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The limitation of the ratio of fecal coliforms to total coliphage as a water pollution index
&i.~te,ResearchVol. I0. pp. 7..t5to 748. PergamonPre~s ig"b Printedira Great Britain.
THE LIMITATION OF THE RATIO OF FECAL COLIFORMS TO TOTAL COLIPHAGE AS A WATER P O L L U T I O N INDEX R. G. BELL Research Station. Agriculture Canada, Lethbridge, Alberta, Canada TiJ 4BI
(Receired 17 September 1975) Abstract--The fecal coliform populations of raw sewage, sewage lagoon effluent, and river water were determined using the most probable number technique. The total coliphage populations of the three water sources were determined using Escherichia coli B (ATCC 11303-1) host ceils. The ratios of fecal coliforms to cotiphage in the three water samples were 87: l. 4.2: 1. and 0.15: I, respectively. The ratio of fecal coliforms to coliphage in stored raw sewage decreased from 87:1 to about I: I within , da~s at 20°C and within 28 days at 4°C. These changing ratios resulted from the greater longevity of the coliphage compared with that of the coliform bacteria. The use of the ratio of fecal coliforms to coliphage is not considered reliable as an index of when a fecal pollution event occurred because the ratio is influenced by prior contamination, presence of sediment, chlorination, and temperature.
INTRODUCTION
MATERIALS AND METHODS
The presence of coliform bacteria in water as an indicator of fecal contamination of water is well established (APHA, 1971). Two methods of determining coliform p o p u l a t i o n - - t h e multiple fermentation tube or most probable number technique (MPN) and the membrane filter technique ( M F ) - - h a v e become accepted as standard procedures (APHA, 1971). The two tests provide comparable results and both differentiate between fecal and non-fecal coliforms, but they suffer from the disadvantage of being slow, the M P N test requiring 48 h and the M F test 24 h. It has been suggested that coliform numbers can be determined in as little as 2 h by enumerating the total coliphage population of a water sample because the fecal coliform bacteria and coliphage are said to exist in a fixed ratio of 1:0.7 (Kenard and Valentine, 1974). This proposal, although very appealing, is surprising in the light of the many observations that in wastewater the enteroviruses persist longer than Escherichia coli or enteropathogenic bacteria (Holden, 1970). Therefore, the ratio of coliform bacteria to virus particles can be expected to decrease with time following fecal contamination of water. Field observations (Poynter, 1966) have shown that the ratio of fecal coliforms to enterovirus plague-forming units declined from 16,000:1 in sewage effluent to 1330:1 in the receiving stream 7.2 km below the sewage outfall. The studies of Kott et al. (1974) suggest that the coliphage may be even more persistent than the enteroviruses in wastewater and receiving streams. The present study was undertaken to determine whether the ratio of fecal coliform bacteria to total coliphage--in raw sewage, sewage lagoon effluent, or river w a t e r - - h a d any significance as a pollution index.
Sample sources Samples of raw sewage and a sewage lagoon effluent, which in summer is used to irrigate alfalfa, were obtained from the sewage treatment facility at Taber, Alberta, Canada. The treatment unit receives domestic waste from a population of 5000 as well as the effluent from a vegetable processing plant. Samples of river water were taken from the Oldman River opposite the Provincial Park at Taber, about 1.5 km upstream from the sewage treatment plant.
Sampliny proced,lre Samples were taken at about weekly intervals from 12 November 1974 to 3 March 1975. Single grab samples of raw sewage were taken at an inspection manhole 3 m upstream from the Taber sewage treatment facility. Single samples of river water and lagoon effluent were taken by opening a sterile 250-ml water sampling bottle 5 cm below the water surface. An ice auger was needed to penetrate up to 105cm of ice on the lagoon and 72cm on the river for part of the sampling period. All samples were taken to the laboratory and analyses started within 2 h.
Laboratory procedures The fecal coliform population of the samples was determined using the EC medium MPN technique at an incubation temperature of 44.5 + 0.2~C (APHA. 1971) with modified McConkey broth (Difco) used for the presumptive count (Cruikshank. 1962). About I0~,~ of those isolates that produced gas in EC medium (Difco) when incubated at 44.5 _+ 0.5~C were stained by Gram's method and examined microscopically to check that they were indeed Gramnegative nonsporing bacilli. All coliphage counts were made using the following procedure: (a) Water samples were shaken vigorously and 1 ml was pipetted into 3.0ml of a semisolid nutrient agar medium (0.87,~, nutrient broth [Difco] and 0.66~ Bacto agar [Difco] maintained at 45°C); where phage numbers were high. dilutions (l:10 in distilled water) were used. (b) To this mixture was added 0.1 ml of host cells prepared by centrifuging 10ml of a 24-h nutrient broth culture of E. coil B (ATCC 11303-1) and resuspending the cells in 745
7z6
R.G. BELL
2 ml of sterile distilled water. IcJ Alter agitation for 30 sec on a vortex mixer, the mixture was poured onto the surface of a petri dish containing nutrient agar 11.57o agar): 10 such plates were prepared ior each water sample. (d) When the agar overla) had solidified, the plates were placed inverted in a polyethylene bag humidor I Bell. 19751 and incubated at 37:C. (el After incubation for 18-24h. the coliphage plaques were counted. The host cells were checked for coliphage contaminatior, upon receipt from the culture collection and weekly during the coliphage enumeration experiments using the above procedure, with sterile distilled water substituted for the water sample, Surviral of coliphaqe in stored sewaqe Single samples of ra~ sewage were collected on 18 November 1974, 6 January 1975, and 10 February 1975 and stored at 20 and 4°C in 600-ml wide-mouthed glassstoppered jars. Samples of the stored sewage were withdrawn periodically (see Fig. 1)and subjected to MPN enumeration for fecal coliform bacteria and total coliphage counts as detailed above
would suggest that the fecal coliform bacteria were better able to survive in the b o t t o m sediments than in the water column. The changing ratio of fecal colitbrms to cotiphage in sewage stored at 20 a n d 4:C is shown in Fig. I. G a s production, which indicates bacterial metabolism. was observed during the first 2 days m samples stored at 20°C a n d for the first 8 days at 4:C. During the first 2 days, the mean fecal coliform population of the samples stored at 20~C apparently declined from 2.07 to 1.40 × 1 0 6 100ml) - l while that of the 4~C sample apparently increased from 0.8 to 2.1 × 106 (100mt)-~ o n the eighth day. However. because of the wide confidence limits associated with the M P N test (APHA, 19711, neither of these changes can be considered significant.
DISCUSSION RESULTS The medians of the fecal coliform to coliphage ratios in raw sewage a n d lagoon effluent (Table t) were found to be significantly different at the 1~o level using the n o n p a r a m e t r i c Wilcoxon signed tank test (Campbell, 1967). However. the difference in the medians of the coliphage populations was shown by the same test not to be significant even at the 5'?,~; level. Therefore. the significant differences in the fecal coliform to coliphage ratios resulted from the 14-fold difference in fecal coliform populations. The results obtained for the river water ITabte 2) represent a much lower level of fecal contamination. It is interesting to note that the turbid samples taken on 13 January, 20 January, a n d 3 March, when the auger struck the riverbed, gave ratios similar to those found in the sewage lagoon effluent. This observation
The choice of a host bacterium is extremely important in any bacteriophage study because of the spectficity of phage acceptor sites on the surface of the bacterium. E. coli B (ATCC 11303-l) was selected because of its wide susceptibility to the coliphage. This organism is used as a host for 39 coliphage by the American Type Culture Collection. some 34 more than the nearest other multi-coliphage host E. coil C (ATCC 13709) (ATCC. 1974). In preliminary experiments. E. coil B (ATCC 11303-1) was found to support a b o u t I5 times as many phage from domestic sewage as a fecal coliform isolated from the sewage lagoon and 200 times as many as a strain isolated from the O l d m a n River, During passage of sewage t h r o u g h the lagoon system, the coliphage population remained almost unchanged while the fecal coliform population declined
Table I. Coliphage, fecal coliforms, and fecal coliform to coliphage ratio in raw sewage (S) and in sewage lagoon e~uent (E)
Date sample taken November 12 November 18 November 25 December 2 December 9 December 16 January 6 January 13 January 20 January 27 February 3 February 10 February 17 February 24 March 3 Median
Coliphage (1130 mll- 1 ( x 105)
Fecal coliforms (100 ml)- ~~x 105)
Ratio fecal coliforms to coliphage
S
E
S
E
S
E
0.25 0.53 0.96 0.73 0.26 0.51 0.12 0.81 0.09 0.28 0.09 0.09 0.42 0.02 0.09 0.25a*
0.t6 0.20 0.32 0.41 0.28 0.49 0.37 0.28 0.35 0,24 0.39 0.32 0.24 0.24 0.13 0.28a
33.0 46.0 49.0 70.0 17.0 11.0 11.0 130.0 l 1.0 6.0 2.0 5.0 2.0 2.0 23.0 1t .0b
3.3 13.0 7.9 1.7 0.8 0.8 0.5 4.9 2.3 0.2 0.8 0.8 4.0 0.5 2.4 0.8c
130:1 88:1 51 : 1 95:1 66:1 27:1 93:1 [60:1 130:1 21 : 1 24:1 57:1 5:1 87: l 270:1 87:1 d
21: t 66:1 25: I 4.2:1 2.9:1 t.6:1 1.4:1 18: I 6.7: t 0.9:1 2.1:1 2.5:1 L9:I
* For pairs of column medians, those followed by the same letter do not differ significantly (P<0.01).
2. t : I
19: t 4.2: I e
Coliform coliphage ratio
/q,I
Table 2. Coliphage. fecal coliforms, and fecal coliform to coliphage ratio in river water Date sample taken
Coliphage ( I00 ml)- * t x 10)
Fecal coliforms ( 100 ml)- ~t x 10)
Ratio fecal coliforms to coliphage
21.0 42.0 19.0 5.0 55.0 56.0 7.0 98.0 49.0 16.0 32.0
1.7 3.3 160.0 17.0 1.1 7.9 7.9 7.9 7.9 35.0 7.9
0.08:1 0.08:1 8.5:1 3.4:1 0.02:1 0.14: I l.l: l 0.0l: t 0.16:1 2.2: l 0.15: I
December 16 January 6 January 13" January 20* January 27 Februar? 3 February 10 February 17 February 24 March 3* Median * Turbid sample,
14-fold (Table 1). Therefore, contrary to the results of Kenard and Valentine (1974). the fecal coliform population cannot be readily calculated from the coliphage population because the ratio between them is not constant but decreases with time (Fig. l). The wide fluctuations in the ratio of fecal coliforms to total coliphage (Tables 1 a n d 2) are similar to those observed by Hilton and Stotzky (1973). Notwithstanding these wide fluctuations, this ratio may in itself be a useful pollution index. Unlike a simple count of fecal coliforms as a measure of fecal pollution, the coliform to coliphage ratio is independent of dilution. Therefore, because of the differential persistence of the coliform bacteria and the coliphage, a high ratio could be considered to indicate recent fecal c o n t a m i n a t i o n while a low ratio would indicate less recent contamination. In support of this hypothesis, it is reported that a ratio of 100:1 (Kott, Buras and Lindman, 1971) occurs in fresh feces while the results presented here show ratios I
o00~200
I:~",./
REFERENCES
,../ - ,
,oo ' .,°
American Public Health Association. {1971) Standard
methods for tlle examination oj" water and wastewater,
so
Ig u. ..a o o c0
...""%.......
~o
~-,.,.
............- \/,_
Io 5
ta.
o
of 8 7 : l , 4.2:1, and 0.15: I as being characteristic of raw sewage, lagoon effluent, and river water, respectively. The declining ratio of coliforms to coliphage observed in the sewage storage experiment (Fig. 1) supports, yet complicates, the use of the coliform to coliphage ratio as an index of when a fecal contamination event took place because the rate of change of the ratio is highly temperature-dependent. Furthermore, because of the differential persistence of fecal coliforms a n d coliphage from previous contamination, the ratio of the most recent event would be decreased and so the event would be considered more distant than it really was. The river water studies (Table 2) show the ratio displacing effect of the presence of sediment in a sample. While it is tempting to place significance on the coliform to coliphage ratio, it c a n n o t be considered as a precise index of the time since fecal pollution occurred because it is influenced by so many factors other than time. including chlorination (Kott et al., 1974), presence of sediment (Table 2). and temperature (Fig. 1).
\
2
4
8
12
OAYS
16
20
24
28
STOREO
Fig. I. Ratios of fecal coliforms to total colipbage in raw sewage stored at 20°C (solid line) and at 4°C (broken fine). Points are means of samples taken on three dates--18 November 1974, 6 January 1975 and 10 February 1975.
13th edition, p. 874. APHA, Washington, D,C. American Type Culture Collection. (1974) Catalogue of strains, 1lth edition, p. 369. ATCC, Rockville, Maryland. Bell R. G. (1975) The influence of NaCI, Ca-' -. and Mg-'* on the growth of a marine Bdellovibrio sp. Estuar. coastal mar. Sci. 3, 381-384. Campbell R. C. (1967) Statisticsfi)r hioloyists, p. 242. Cambridge University Press. Cruikshank R. (1962} Mackie and McCartney's Handbook of Bacteriology, 10th edition, p. 359. Livingstone. Edinburgh. Hilton M. C. & Stotzky G. (1973) Use of coliphages as indicators of water pollution. Can. J. Microbiol. 19. 747-751. Holden W. S. (1970) Water Treatment and Examination. p. 513, Churchill. London. Kenard R. P. & Valentine R. S. (1974) Rapid determination of the presence of enteric bacteria in water. Appl. Microbiol. 27, 484-487.
748
R.G. BELL
Kott Y., Buras N. & Lindman S. (t971) Coliphages a s virus indicators in water and wastewater. Sec. Annu. Rep. FWQA Res. G. 16030 DQN. FWPCA and Technion Res. and Dev. Found.. Haifa. Kott Y.. Roze N.. Sperber S. & Betzer N. (1974} 8acterio-
phages as viral pollution indicators. Water Res. 8. 165-171. Poynter S. F. B. (1966) Studies in the access of enteroviruses to water supplies. Ph.D. Thesis, Univ~ of London.
THE LIMITATION OF THE RATIO OF FECAL COLIFORMS TO TOTAL COLIPHAGE AS A WATER P O L L U T I O N INDEX R. G. BELL Research Station. Agriculture Canada, Lethbridge, Alberta, Canada TiJ 4BI
(Receired 17 September 1975) Abstract--The fecal coliform populations of raw sewage, sewage lagoon effluent, and river water were determined using the most probable number technique. The total coliphage populations of the three water sources were determined using Escherichia coli B (ATCC 11303-1) host ceils. The ratios of fecal coliforms to cotiphage in the three water samples were 87: l. 4.2: 1. and 0.15: I, respectively. The ratio of fecal coliforms to coliphage in stored raw sewage decreased from 87:1 to about I: I within , da~s at 20°C and within 28 days at 4°C. These changing ratios resulted from the greater longevity of the coliphage compared with that of the coliform bacteria. The use of the ratio of fecal coliforms to coliphage is not considered reliable as an index of when a fecal pollution event occurred because the ratio is influenced by prior contamination, presence of sediment, chlorination, and temperature.
INTRODUCTION
MATERIALS AND METHODS
The presence of coliform bacteria in water as an indicator of fecal contamination of water is well established (APHA, 1971). Two methods of determining coliform p o p u l a t i o n - - t h e multiple fermentation tube or most probable number technique (MPN) and the membrane filter technique ( M F ) - - h a v e become accepted as standard procedures (APHA, 1971). The two tests provide comparable results and both differentiate between fecal and non-fecal coliforms, but they suffer from the disadvantage of being slow, the M P N test requiring 48 h and the M F test 24 h. It has been suggested that coliform numbers can be determined in as little as 2 h by enumerating the total coliphage population of a water sample because the fecal coliform bacteria and coliphage are said to exist in a fixed ratio of 1:0.7 (Kenard and Valentine, 1974). This proposal, although very appealing, is surprising in the light of the many observations that in wastewater the enteroviruses persist longer than Escherichia coli or enteropathogenic bacteria (Holden, 1970). Therefore, the ratio of coliform bacteria to virus particles can be expected to decrease with time following fecal contamination of water. Field observations (Poynter, 1966) have shown that the ratio of fecal coliforms to enterovirus plague-forming units declined from 16,000:1 in sewage effluent to 1330:1 in the receiving stream 7.2 km below the sewage outfall. The studies of Kott et al. (1974) suggest that the coliphage may be even more persistent than the enteroviruses in wastewater and receiving streams. The present study was undertaken to determine whether the ratio of fecal coliform bacteria to total coliphage--in raw sewage, sewage lagoon effluent, or river w a t e r - - h a d any significance as a pollution index.
Sample sources Samples of raw sewage and a sewage lagoon effluent, which in summer is used to irrigate alfalfa, were obtained from the sewage treatment facility at Taber, Alberta, Canada. The treatment unit receives domestic waste from a population of 5000 as well as the effluent from a vegetable processing plant. Samples of river water were taken from the Oldman River opposite the Provincial Park at Taber, about 1.5 km upstream from the sewage treatment plant.
Sampliny proced,lre Samples were taken at about weekly intervals from 12 November 1974 to 3 March 1975. Single grab samples of raw sewage were taken at an inspection manhole 3 m upstream from the Taber sewage treatment facility. Single samples of river water and lagoon effluent were taken by opening a sterile 250-ml water sampling bottle 5 cm below the water surface. An ice auger was needed to penetrate up to 105cm of ice on the lagoon and 72cm on the river for part of the sampling period. All samples were taken to the laboratory and analyses started within 2 h.
Laboratory procedures The fecal coliform population of the samples was determined using the EC medium MPN technique at an incubation temperature of 44.5 + 0.2~C (APHA. 1971) with modified McConkey broth (Difco) used for the presumptive count (Cruikshank. 1962). About I0~,~ of those isolates that produced gas in EC medium (Difco) when incubated at 44.5 _+ 0.5~C were stained by Gram's method and examined microscopically to check that they were indeed Gramnegative nonsporing bacilli. All coliphage counts were made using the following procedure: (a) Water samples were shaken vigorously and 1 ml was pipetted into 3.0ml of a semisolid nutrient agar medium (0.87,~, nutrient broth [Difco] and 0.66~ Bacto agar [Difco] maintained at 45°C); where phage numbers were high. dilutions (l:10 in distilled water) were used. (b) To this mixture was added 0.1 ml of host cells prepared by centrifuging 10ml of a 24-h nutrient broth culture of E. coil B (ATCC 11303-1) and resuspending the cells in 745
7z6
R.G. BELL
2 ml of sterile distilled water. IcJ Alter agitation for 30 sec on a vortex mixer, the mixture was poured onto the surface of a petri dish containing nutrient agar 11.57o agar): 10 such plates were prepared ior each water sample. (d) When the agar overla) had solidified, the plates were placed inverted in a polyethylene bag humidor I Bell. 19751 and incubated at 37:C. (el After incubation for 18-24h. the coliphage plaques were counted. The host cells were checked for coliphage contaminatior, upon receipt from the culture collection and weekly during the coliphage enumeration experiments using the above procedure, with sterile distilled water substituted for the water sample, Surviral of coliphaqe in stored sewaqe Single samples of ra~ sewage were collected on 18 November 1974, 6 January 1975, and 10 February 1975 and stored at 20 and 4°C in 600-ml wide-mouthed glassstoppered jars. Samples of the stored sewage were withdrawn periodically (see Fig. 1)and subjected to MPN enumeration for fecal coliform bacteria and total coliphage counts as detailed above
would suggest that the fecal coliform bacteria were better able to survive in the b o t t o m sediments than in the water column. The changing ratio of fecal colitbrms to cotiphage in sewage stored at 20 a n d 4:C is shown in Fig. I. G a s production, which indicates bacterial metabolism. was observed during the first 2 days m samples stored at 20°C a n d for the first 8 days at 4:C. During the first 2 days, the mean fecal coliform population of the samples stored at 20~C apparently declined from 2.07 to 1.40 × 1 0 6 100ml) - l while that of the 4~C sample apparently increased from 0.8 to 2.1 × 106 (100mt)-~ o n the eighth day. However. because of the wide confidence limits associated with the M P N test (APHA, 19711, neither of these changes can be considered significant.
DISCUSSION RESULTS The medians of the fecal coliform to coliphage ratios in raw sewage a n d lagoon effluent (Table t) were found to be significantly different at the 1~o level using the n o n p a r a m e t r i c Wilcoxon signed tank test (Campbell, 1967). However. the difference in the medians of the coliphage populations was shown by the same test not to be significant even at the 5'?,~; level. Therefore. the significant differences in the fecal coliform to coliphage ratios resulted from the 14-fold difference in fecal coliform populations. The results obtained for the river water ITabte 2) represent a much lower level of fecal contamination. It is interesting to note that the turbid samples taken on 13 January, 20 January, a n d 3 March, when the auger struck the riverbed, gave ratios similar to those found in the sewage lagoon effluent. This observation
The choice of a host bacterium is extremely important in any bacteriophage study because of the spectficity of phage acceptor sites on the surface of the bacterium. E. coli B (ATCC 11303-l) was selected because of its wide susceptibility to the coliphage. This organism is used as a host for 39 coliphage by the American Type Culture Collection. some 34 more than the nearest other multi-coliphage host E. coil C (ATCC 13709) (ATCC. 1974). In preliminary experiments. E. coil B (ATCC 11303-1) was found to support a b o u t I5 times as many phage from domestic sewage as a fecal coliform isolated from the sewage lagoon and 200 times as many as a strain isolated from the O l d m a n River, During passage of sewage t h r o u g h the lagoon system, the coliphage population remained almost unchanged while the fecal coliform population declined
Table I. Coliphage, fecal coliforms, and fecal coliform to coliphage ratio in raw sewage (S) and in sewage lagoon e~uent (E)
Date sample taken November 12 November 18 November 25 December 2 December 9 December 16 January 6 January 13 January 20 January 27 February 3 February 10 February 17 February 24 March 3 Median
Coliphage (1130 mll- 1 ( x 105)
Fecal coliforms (100 ml)- ~~x 105)
Ratio fecal coliforms to coliphage
S
E
S
E
S
E
0.25 0.53 0.96 0.73 0.26 0.51 0.12 0.81 0.09 0.28 0.09 0.09 0.42 0.02 0.09 0.25a*
0.t6 0.20 0.32 0.41 0.28 0.49 0.37 0.28 0.35 0,24 0.39 0.32 0.24 0.24 0.13 0.28a
33.0 46.0 49.0 70.0 17.0 11.0 11.0 130.0 l 1.0 6.0 2.0 5.0 2.0 2.0 23.0 1t .0b
3.3 13.0 7.9 1.7 0.8 0.8 0.5 4.9 2.3 0.2 0.8 0.8 4.0 0.5 2.4 0.8c
130:1 88:1 51 : 1 95:1 66:1 27:1 93:1 [60:1 130:1 21 : 1 24:1 57:1 5:1 87: l 270:1 87:1 d
21: t 66:1 25: I 4.2:1 2.9:1 t.6:1 1.4:1 18: I 6.7: t 0.9:1 2.1:1 2.5:1 L9:I
* For pairs of column medians, those followed by the same letter do not differ significantly (P<0.01).
2. t : I
19: t 4.2: I e
Coliform coliphage ratio
/q,I
Table 2. Coliphage. fecal coliforms, and fecal coliform to coliphage ratio in river water Date sample taken
Coliphage ( I00 ml)- * t x 10)
Fecal coliforms ( 100 ml)- ~t x 10)
Ratio fecal coliforms to coliphage
21.0 42.0 19.0 5.0 55.0 56.0 7.0 98.0 49.0 16.0 32.0
1.7 3.3 160.0 17.0 1.1 7.9 7.9 7.9 7.9 35.0 7.9
0.08:1 0.08:1 8.5:1 3.4:1 0.02:1 0.14: I l.l: l 0.0l: t 0.16:1 2.2: l 0.15: I
December 16 January 6 January 13" January 20* January 27 Februar? 3 February 10 February 17 February 24 March 3* Median * Turbid sample,
14-fold (Table 1). Therefore, contrary to the results of Kenard and Valentine (1974). the fecal coliform population cannot be readily calculated from the coliphage population because the ratio between them is not constant but decreases with time (Fig. l). The wide fluctuations in the ratio of fecal coliforms to total coliphage (Tables 1 a n d 2) are similar to those observed by Hilton and Stotzky (1973). Notwithstanding these wide fluctuations, this ratio may in itself be a useful pollution index. Unlike a simple count of fecal coliforms as a measure of fecal pollution, the coliform to coliphage ratio is independent of dilution. Therefore, because of the differential persistence of the coliform bacteria and the coliphage, a high ratio could be considered to indicate recent fecal c o n t a m i n a t i o n while a low ratio would indicate less recent contamination. In support of this hypothesis, it is reported that a ratio of 100:1 (Kott, Buras and Lindman, 1971) occurs in fresh feces while the results presented here show ratios I
o00~200
I:~",./
REFERENCES
,../ - ,
,oo ' .,°
American Public Health Association. {1971) Standard
methods for tlle examination oj" water and wastewater,
so
Ig u. ..a o o c0
...""%.......
~o
~-,.,.
............- \/,_
Io 5
ta.
o
of 8 7 : l , 4.2:1, and 0.15: I as being characteristic of raw sewage, lagoon effluent, and river water, respectively. The declining ratio of coliforms to coliphage observed in the sewage storage experiment (Fig. 1) supports, yet complicates, the use of the coliform to coliphage ratio as an index of when a fecal contamination event took place because the rate of change of the ratio is highly temperature-dependent. Furthermore, because of the differential persistence of fecal coliforms a n d coliphage from previous contamination, the ratio of the most recent event would be decreased and so the event would be considered more distant than it really was. The river water studies (Table 2) show the ratio displacing effect of the presence of sediment in a sample. While it is tempting to place significance on the coliform to coliphage ratio, it c a n n o t be considered as a precise index of the time since fecal pollution occurred because it is influenced by so many factors other than time. including chlorination (Kott et al., 1974), presence of sediment (Table 2). and temperature (Fig. 1).
\
2
4
8
12
OAYS
16
20
24
28
STOREO
Fig. I. Ratios of fecal coliforms to total colipbage in raw sewage stored at 20°C (solid line) and at 4°C (broken fine). Points are means of samples taken on three dates--18 November 1974, 6 January 1975 and 10 February 1975.
13th edition, p. 874. APHA, Washington, D,C. American Type Culture Collection. (1974) Catalogue of strains, 1lth edition, p. 369. ATCC, Rockville, Maryland. Bell R. G. (1975) The influence of NaCI, Ca-' -. and Mg-'* on the growth of a marine Bdellovibrio sp. Estuar. coastal mar. Sci. 3, 381-384. Campbell R. C. (1967) Statisticsfi)r hioloyists, p. 242. Cambridge University Press. Cruikshank R. (1962} Mackie and McCartney's Handbook of Bacteriology, 10th edition, p. 359. Livingstone. Edinburgh. Hilton M. C. & Stotzky G. (1973) Use of coliphages as indicators of water pollution. Can. J. Microbiol. 19. 747-751. Holden W. S. (1970) Water Treatment and Examination. p. 513, Churchill. London. Kenard R. P. & Valentine R. S. (1974) Rapid determination of the presence of enteric bacteria in water. Appl. Microbiol. 27, 484-487.
748
R.G. BELL
Kott Y., Buras N. & Lindman S. (t971) Coliphages a s virus indicators in water and wastewater. Sec. Annu. Rep. FWQA Res. G. 16030 DQN. FWPCA and Technion Res. and Dev. Found.. Haifa. Kott Y.. Roze N.. Sperber S. & Betzer N. (1974} 8acterio-
phages as viral pollution indicators. Water Res. 8. 165-171. Poynter S. F. B. (1966) Studies in the access of enteroviruses to water supplies. Ph.D. Thesis, Univ~ of London.