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Applied and Environmental Microbiology, February 2004, p. 814-821, Vol. 70, No. 2
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.2.814-821.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Effect of Environmental Factors on the Relationship between Concentrations of Coprostanol and Fecal Indicator Bacteria in Tropical (Mekong Delta) and Temperate (Tokyo) Freshwaters

Kei O. Isobe,¹ Mitsunori Tarao,¹ Nguyen H. Chiem,² Le Y. Minh,² and Hideshige Takada^1*

Faculty of Agriculture, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183-8509, Japan,¹ College of Agriculture, Can Tho University, Can Tho City, Can Tho Province, Vietnam²

Received 29 September 2003/ Accepted 30 October 2003

Top Abstract Introduction Materials and Methods Results Discussion References

 
A reliable assessment of microbial indicators of fecal pollution (total coliform, Escherichia coli, and fecal streptococcus) is critical in tropical environments. Therefore, we investigated the relationship between concentrations of indicator bacteria and a chemical indicator, coprostanol (5ß-cholestan-3ß-ol), in tropical and temperate regions. Water samples were collected from the Mekong Delta, Vietnam, during wet and dry seasons, and from Tokyo, Japan, during summer, the aftermath of a typhoon, and winter. During the wet season in the Mekong Delta, higher bacterial densities were observed in rivers, probably due to the higher bacterial inputs from soil particles with runoff. In Tokyo, higher bacterial densities were usually observed during summer, followed by those in the typhoon aftermath and winter. A strong logarithmic correlation between the concentrations of E. coli and coprostanol was demonstrated in all surveys. Distinctive seasonal fluctuations were observed, as concentrations of coprostanol corresponding to 1,000 CFU of E. coli/100 ml were at their lowest during the wet season in the Mekong Delta and the typhoon aftermath in Tokyo (30 ng/liter), followed by the dry season in the Mekong Delta and the summer in Tokyo (100 ng/liter), and they were much higher during the winter in Tokyo (400 ng/liter). These results suggested that E. coli is a specific indicator of fecal contamination in both tropical and temperate regions but that the densities are affected by elevated water temperature and input from runoff of soil particles. The concurrent determination of E. coli and coprostanol concentrations could provide a possible approach to assessing the reliability of fecal pollution monitoring data.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Sewage pollution in tropical Asian regions is a severe health risk to people that live near rivers and waterways. Direct discharge of domestic waste, leaching from poorly maintained septic tanks, and improper management of farm waste are suspected as the major sources of waterborne disease (19). Appropriate countermeasures against water pollution necessitate the availability of reliable indicators of sanitary risk. In tropical Asia, however, these factors have been inadequately studied.

The rapid and simple enumeration of fecal bacteria, such as coliforms and fecal streptococci, is beneficial in fecal pollution monitoring in geographically large areas, and thus several water quality guidelines based on bacterial indicator densities have been established (3, 28). However, the use of these indicators is controversial, especially in tropical regions characterized by high temperatures and frequent rainstorms that facilitate erosion of soils. Several studies have reported that the growth and survival of fecal indicator bacteria are susceptible to environmental factors, such as water temperature (4, 13), sunlight (5, 30), and rainfall (9, 22). Therefore, a more reliable indicator of water pollution than fecal bacterial enumeration needs to be developed.

For determination of the fecal pollution status in tropical waters by monitoring of indicator bacteria, it is important to assess the reliability of the method. In this report, we propose the exploitation of the relationship between bacterial indicators and a chemical indicator, coprostanol (5ß-cholestan-3ß-ol), to verify the reliability of fecal indicator bacteria in tropical regions. Coprostanol is a major sterol found in human feces (24 to 89% of total steroids) (12, 23) and has been proven to be a very promising indicator of fecal pollution by numerous studies of coprostanol concentrations in the waters and sediments of rivers, lakes, and estuaries (10, 11, 26, 34). Reported half-lives of coprostanol in aerobic conditions are generally <10 days at 20°C (29). Thus, the presence of coprostanol in an aerobic environment can be considered an indication of recent fecal input to the waters.

Positive correlations between fecal indicator bacteria and coprostanol concentrations have been reported for several temperate and cold climate regions (8, 11, 14, 24, 27). Previously, we conducted an intensive survey in the Mekong Delta and in western Malaysia and demonstrated a strong correlation between fecal indicator bacteria and coprostanol concentrations in tropical regions for the first time (20). However, none of the previous studies, to our knowledge, have directly compared this relationship in different climatological settings. In this study, we present the results of surveys conducted during the wet and dry seasons in the Mekong Delta and during the summer, the aftermath of a typhoon, and winter in the Tokyo metropolitan area. The objectives are (i) to provide fecal pollution monitoring data based on three bacterial indicators, namely total coliform (TC), Escherichia coli, and fecal streptococcus (FS), and a chemical indicator, coprostanol; and (ii) to assess the reliability of using indicator bacteria to assess sewage contamination in tropical regions by directly comparing variations in their relationships with coprostanol concentrations in different climates.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Site description.
The climate in the Mekong Delta (Fig. 1a) is tropical, with wet (May to November) and dry (December to April) seasons. The average monthly temperatures have little seasonal variation, ranging from 25.5°C (December) to 28.7°C (February). The total average annual precipitation is 1,663 mm, with the maximum monthly precipitation in October (280 mm) and the minimum in February (2 mm). On the other hand, Tokyo (Fig. 1b) is characterized by a temperate climate with four seasons. The average monthly temperatures are highest during summer (August, 26.6°C) and lowest during winter (January, 5.4°C). The total average annual precipitation is 1,467 mm, with the maximum monthly precipitation in June (185 mm) and the minimum in January (45 mm).

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FIG. 1. Map showing location of study sites in Mekong Delta, Vietnam (a), and Tokyo, Japan (b). Solid circles indicate sampling sites and double circles indicate STPs.

The Mekong River, an international river in Southeast Asia, has a total length of 4,620 km, and the Mekong Delta lies in southern Vietnam. The catchment area (65,170 km²) constitutes 19.6% of the total land area of Vietnam (331,700 km²) and has a population of approximately 15 million. Almost no sewage treatment plants (STPs) had been established in the Mekong Delta at the time of this survey.

In Tokyo, three rivers with different characteristics were surveyed. The Sumidagawa River, with a total length of 23.5 km, has a population of approximately 6.2 million in its catchment area (690 km²). It receives approximately 21 m³ of secondary effluent/s from several STPs which comprise up to 70% of the river water (36). The Tamagawa River, with a total length of 138 km, has a population of approximately 3.4 million in its catchment area (1,240 km²) and also receives secondary effluents from STPs, with a minor input from septic tank effluents. The Minamiasakawa River, with a total length of 8 km, receives effluents from septic tanks, and its headwaters are considered to be control sites because they have no known source of human impact.

Sample collection.
Water samples were collected from 44 sites (23 river, 12 canal, and 9 groundwater) and 29 sites (17 river, 9 canal, and 3 groundwater) during the wet season (October 2000) and dry season (March 2002), respectively, in the Mekong Delta and from 24 sites during the summer (July 2001), the aftermath of a typhoon (September 2001), and the winter (December 2001) in Tokyo. During the typhoon aftermath survey in Tokyo, the rivers were heavily swollen due to the passage of a typhoon with a total rainfall of 290 mm. In addition, for examination of removal efficiencies, grab samples of raw sewage (influents) and secondary effluents were taken from five STPs located in the Tokyo metropolitan area.

All samples for bacteriological analysis were collected in sterile glass bottles, immediately placed on ice in dark cooling boxes, and processed within 8 h of collection. All samples for sterol analysis were collected in a stainless steel bucket and stored in solvent-rinsed 3-liter amber glass bottles. The samples (0.2 to 1 liter) were filtered with prebaked 0.7-µm-pore-size glass fiber filters (GF/F; Whatman, Kent, United Kingdom) within 6 h of collection. The filters (with trapped particulate matter) were then stored at -30°C until analysis. The dry weight of particles on the filters was measured for all samples.

Bacteriological analysis.
The membrane filter technique (2) was used for the enumeration of indicator bacteria. The water samples were diluted, and each dilution was filtered through a sterile 0.45-µm-pore-size nitrocellulose filter (Millipore Corp., Bedford, Mass.). The filters were placed on a plate of Chromocult coliform agar (Merck, Darmstadt, Germany) with 5 µg of cefsulodin (Sigma Chemical Co., St. Louis, Mo.)/ml for the enumeration of TC and E. coli (1). The plates were incubated at 36°C for 24 h. All salmon to red colored colonies were counted as TC except for E. coli, and all blue to violet colored colonies were counted as E. coli. m-Enterococcus agar for FS was used for the enumeration of FS (2) and was incubated at 36°C for 48 h. All light and dark red colored colonies were counted as FS.

Sterol analysis.
Sterol standards were purchased from Sigma-Aldrich, Co. (coprostanol, 5ß-cholestan-3ß-ol; epicoprostanol, 5ß-cholestan-3-ol; ß-sitosterol, 24-ethylcholest-5-en-3ß-ol; stigmastanol, 24-ethyl-5-cholestan-3ß-ol), Applied Science Labs, State College, Pa. (coprostanone, 5ß-cholestanone; cholesterol, cholest-5-en-3ß-ol; campesterol, 24-methylcholest-5-en-3ß-ol), and Tokyo Kasei Kogyo Co., Ltd., Tokyo, Japan (cholestanol, 5-cholestan-3ß-ol; stigmasterol, 24-ethylcholesta-5,22E-dien-3ß-ol; fucosterol, (3ß,24E)-stigmasta-5,24(28)-dien-3-ol). Sterol names correspond to the notation used in the Merck Index (25). Cholesterol-2,2,3,4,4,6-d₆ purchased from Sigma-Aldrich was used as a recovery surrogate, and perylene-d₁₂ purchased from Supelco was used as a gas chromatography injection internal standard.

The detailed procedure for sterol analysis has been previously reported (20). Briefly, the samples were consecutively ultrasonically extracted with three solvents, namely methanol, dichloromethane-methanol (1:1 [vol/vol]), and dichloromethane, and then were purified with silica gel column chromatography. The sterol fraction eluted with dichloromethane and acetone-dichloromethane (3:7 [vol/vol]) was acetylated with acetic anhydride and pyridine before instrumentation. A Hewlett-Packard 5973 quadrupole mass spectrometer fitted with an HP 6890 gas chromatograph equipped with a split/splitless injector operated in splitless mode under isothermal conditions (300°C) was used for sterol analysis. The analysis was performed on an HP-5ms, 30 m by 0.25 mm (inside diameter), 0.25-µm film thickness capillary column (Hewlett Packard). Chromatographic and operational conditions of the mass spectrometer have been previously described (20).

Statistical analysis.
The concentrations of indicator bacteria and sterols were logarithmically transformed, and the samples in which microorganisms or sterols occurred below the limit of detection were recorded as zero for statistical analysis. Analyses were performed using the statistical software package StatView v. 5.0 (SAS Institute Inc., Cary, N.C.). In all cases, significance was determined at the 95% confidence level. One-way analysis of variance was performed to assess the differences among surveys, with a significance level of 5% (P < 0.05). The coefficients of determination (R²) were calculated to express the percentage variation that can be explained based on the regression equation indicating coprostanol concentrations corresponding to bacterial standards. The regressions presented use the bacterial number as the independent variable and the sterol concentration as the dependent variable, and F significance (P) values were determined.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Physical characteristics of the field surveys.
The water temperatures in the Mekong Delta were 27 to 30°C throughout the surveys. In Tokyo, the water temperature during summer was above 25°C, while the temperature dropped to below 10°C during winter. The geometric mean concentrations (in milligrams per liter) of suspended solids (SS) in the Mekong Delta during the wet and dry seasons were 68.3 (maximum, 415.5) and 31.8 (maximum, 477.5), respectively. All the river water samples had higher SS concentrations in the wet season, while most of the canal water samples exhibited lower SS concentrations in the wet season (P < 0.05). Higher SS concentrations during the wet season in the river waters were probably due to inputs of soil and other terrestrial particles carried by runoff, riverside soils eroded by strong and frequent rains, and resuspended bottom sediments. The geometric mean concentration of SS in Tokyo during the aftermath of a typhoon was close to the value observed in the Mekong Delta, at 65.9 mg/liter (maximum, 241.8 mg/liter). The respective geometric mean concentrations (in milligrams per liter) of SS in the summer and winter surveys were 6.1 (maximum, 26.7) and 5.7 (maximum, 49.9), approximately 1/10 those after the typhoon survey.

STP surveys.
Influent and effluent samples taken from STPs located in the Tokyo metropolitan area were analyzed for TC, E. coli, FS, and 10 sterols, including coprostanol, to examine the removal efficiencies of the plant (Table 1). All STPs used activated sludge treatment followed by physical treatment (settlement) for their treatment process. As a result, high removal efficiencies were confirmed for both indicator bacteria (>99.9%) and all sterols, including coprostanol (99.4 ± 0.5%).

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TABLE 1. Bacterial densities, coprostanol concentrations, and %Cop in sewage samplesa

Bacterial indicators.
Densities of TC, E. coli, and FS isolated from water samples collected from the Mekong Delta and Tokyo are illustrated in Fig. 2. One-way analysis of variance demonstrated a significant difference in TC, E. coli, and FS densities between the surveys (P < 0.05). The highest number of E. coli per 100 ml was found during the dry season (2.4 x 10⁶), followed by the wet season in the Mekong Delta (1.1 x 10⁶) and the summer (4.8 x 10⁵), the aftermath of a typhoon (9.0 x 10⁴), and the winter (1.9 x 10⁴) in Tokyo. In the Mekong Delta, E. coli numbers were found to increase by 1 to 2 orders of magnitude in 76% of the river sites during the wet season compared to the dry season. Meanwhile, in Tokyo, E. coli numbers were found to decrease by 1 to 2 orders of magnitude in 75% of the sites during the winter compare to the summer.

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FIG. 2. Densities of TC (a), E. coli (b), and FS (c) in water collected during the dry season (n = 29) and wet season (n = 44) in the Mekong Delta and during the summer (n = 24), typhoon aftermath (n = 24), and winter (n = 24) in Tokyo.

E. coli was detected in the groundwater at six of nine sites during the wet season survey in the Mekong Delta (maximum CFU, 1.9 x 10⁴). FS was also detected at the same sites (maximum, 4.2 x 10²). TC, E. coli, and FS were detected at control sites in Tokyo irrespective of season, and the maximum numbers per 100 ml were 2.6 x 10³, 2.0 x 10², and 1.9 x 10³, respectively. Coprostanol concentrations at these sites were below the detection limit (0.1 ng/liter), indicating no input of human feces. Therefore, E. coli and FS may be naturally present in soil particles or feces of animal origin (16) at the sources of these sites.

Chemical indicators.
The coprostanol concentrations in the particulate phase of water samples are illustrated in Fig. 3a. Significant differences in coprostanol were observed among all seasons and sites (P < 0.05). The highest concentrations of coprostanol were observed during the dry season (0.001 to 97.1 µg/liter), followed by the wet season in the Mekong Delta (<0.0001 to 13.5 µg/liter), the winter (<0.0001 to 3.77 µg/liter), the typhoon aftermath (<0.0001 to 2.94 µg/liter), and the summer in Tokyo (0.0002 to 1.52 µg/liter). The highest coprostanol concentration observed during the dry season in the Mekong Delta was higher than concentrations reported in Japan in the 1970s (24.0 µg/liter) (21) or in Canada (22.0 µg/liter) (10).

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FIG. 3. Concentrations of coprostanol (a) and %Cop (b) in water collected during the dry season (n = 29) and wet season (n = 44) in the Mekong Delta and during the summer (n = 24), typhoon aftermath (n = 24), and winter (n = 24) in Tokyo.

Similarly, the highest total sterol concentrations (summed concentrations of 10 analyzed sterols) were observed during the dry season (0.17 to 308.9 µg/liter), followed by the wet season in the Mekong Delta (0.04 to 57.6 µg/liter), the winter (0.16 to 17.9 µg/liter), the typhoon aftermath (0.11 to 12.4 µg/liter), and the summer in Tokyo (0.06 to 11.9 µg/liter). Extremely high concentrations of total sterols were observed during the dry season in the Mekong Delta (308.9, 186.55, and 85.03 µg/liter). The maximum concentrations of total sterols were approximately 2 to 5 times higher in the Mekong Delta than in Tokyo.

The percentage of coprostanol relative to total sterols (%Cop) was calculated (Fig. 3b). Since the source of coprostanol is considered to be mainly human feces (12, 23), %Cop indicates the contribution of human feces to sterol inputs to the aquatic environment and has been examined in several studies (17, 18, 20). The highest %Cop was observed during the dry season (34%), followed by the wet season in the Mekong Delta (27%), the typhoon aftermath (24%), the winter (22%), and the summer in Tokyo (17%).

Correlation between indicator bacteria and coprostanol.
The coefficients of determination (R²) were calculated for log-transformed TC, E. coli, and FS versus coprostanol concentrations. The strongest correlation was demonstrated for E. coli and coprostanol concentrations throughout the surveys, as shown in Fig. 4 (R² = 0.82 to 0.91; P < 0.001). TC also showed a strong correlation with coprostanol (R² = 0.75 to 0.90; P < 0.001), whereas lower correlation coefficients were demonstrated for FS (R² = 0.17 to 0.86; P < 0.001).

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FIG. 4. Relationship between concentrations of E. coli and coprostanol in water collected during the dry season (a) and wet season (b) in the Mekong Delta and during the summer (c), typhoon aftermath (d), and winter (e) in Tokyo. Each open circle represents a sampling event. Log-transformed E. coli and coprostanol concentrations are linearly regressed (solid lines), and coefficients of determination (R2) are indicated. Coprostanol concentrations corresponding to an E. coli concentration of 1,000 CFU/100 ml (secondary contact limit) are indicated by dotted lines.

FS in the Mekong Delta was more susceptible to climate changes than fecal coliforms, as the correlation was weaker during the wet season (R² = 0.27) than during the dry season (R² = 0.86). Figure 5 emphasizes that the relationship in river waters was more dispersed than that in canal waters (P < 0.05). It is likely that rivers receive fecal matters from various natural sources, such as wild and/or domestic animals, compared to urban canals, in which the majority of fecal inputs are of human origin.

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FIG. 5. Relationship between concentrations of FS and coprostanol in water collected during the dry season (a) and wet season (b) in the Mekong Delta. Each open circle represents a sampling event. Log-transformed FS and coprostanol concentrations are linearly regressed (solid lines), and coefficients of determination (R2) are indicated. Solid circles, open circles, and crosses indicate the canal water, river water, and groundwater samples, respectively.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Fecal pollution monitoring.
The rapid population growth and urbanization in tropical Asia have led to serious water pollution problems in the region. Intensive monitoring, especially of fecal pollution, is important for understanding the extent of this problem. According to the water quality-based limit established by the Ministry of the Environment in Japan, no detection of fecal coliform is desirable, but up to 1,000 CFU/100 ml is permissible for bathing water, although water with >400 CFU/100 ml requires improvement. Among the surveyed river and canal sites in the Mekong Delta, 91 and 77% in the wet and dry seasons, respectively, exceeded E. coli limits of 1,000 CFU/100 ml. This indicates a pressing need for improved sanitation to halt the potential spread of waterborne diseases in this region.

Despite the high coverage of sewage treatment in Tokyo, E. coli was detected at densities higher than the guideline limit at 47, 63, and 54% of the surveyed sites in the summer, after a typhoon, and in the winter, respectively. Since the geometric mean density of E. coli in STP effluents was 1.3 x 10² CFU/100 ml (Table 1), E. coli densities in the rivers that exceed 10³ CFU/100 ml cannot be explained solely by inputs via STP effluents. The higher E. coli densities may be attributed to combined sewage overflows during extreme runoff situations as well as %Cop during the aftermath of a typhoon (22). The results suggest that urban rivers receiving large amounts of effluent from STPs also require frequent monitoring to ensure that favorable sanitary water quality is maintained.

Correlation of indicator bacteria and coprostanol.
This study has emphasized the necessity of reappraising the use of fecal indicator bacteria in tropical regions to account for distinctive seasonal variations in bacterial counts in different climatological settings. Strong correlations existed between E. coli and coprostanol concentrations throughout our surveys; however, the relationship appeared to be affected by water temperature and SS concentrations.

Similar studies conducted in the Derwent Estuary (24) and in Sydney (27) did demonstrate a strong correlation between fecal coliform and coprostanol concentrations. These studies concluded that a coprostanol concentration of 400 ng/liter was equivalent to the secondary contact limit (1,000 CFU/100 ml) of fecal coliforms (24). As shown in Fig. 4, this concentration criterion for coprostanol was consistent with the result of our winter survey in Tokyo. However, the calculated coprostanol concentrations which corresponded to E. coli concentrations of 1,000 CFU/100 ml were much lower during the wet (30 ng/liter) and dry seasons (100 ng/liter) in the Mekong Delta and the summer (30 ng/liter) and typhoon aftermath (100 ng/liter) in Tokyo.

The observed differences in the corresponding coprostanol concentrations can probably be ascribed to variations in water temperature and soil particle concentration. It is likely that lower water temperatures during the winter in Tokyo inhibited in situ growth of E. coli (31), while coprostanol concentrations remained high in the winter because microbial degradation was lower. Similar observations have been reported for other organic compounds, such as linear alkylbenzenes (LABs), whose concentrations were higher in winter due to decreased water flow and lower rates of biodegradation due to lower water temperatures (35). Thus, the higher coprostanol concentration (400 ng/liter), which corresponded to an E. coli concentration of 1,000 CFU/100 ml, in the winter agrees with observations from previous studies conducted in moderate or cold climate regions. On the other hand, in the Mekong Delta and in Tokyo during the summer and after a typhoon, the densities of E. coli reached the critical level of bathing water (1,000 CFU/100 ml) at lower coprostanol concentrations (<100 ng/liter), probably due to the higher survival or growth rates of microbiota in waters with a higher ambient temperature.

The higher soil particle concentrations during the wet season in the Mekong Delta and in the aftermath of a typhoon in Tokyo probably resulted in even lower coprostanol concentrations (30 ng/liter), as suggested by Byappanahalli and Fujioka (7), because tropical soil environments provide sufficient means to support the growth of fecal coliforms and E. coli. Kistemann et al. (22) found considerably elevated numbers of bacteriological parameters, such as TC, E. coli, and FS, during extreme runoff events. Other studies have documented in detail that the sources of coliforms and enterococci detected after heavy rain, such as from a typhoon, can probably be attributed to soils along the river bank (9, 16, 32). Solo-Gabriele et al. (33) performed field measurements and laboratory experiments to evaluate the sources of E. coli in a coastal waterway and concluded that the higher concentrations of E. coli observed after episodes of heavy rain were caused by the growth of E. coli within riverbank soils which were subsequently washed away during high tide. The resuspension of sediments, which can act as reservoirs of enteric bacteria, in response to heavy rain can also be a source of E. coli (15). Moreover, the detection of E. coli at groundwater sites in the Mekong Delta and at control sites in Tokyo suggested the possible input of fecal indicator bacteria that has a nonhuman origin. The possible presence of E. coli in soil particles in addition to soil bacteria mistakenly counted as E. coli has also been reported (6, 16). Thus, we observed a lower coprostanol concentration (30 ng/liter) which corresponded to an E. coli concentration of 1,000 CFU/100 ml during the wet season in the Mekong Delta and after a typhoon in Tokyo.

On the basis of our results, we suggest that the concurrent determination of E. coli densities and coprostanol concentrations can provide a possible approach for the assessment of the reliability of fecal pollution monitoring data. The strong correlations demonstrated throughout the surveys suggest that E. coli can be considered an efficient indicator of fecal contamination in tropical regions, but because the densities are affected by water temperature and inputs of soil particles during heavy rains, some caution should be observed in their interpretation. Further studies are necessary to examine accurately the relationship between fecal indicator bacteria and the occurrence of pathogens and to reexamine the recommended guidelines for the microbiological quality of tropical freshwater.

 
This work was financially supported by the "Research Project for Sustainable Coexistence of Humans, Nature and the Earth" (Research Revolution 2002) of the Japanese Ministry of Education, Culture, Sports, Science and Technology (MEXT). Our field trips were supported by the "JICA/Can Tho University Project for Improvement of Environmental Education in Agricultural Sciences" of the Japanese International Cooperation Agency (JICA).

We acknowledge the invaluable efforts of R. Leeming, H. Hayashidani, and T. Okatani in the preparation of the manuscript and thank them for their expertise and guidance. We also thank several graduate students and undergraduates in our laboratories for their helpful assistance with fieldwork.

 
* Corresponding author. Mailing address: Faculty of Agriculture, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183-8509, Japan. Phone: 81-42-367-5825. Fax: 81-42-360-8264. E-mail: shige{at}cc.tuat.ac.jp.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Applied and Environmental Microbiology, February 2004, p. 814-821, Vol. 70, No. 2
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.2.814-821.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

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