Survival of bacterial indicator organisms and enteric viruses in the Mediterranean coastal waters off Tel-Aviv

Water Res. Vol. 17, No. 4, pp. 397-402, 1983 Printed in Great Britain. All rights reserved

0043-1354/83/04039%06503.00/0 Copyright © 1983 Pergamon Press Ltd

SURVIVAL OF BACTERIAL INDICATOR ORGANISMS A N D ENTERIC VIRUSES IN THE MEDITERRANEAN COASTAL WATERS OFF TEL-AVIV BADR! FATTAL, ROBERT J. VASL, ELIYAHU KATZENELSONand HILLEL I. SHUVAL Environmental Health Laboratory, Hebrew University-Hadassah Medical School, Jerusalem, Israel (Received January 1982)

Abstract--Field and laboratory studies were initiated on the die-away kinetics of coliforms and enteric viruses in coastal waters off Tel-Aviv. Samples were taken from the sewage boil located approx. 880 m out to sea and from three main Tel-Aviv beaches designated as sampling stations, ranging 3 km to the north and 2 km to the south. Routine bacteriological tests were made from these stations; in addition, approx. 50~o of the samples were also examined for enteroviral content and occasionally, samples were taken from most of the other Tel-Aviv beaches for comparison of bacterial and viral content. The concentrations of total coliforms, fecal coliforms and fecal streptococci were correlated with the concentrations of enteroviruses. In general, the data suggests that the number of enteric bacteria in the sea was reduced relatively more rapidly than that of the enteroviruses, while fecal streptococci displayed a die-away rate similar to that of enteroviruses. The percentage of samples positive for enteroviruses was found to be lower during the summer months of the year than in the remaining months. The concentration of coliforms was also significantly lower during the summer months. This suggests that the decreased microbial level is associated with increased summertime daylight and solar radiation which affect the micro-organisms' die-away rate. About 76~o of the positive enteroviral samples were found at beaches with a fecal coliform bacterial level considered safe for bathing beaches.

INTRODUCTION Pathogenic bacteria and viruses are discharged in large numbers into the sea through sewage outfalls and pose a potential health risk to consumers of shellfish bred in adjacent waters, or to bathers at contaminated beaches. Although the concentration of bacteria is rapidly reduced in the sea as a result of dilution and other factors, it has been demonstrated that the pathogens can survive long enough and in sufficiently high concentration to lead to disease among bathers at contaminated beaches. The introduction of uniform standards and test procedures is an essential aspect of marine pollution control. This uniformity has been achieved recently through the U N E P / W H O Mediterranean pollution monitoring and control program, in which the Environmental Health Laboratory in Israel is an active participant. The purpose of this research project is to study the die-away of coliforms and enteric viruses in coastal waters and to test the concentration of the total coliforms, fecal coliforms and fecal streptococci in order to determine if there is any correlation with concentrations of enteroviruses at three Mediterranean beaches off the coast of Tel-Aviv, near one of its sewage outfalls.

main beaches (Tel Baruch No. 3, Nachshon-Hilton Nos 5 and 6, and Country Club No. 2) designated as sampling stations in Tel-Aviv, and an additional 27 samples were examined for enteroviral content. Occasionally, however, samples were taken from most of the other Tel-Aviv beaches for comparison of bacterial and viral content (see Map). Coliform data based on tests carried out by the Ministry of Health between the years 1963 and 1974 are also analyzed in this report. At a point about 880m out into the sea, the untreated sewage of Tel-Aviv is discharged through a pipe. The closest beaches are the Tel Baruch beach No. 3, 2 km north of the Reading power plant sewage outfall, Nachson and Hilton Nos 5 and 6, located 3 km southward and Country Club No. 2, which is further north of the Reading power plant sewage outfall. It should be noted that there are other points at which sewage enters the sea. One major source is the Yarkon River into which cities further inland release their sewage. Another smaller outlet is at Feingold Street No. 12 and near Bassa No. 13, both of which are now closed. MATERIALS AND METHODS

THE AREA

STUDIED

In accordance with the M E D VII plan during the study period October 1977-July 1980, 50 routine bacteriological tests were made on samples taken at three 397

For measurement of all bacterial parameters, the membrane filter methods used were those recommended i n "Guidelines for Health Related Monitoring of Coastal Water Quality", WHO (Copenhagen, 1977). The organic flocculation method was used for concentrating and testing

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13 Basso 14 Jaffa Port

JAFFA

BA T - YAM

Map 1. Tel-Aviv's coastal region in which samples were taken.

viruses from sea-water (Katzenelson et al., 1976; Katzenelson, 1978). These tests yielded a mean virus recovery of 65% with seeded polio virus in 351. samples of sea-water. RESULTS AND DISCUSSION

Data on wind velocity and direction, temperature and conductivity were collected for the above mentioned beaches. The mean wind velocity was 10 m s- 1 and the wind direction was generally from south to north. The temperature of the sea-water studied ranged between 15 and 29°C. with a summertime mean of 25°C. The mean for the remaining months of the year was 19°C. For the purposes of this study, summertime was defined as the months of May through October. inclusive. The mean conductivity for the entire period was 47 mmho. It should be noted, however, that the summertime mean was slightly higher (49 mmho).

Tables 1-4 summarize the data on total coliforms, fecal coliforms, fecal streptococci and enteroviruses at three beaches. F r o m these tables, the following can be seen: (1) The mean value of the coliform count is lower in the summer than in the remaining months of the year (presented as "others" in the tables). (2) Out of 27 enterovirus samples, 67% were positive. The percentage of positive enterovirus samples in the summer was 44 as against 56% in the remaining months. (3) About 76% of the positive enterovirus samples were found at beaches in which the level of bacterial pollution was considered to be within the acceptable range (less than 1000 fecal coliforms/lO0 ml) The U N E P / W F I O Int~,im Guideline for batMng water quality has established that aozepmble beaches should have no more than 109/o of the samples tested

Survival of bacterial indicator organisms

399

Table 1. Total coliforms--sea-surface (No./100 ml). Log mean values and SD Country Club Summer Others Total No. of cases

38 _+ 4 537 +_ 10 91 _+ 8 15

Distribution (all seasons) 0-100 101-1000 + 1000

No. 10 2 3

% 67 13 20

Tel Baruch

Hilton and Nachshon

All points

186 _+ 6 2512 ___5 468 _+ 9 17

36 _+ 6 120 _+ 8 59 _+ 7 18

51 + 4 269 _+ 8 110 _+ 7 50

No. 10 7 1

No. 25 14 11

No. 5 5 7

showing a fecal coli count greater than 1000/100 ml. Based on these guidelines, all three of the beaches studied met these criteria during the official summer b a t h i n g season (no samples greater than 1000/100 ml) a n d one, Tel Baruch (No. 3) showed one sample out of 11 had greater than 1000/100 ml. However, based on high total coliform counts, the Israeli health authorities have declared the beach unsafe for swimming for a n u m b e r of seasons. All of the results b u t one over 1000. F. coli/100ml shown in Table 2, were samples from the n o n - b a t h i n g season. Correlation analyses between the various bacteria a n d the enteroviruses are presented in Figs 1-3, with details of the regression line. A significant correlation was found ranging from r = 0.64--0.77 with p ~< 0.10.01.

% 29 29 42

% 56 39 5

% 50 28 22

The mean of the ratios of positive enteric virus samples vs other positive samples were as follows: (a) enteric viruses vs total coliforms = 1:3.0 x 106; (b) enteric viruses vs fecal coliforms = 1 : 1.8 x 106; (c) enteric viruses vs fecal streptococci = 1:4.3 x 104 . A total of 15 different enteroviruses was identified, including types of Poliovirus, Echovirus a n d Coxsackie (Fig. 4). These findings concur with those of Goyal et al. (1978). In the sea, total coliforms and fecal coliforms were shown to have more rapid dieaway rates than enteroviruses. The die-away rate of the fecal streptococci more closely paralleled that of the enteroviruses (Fig. 5), although the n u m b e r of samples was small and the scatter large.

Table 2. Fecal coliforms--sea surface (No./100 ml). Log mean values and SD Country Club Summer Others Total No. of cases Distribution (all seasons) 0-100 101-1000 + 1000

25 -+ 5 208 _+ 8 65 _+ 10 14 No. 10

% 71

Tel Baruch 79 -+ 11 2089 _+ 8 219 _+ 16 18 No. 7

% 44

Hilton and Nachshon

All points

14 _+ 6 28 _+ 12 18 _+ 9 18

25 _+ 6 89 _+ 14 43 _+ 10 48

No. 13

No. 30 10 8

I

7

5

31

4

3

22

4

25

1

% 72 22 6

% 62 21 17

Table 3. Fecal streptococci--sea surface (No./100 ml). Log mean values and SD

Summer Others Total No. of cases: Distribution (all seasons) 0-100 101-i000 +1000

Country Club

Tel Baruch

Hilton and Nachshon

All points

38 _+ 4 589 ___2 76 -+ 6 12

85 _+ 3 708 + 7 182 ___6 14

31 _+3 16+8 24 _+ 5 15

45_+3 85_+4 58 _+ 4 41

No. 6 5 1

% 50 42 8

No. 4 8 2

% 28 58 14

No. 13 2 0

% 86 14 0

No. 23 15 3

% 56 37 7

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Table 4. Comparison of the concentration of total coliforms (TC}, fecal coliforms {FC} and fecal streptococci (FS) in bathing water samples found positive for enterovirus Enterovirus (Pill I ~)

Date 26.10.77 22.01.78 22.01.78 14.02.78 15.03.78 15.03.78 03.04.78 03.04.78 18.09.79 16.10.79 16.10.79 14.04.80 14.04.80 14.04.80 26.05.80 01.07.80 01.07.80 01.07.80

2/7/) 1/511 4/70 1/85 7/85 8/50 7/80 7/80 2/34 1/80 4/70 28/50 40/65 36/65 5/75 13/78 5/80 4/78

TC (No./l(~ ml)

FC (No./100 ml)

0 20 380 10 8.9 x 103 980 7.4 x 103 250 23 50 23 1.4 × 1 0 3 1.8 x 103 210 24 4 400 130

0 20 0 4.6 x 530 5.1 × 40 t4 1 0 2.6 x 3.1 x 155 23 6 60 0

The finding that coliform counts are higher in winter than in summer has been noted by us for some time. In an effort to elucidate this question, several h u n d r e d coliform tests at the Country Club Beach between 1963-1974 were analyzed, as shown in Fig. 6 (Shuval, 1978). There is a very strong negative correlation between mean m o n t h l y log coliform c o u n t and mean m o n t h l y duration of sunshine (r = 0,93). F r o m this data, it can be seen that coliform counts in the sea were almost two log cycles higher during the winter period of low sunshine duration than in the summer m o n t h s of m a x i m u m sunshine duration. O t h e r studies ( G a m e s o n & Gould, 1975) have shown that light plays a major role in coliform dieaway rates in sea-water. This might provide a reasonable explanation for the significantly lower coliform counts during the summer at beaches in the vicinity of outfalts. It should be noted, however, t h a t most beach monitoring tests in Israel and in m a n y Mediterranean countries are carried out during the official summer bathing season. This could lead to the approval of beaches which might not meet the criteria during the winter "off season" period. M a n y of these Mediterranean beaches are frequented in the winter as well, particularly by tourists from n o r t h e r n E u r o p e a n

8 --

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FS (No./100 ml)

Hilton and Nachshon Country Club Tel Baruch Hilton and Nachshon Country Club Tel Baruch Tel Baruch Hilton and Nachshon Hilton and Nachshon Country Club Hilton and Nachshon Country Club Tel Baruch Hilton and Nachshon Country Club Tel Baruch Hilton and Nachshon Hilton and Nachshon

0 1.2 x 103 130 1.2 x 103 20 9 8 l5 880 450 65 70 600 190 60

103 l03

103 103

countries who find swimming in 18"C sea-water quite acceptable. An operative conclusion from these findings might be to require bacteriological monitoring of such tourist beaches o n a year-round basis with decisions as to their acceptability based both on winter and summer test results. In a mathematical analysis of Figs 1-3, the volume of sea-water in which one would expect to find one enterovirus and one of the indicator bacteria can be theoretically obtained b y either of two methods, first by solving the two simultaneous equations of x and the linear regression equation of the particular bacteria vs enterovirus graph. The point of intersec-I

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Fig. 3. Correlation between fecal streptococci and viruses found at beaches.

Survival of bacterial indicator organisms 18.0

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Fig. 4. Types of viruses found at beaches.

tion of the two lines is that fraction of the virus or bacterium which can be found in 100 ml of sea-water. Secondly, the point of intersection could be found graphically. Extrapolating further to the left from this point, the number of viruses found would be greater than the number of bacteria. This may explain the findings in this report in which some samples containing zero or very few bacteria were still found to contain enteroviruses. Table 5 shows that the volumes of the sea-water samples in which there were equal concentrations of bacteria and viruses in beach samples were: 495, 170 and 4521. for virus:coliforms, virus:fecal coliforms and virus:fecal streptococci respectively.

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These volumes are much larger than the 2.31. calculated in Italy (Petrilli & deFlora, 1977). This could be explained by the fact that Petrilli's work was performed in very polluted water close to a sewage outfall in the sea and any extrapolation to any less contaminated waters would be more reliably reflected when samples are indeed taken from less contaminated sea-water. CONCLUSIONS

I. In general, the finding of this study, although limited, suggest that the number of enteric bacteria in the sea is reduced relatively more rapidly than that of enteroviruses. 2. Fecal streptococci display a die-away rate which appears similar to that of enteroviruses. This may be a useful indicator of the degree of viral contamination in the sea. 3. A wide range of enteroviruses can be detected in the sea at a distance of up to 5 km from the point of sewage discharge into the sea. 4. The microbial concentration in the sea is low in the summer as compared to the remaining months of the year. This is apparently due to increased summertime daylight and solar radiation which affects the rate of microbial die-away.

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Fig. 5. Disappearance of enteric microorganisms at various distances from the point of discharge to sea.

' 400

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, 500

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Mean monthly duration of sunshine,

rain day-'

Fig. 6. Relationship between duration of sunshine and coliform count at the Tel-Aviv Country Club Beach, 1963-1974.

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BADRI ]"AIIAI el u]. Table 5. Mathematical analysis of samples taken at beaches

Coliforms vs viruses Fecal coliforms vs viruses Fecal strep. vs viruses

Vol. 1l.) Intersect containing pt. with 1 vir. and y=x(log) I bact

Figure No.

Lin. corr.

No. of points

Level of signif.

Regression line equation Log y = m log x - i

1

0.7741

14

<0.01

log y = 0.2046 log x - 2.9392

3.695

495

2

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8

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3.232

170

3

0.6412

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log y = 0.2583 log x - 2.7116

3.656

452

x = bacteria concentration; y = virus concentration; m = slope; i = intercept.

5. In view o f our finding that enteroviruses were detected at all three beaches, even w h e n the coliform levels were acceptable, health authorities s h o u l d n o t ignore the possible risk o f b a t h i n g in sea-water slightly c o n t a m i n a t e d with sewage. This finding e m p h a s i z e s the i m p o r t a n c e o f impleanenting the prop o s e d study on disease r a t e s a m o n g b a t h e r s a n d n o n b a t h e r s at b e a c h e s o f varying levels o f bacterial cont a m i n a t i o n (Cabelli et al:, 1976). It also suggests that in the future there m i g h t be a need to d e v e l o p a virus s t a n d a r d for b a t h i n g beaches.

Acknowledgement--We wish to express our appreciation and thanks to the W H O / U N E P coordinated Mediterranean Pollution Monitoring and Research Programme (MED VII) who have made this study possible. REFERENCES

Cabelli V., Levin A. P., McCabe M. A. & Habermann

B. W. (1976) Relationship of microbial indicators to health effects at marine bathing beaches. Annual Meeting of American Public Health Association. Chicago. Gameson A. L. H. & Gould O. J. (1975) Effects of solar radiation on the mortality of some terrestrial bacteria in sea-water. In Discharge of Sewage from Sea Outfalls (Edited by Gameson A. L. H.), pp. 209-211. Supplement to Progress in Water Technology, Pergamon Press. Oxford. Goyal S., Gerba C. & Melnick J. (1978) Prevalence of human enteric viruses in coastal canal communities, d. Wat. Pollut. Control Fed. 50, 2247-2256. Katzenelson E. (1978) Concentration and identification of viruses in sea-water. Rev. Int. Oceanogr. Med. 48, 9-16. Katzenelson E., Fattal B. & Hostovesky T. (1976) Orgamc flocculation as an efficient second step in virus concentration. Appl. envir. Microbiol. 32. 638-639. Petrilli F. L. & deFlora S. (1977) Correlation between animal viruses and bacteria in coastal sea-waters and sediments. Rev. Int. Oceanogr. Med. 47, 33-36 Shuval H. I. (1978l Studies on bacterial and viral contamination of the marine enwronment. Rev. Int. Oceanogr. Med. 48, 43-50.