Seasonal fluctuation of bacterial indicators in coastal waters

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Taylor & Francis 2001. ISSN 0891-060X ..... Pike EB. Recreational use of coastal waters: development of health related standards. J IWEM 1993; 7: 162–7. 23.
ORIGINAL ARTICLE

Seasonal  uctuation of bacterial indicators in coastal waters Vasiliki Maipa1 , Yannis Alamanos1 and Eugenia Bezirtzoglou2 From the 1 Department of Hygiene, Medical School, University of Ioannina, Greece and 2 Department of Microbiology, Medical School, University of Ioannina, Greece Correspondence to: E. Bezirtzoglou, Department of Microbiology, University of Ioannina, 45110 Ioannina, Greece. Tel.: »30 651 97590:1; Fax: »30 651 46480; E-mail: [email protected]

Microbial Ecology in Health and Disease 2001; 13: 143 – 146 The relationships between number of coliforms, fecal coliforms, E. coli, fecal streptococci, location, time and seasonal factors in marine environments of northwest Greece were investigated over a period of 4 years. Research focused on measuring and comparing bacteria in coastal marine waters undergoing heavy bacterial charge during the tourist season. Microbiological pollution was increased during the summer period. Seasonal  uctuation of the fecal indicator bacteria was noted and concerned mainly E. coli and fecal coliforms, which are the most sensitive indicators in the marine environment. Key words : bacterial indicators, fecal coliforms, fecal streptococci, recreational water, seasonal  uctuation, sea water, water pollution.

INTRODUCTION Many directives relating to the prevention of pollution also relate to a potential health risk associated with marine coastal waters, since these waters ultimately inherit much of the pollution that enters fresh water. It is therefore necessary to estimate the quality of water which could be a risk for the health of people using beaches with polluted bathing waters. Although there is not sufŽ cient epidemiological data to support a precise relationship between swimming-related illness and the levels of bacteriological indicators of water pollution, it is accepted that there is some degree of health risk associated with high levels of contamination. Epidemiological studies indicate that certain health effects correlate with swimming in polluted water and the degree of microbiological pollution (1– 3). Among several microbiological indicators used to deŽ ne the quality of bathing water are fecal coliforms, enterococci, fecal streptococci, staphylococci, pseudomonas and other bacterial species. Concentrations of bacterial indicators in bathing waters are considered signiŽ cant in relation to parameters such as time of sampling, season and location of collection (4– 6). Mediterranean coasts are of great interest for such studies because of the particular conditions of the ecosystem and the increased pressures and microbial charges that this system receives every year, especially during the summer period under anthropogenic in uence. © Taylor & Francis 2001. ISSN 0891-060 X

The concerted degradation of microbial associations in this dense ecosystem can result in a leached out production of high organic strength, which, if left untreated, may have adverse environmental effects (7). Process optimization depends on an understanding of the interactions between the microbial physiological groups (8). This suggests that there may be a seasonal pollution problem, mainly in coastal waters undergoing increased seasonal pressure during the summer months because of an in ux of tourists. The aim of the present study is to describe and compare the  uctuation of different microbiological indicators in relation to time and location in an area of Greece considered as a low pollution area, but undergoing considerable pressure every summer because of the increasing numbers of tourists visiting the region. Clearly, there are certain factors that affect the fecal indicator bacteria more in very eutrophic areas than in other sampling areas (9) and so it is necessary to use very sensitive indicators to evaluate the quality of the water being used. MATERIAL AND METHODS Sampling area During the course of 4 years and within a program of bathing waters control carried out by the Laboratory of Hygiene of Ioannina University, we estimated the degree of pollution along the northwestern coasts of Greece. Four Microbial Ecology in Health and Disease

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beaches were selected for study, two located in Thesprotia and two in Preveza (Fig. 1). Sample handling and analyses Between Ž ve and eight samples of coastal water were collected every month from each beach, starting in May and ending in September. They were all collected in a ‘sampling column’ equipped with a thermometer and a measuring scale that allowed the depth to be selected (10). When the column is immersed in the water an abrupt movement causes water to pass through the entrance. Duplicate samples were collected at a depth of 30 cm below the surface at selected times of the day (between 8 a.m. and 5 p.m.). Water samples were thoroughly mixed, placed in sterile 100 ml bottles and transported to the laboratory on ice within an hour of collection. They were shaken vigorously by hand before analysis. At the laboratory, all samples were stored at 59 2°C until analyses were complete, which was always within 24 h of sample collection. Each water sample was analyzed for total coliforms, fecal coliforms, E. coli and fecal streptococci. Samples (100 ml) were diluted (1:10) in accordance with level of pollution, and analysis was performed using the membrane Ž ltration culture method in accordance with the standard methods proposed by APHA (1992) (11).

Fig. 2. Seasonal  uctuation of bacterial indicators.

The growth media used were the following : Total coliforms (Ž ltration method) m-ENDO Agar (Difco) incubated at 36°C for 24 h. ConŽ rmation was made by selection and culturing of 10 characteristic colonies in BGLB (Brilliant Green Lactose Broth) at 36°C for 24 h. Fecal coliforms-E. coli (Ž ltration method) mFC Agar (Difco) incubated at 44°C for 24 h. ConŽ rmation was made by selection and culturing of 10 characteristic colonies in LTLSB (Lactose-Tryptone-Lauryl-SylphateBroth) at 44°C for 24 h. Fecal streptococci (Ž ltration method) Slanetz and Bartley (Oxoid) incubated at 36°C for 48 h. ConŽ rmation was made by transport of the membrane in Esculin bile Agar at 36°C for 1 h.

STATISTICAL ANALYSIS Multiple linear regression analyses were carried out using the SPSS:PC program, with microbiological parameters as dependent variables. Time parameters were included in the analysis as independent variables. Location was also included in the analysis as an independent variable (each sampling station was assigned a number from one to four). RESULTS

Fig. 1. Four sampling stations on the northwestern coasts of Greece.

The four sampling stations in the present study are located along the coast of the Ionian Sea within two different districts of Epirus, a northwestern region of Greece (see Fig. 1), i.e. between 5 and 12 km from the district capital cities. The seasonal  uctuation of fecal coliforms, E. coli and fecal streptococci concentrations is presented in Fig. 2. All bacterial indicators re ect increased microbiological pollution in the months of July and August. The mean monthly

Seasonal  uctuation of bacterial indicators in coastal waters

concentration of fecal coliforms ranges from 12.2 colonies per 100 ml in May to 29.7 in August; of E. coli from 6.1 in May to 13.9 in August; and of fecal streptococci from 2.0 in June to 9.2 in August. Table I gives the results of multiple regression analysis. Microbiological indicators are considered as dependent variables, and time and location parameters as independent variables. Month and year of sampling were independently associated with total coliforms and E. coli. The hour of sampling correlated independently with total coliforms and fecal streptococci. The pH ranged from 8.1 to 8.3 at all sampling stations and the value of conductivity was approximately 66 mS: cm. Algae were present at all locations. DISCUSSION The health effect of bathing in coastal waters is a subject of scientiŽ c controversy. Although no consistent relation between any precise microbiological indicator of water quality and disease has been conŽ rmed, many studies undertaken on the subject indicate a possible link between microbiological water pollution and measurable health effects. Swimming-related illness seems to manifest mainly in upper respiratory and gastrointestinal illness, as well as in diseases of the eyes, ears and skin (12– 14). It is unlikely that one bacterial indicator will sentinal all possible health hazards related to recreational waters. Total coliform bacteria concentrations are generally assumed to be replaced by fecal coliform and other fecal bacteria concentrations as indicators of water quality (14). It is also unlikely that all bacterial indicators are sensitive indicators of sea water pollution  uctuation in relation to seasonal parameters. The results of the present study suggest that fecal coliforms are the most sensitive indicators of the seasonal  uctuation of sea water pollution. However, it is interesting that these bacterial indicators, which according to the present study are the most sensitive concerning variation in time, are unlikely always to be the best indicator of health risks related to polluted water. A recent randomized trial study suggests that fecal streptococci concentration is the best indicator of water quality when it comes to the risk of gastroenteritis (3). Fecal streptococci are unlikely to be the most sensitive indicator of the seasonal  uctuation of sea water pollution according to the results of this study, although their survival in the environment is generally characterized as limited. Fecal streptococci originate from the intestine of warm-blooded animals, so they are an indicator of water contamination by fecal material. Other similar streptococci originating from plants or plant products can also be found in the sea water, reacting positively when tested with the membrane Ž lter culture method. So data on fecal streptococci concentration are considered more valid in relation to sea water pollution when combined with fecal coliform data (15).

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Fecal coliforms indicate a strong correlation with fecal contamination from warm-blooded animals. Their presence in sea water re ects recent contamination by fecal material, because fecal coliforms die within hours of being exposed to sunlight in sea water at temperatures above »40°C (15). The survival of most bacteria in sea water is also dependent on salinity, temperature, pH, solar radiation and other factors, all of which have to be taken into consideration when interpreting results. In this study no speciŽ c measurements of these parameters were taken, which could have an in uence on our results. According to the Ž ndings of this study there are signiŽ cant differences in bacterial indicator levels between different sampling times. These are most obvious in the case of total coliforms and fecal streptococci (Table I), and could probably be attributed to water temperature, wind speed and direction, and waves. A signiŽ cant between-year  uctuation during the study period can also be concluded from Table I in relation to the mean concentrations of fecal coliforms and E. coli. The increased microbiological pollution during the summer months, mainly in the months of July and August, is the most interesting Ž nding of the study. It is in these months that the coasts of Greece are subjected to enormous environmental pressure from the movement of tourists. And the fact that this seasonal  uctuation is shown mainly by increased fecal coliforms and E. coli concentrations suggests that the increased microbiological pollution can be attributed to the increased human presence during July and August in those areas. It has to be said that other studies carried out in Mediterranean coastal waters suggest a lower level of microbiological pollution during the summer months of the year than in other months, probably associated with increased summertime daylight and solar radiation, both of which affect the mortality of the microorganisms (16). Similar results have been reported by another group of scientists (6), who show that fecal indicator bacteria survive better in water during the winter than the summer. Studies on the survival of fecal indicator bacteria in rivers suggest that maximum survival under natural conditions occurs in waters at 0°C (17). But these two studies derive from northern countries, where waters are ice-covered during the winter months. The numbers of indicator bacteria decrease very soon after stratiŽ cation in the spring and remain relatively low during the summer. In the autumn, bacteria counts begin to rise again (17). These seasonal  uctuations seem to be related to water temperature. It has been reported (18) that the temperature of water correlates signiŽ cantly with the die-off of E. coli, and is governed by the insolation. A lower rate of bacterial mortality due to protozoan grazing, which features intense and selective grazing pressure on those bacteria cells which are near to, or in,

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division, might also effectively control bacterial abundance in marine waters (19). Algae were found at all the studied locations. These are phototrophic organisms whose growth is directly dependent on light; they can also be chemo-organotrophic under certain conditions. Fecal indicator bacteria are reported as chemo-organotrophic bacteria and require the decomposition of organic compounds for their nutrition. These organic compounds can be provided by the phototrophic algae (20), so the growth of fecal bacterial indicators may be interrelated with the development of the aquatic biological chain. Sea water quality criteria proposed by the World Health Organisation and other international foundations are usually suggested as microbiological pollution indicators, total coliforms, fecal coliforms, enterococci and fecal streptococci (15, 21, 22). The combination of several indicators is likely to offer a global picture of water quality (23). The Ž ndings of this study suggest that fecal coliforms represent the most sensitive bacterial indicator when it comes to seasonal  uctuation related to increased human presence during the summer months. This study suggests that bathing waters may be subject to increased microbiological pollution during months of increased tourist movement, and that this could be attributed to the excessive human presence despite the increased daylight and solar radiation during the summer months which affect the die-off rate of microorganisms. Fecal coliforms represent the most sensitive indicator of this seasonal pollution. The known diversity of marine microbial life in waters has been greatly expanded by studies during the past few years because of the heterogeneity of the ecosystem, which is characterized by relatively small mean cell size bacteria and low stock bacterial abundance (19). It is clear, however, that we need a better understanding of the mechanisms of action of preservative systems, particularly factors commonly used to control the growth of microorganisms. These studies will include identiŽ cation of sites of action, different stages of the spore-vegetative cell cycle, biovariability, distribution of germination rates, lag times and rates of outgrowth. REFERENCES 1. Seyfried PL, Tobin RS, Brown NE, Ness PF. A prospective study of swimming-related illness. I. Swimming-associated health risk. Am J Public Health 1985; 75: 1068 – 70. 2. Corbett SJ, Rubin GL, Curry GK, Kleinbaum DG. The health effects of swimming at Sydney beaches. Am J Public Health 1993; 83: 1701 – 6. 3. Kay D, Fleisher JM, Salmon RL, Wyer MD, Godfree AF, Zelenauch-Jacquotte Z, Shore R. Predicting likelihood of gastroenteritis from sea bathing: results from randomised exposure. Lancet 1994; 344: 905 – 9. .

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