar. Salmonellae were detected by filtering 41. of water through a layer of Celite and a Millipore HC membrane before enrichment and growth as described in Standard Methods.
club. Most of our bacteriological samples revealed low bacterial counts, except on August 14, when high bacterial counts were detected and viruses isolated. Six of the 18 samples were positive for virus, five dur-
Statistical analysis The statistical evaluation of the relationships between the presence or absence of virus (in the water) and the bacterial parameters was performed using a corrected chisquare analysis.
ing July, one of which (July, 16) was positive for salmonellae and viruses. Turbidity ranged from 0.8 to I00 N T U with an average value of 18 N T U (Table 1).
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Bacteriological and virological analysis of water PL A G [
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Fig. 5. Figs 2-5. B~eriological and virological data from the water of 4 l~actms. The presence of a circle at a given date indicates that a sample was taken for virus or salmonella isolation. An open circle indicates a negative result while a solid circle indicates a positive result. The asterisks on 4, 5, 9 and 10 July indicate that the samples were not analysed for the presence of fecal streptococci.
'~42
PT~RRE P~,'~'MI:Nrct ,L Table I, Results o( the ',iroIog~cal anal2sls of the ~ater from the 4 beaches
Date
Paul-Sau',e Turb.* A1Cl3t MgCl_,~ Turb.
04.07.79 ND 05,07,79 0.8 -09.07.79 1 4 . 0 +(P3~ 10.07.79 II.0 -12.07.79 8.5 16.07.79 24.5 +(NP) 17.07.79 1 8 . 0 +(P3) 19.07.79 1 5 . 0 +(PI) 30.07.79 II.0 +(NP) 31.07.79 1 3 . 0 +(PI) 02.08.79 6.4 06.08.79 11.0 07.08.79 8.2 09.08.79 6.2 13.08.79 11.0 14.08.79 26.0 +(P3) 16.08.79 8.2 -21.08.79 16.0 12.0 7/18 (mean)
-(NP)§ --, (P3) --
ND 0.9 19.5 8.7 13.0 +(NP) 16.0 +(P3} 12.0 +(P1) 24.0 11.0 +(PI) 10.0 5.8 -8.0 12.0 4.8 -4g -28.0 6.0 7.0 6/18 11.0 tmean~
Roger AICI 3
MgCI:
Turb.
-tP3~ t(P3) ...... . . . . . . . . . . +(PI) +(PI) +(NPJ +(NP) +(NPI +(PI) +(PI) -----+(P3) --
ND 0.8 19.5
-+(P3) 7/18
5'18
6.2
14.0 52.0 18.0 26.0 6.8 22.0 5.4 5.0 8.0 6.4 5.3 100.0 11.0 10.0 18.0 (meanl
El Paralso AtCI 3 MgCI,
Deux-Montagncs Turb. AICI3 MgCI,
ND ND ND 2.7 ---IP3) ~IP3) 37.0 ........ 12.0 . . . . . 52,0 +(NP) +(NP) 3 0 . 0 +{NP) +(NP) +{NP) +(NP} 12.0 +(NP} +(NP) +(NP) 22.0 a-INP) --6.4 -+(NP) +(PI) +(P1) 29.0 +(Pt) +{PI) 104.0 . . . . -18.0 -12.0 . . . . 22.0 ...... 13.0 +(P3) 104.0 +{P3) ND ND ND ND ND ND 5/'18 4/17 32.0 6, 15 6/15 rmeanl . . . . .-{P3) ....
* Turbidity in nephelo metric turbidity units INTU}. t Virus concentrated by adsorption at pH 3.5 in the presence of 1.5 mM aluminium chloride (AICI3). ++Virus concentrated by adsorption at pH 6.0 in the presence of 10 mM magnesium chloride (MgClz). §PI: Polio I; P3: Polio 3; NP: non-Polio: ND: not done. Figure 5 represents samples taken from Camping Deux-Montagnes a private camping ground where the beach was closed for bathing. It has very turbid waters and bacterial counts were more erratm than at the other beaches with occasionally very high values, During our study 8/15 samples were positive for viruses and 1/8 positive for salmonellae. This last sample was also positive for viruses and had high bacterial counts. Again most samples from which viruses were isolated were collected during July. Turbidity ranged from 2.7 to 104 N T U with an average value of 32 N T U (Table 1). Detailed results of the virological analysis are reported in Table 1. As reported, the a l u m i n u m chloride method permitted the detection of 1 or 2 more positive samples than did the magnesium chloride method. A few isolates were detected by only one of the methods but not at a slgnificatively higher rate. Thirteen isolates were identified as poliovirus type 1. Table 2. Correlation between turbidity and the presence of viruses Virus isolation Turbidity (NTU) >5 > I0 > 15 > 20 > 30
Negative (n = 34)
Positive (n = 32)
Chi-square analysist
32 17 7 4 I
29 25 17 12 a
0.941 (NS) 0.033(S) 0.012(S) 0.030 (S) 0.319 (NS)
* Nephelometric turbidity units. ~"Corrected chi-square analysis value; S: significative, NS: non-significative,
14 as poliovirus type 3 and 19 were classified as nonpolioviruses. In order to d e m o n s t r a t e a relationship between the presence of viruses a n d the bacterial indicators a cotrected chi-Square analysis was performed. There was no correlation between the presence of viruses and the presence of bacteria at any level of contamination. The same analysis was performed on the data related to the isolation of salmonellae and viruses, again without correlation. There was also no correlation between the presence of viruses and the temperature or pH of the water. A correlation was found between increased t u r b i d i t y a n d t h e p r e s e n c e o f v i r u s e s ( T a b l e 2 ) . Turbidity in excess of 10 N T U correlated with the isolation of viruses in the sample.
DISCUSSION Water quality in C a n a d a as well as in most countries is m o n i t o r e d by bacterial indicators, usually the coliforms a n d the fecal streptococci. These indicators have been adequate to insure the disappearance of most water-related diseases from the countries that have adopted these standards. Because viruses were not easy to detect in surface waters and because they were usually p r ¢ ~ n t in low numbers most felt that they were not a public health problem, Recent epidemiologic studies have shown that bathing in seven slightly polluted waters resulted in a higher incidence of gastroenteritis (Cabelli e¢ a l . , 1979). O t h e r studies have s h o w n that viruses were frequently isolated from swimming pool or wading pool water meeting all bacteriological water quality criteria and containing
Bacteriological and virological analysis of water residual chlorine (Keswick et al., abstract Q-43, 81st Annual Meeting of the ASM, Dallas, 1981). The results of our study clearly indicate that none of the standard bacteriological indicators and even the salmonellae, are sui~cient to assess the virological quality of fresh surface water. Because viruses can surrive for longer periods of time than any of the indicator bacteria, any human fecal pollution can be the source of viral pathogens that will be present for some time after the bacterial indicators have been inactivated (Gerba et at., 1979). The correlation between turbidity and the presence of viruses in the water was expected as it is known that sediments can contain viable microorganisms and are easily put back in suspension by any water movement (Gerba et al., 1979). The beaches studied are known to be contaminated by untreated sewage effluents which have not been located or from leaking septic tanks. Because of these ponctual contaminations and because turbidity cannot be normally correlated with pathogens if the water is not contaminated by fecal material, the correlation found with turbidity appears to be fortuitous, Similar studies in marine waters have also failed to demonstrate a correlation between viral and bacterial indicators (Hugues et al., 1979; Marzook et al., 1980; Gerba et al., 1980). Because of the accumulated data on the presence of viruses in water that are considered safe for bathing and because of the incidence of gastroenteritis in swimmers, further studies are needed to determine if these gastroenteritis are of viral origin
943
and if so what is the correlation between viral concentration and disease incidence. Acknowledgements--We thank Robert Plante and the technical staff of the laboratory of the Quebec Ministry of Environment for performing all bacteriological analysis. This work was supported by grant EQ-1422 of the Quebec Ministry of Education (FCAC program).
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