Page 9: Guidelines for Canadian Recreational Water Quality – Third Edition

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Part II: Guideline Technical Documentation

4.0 Recommended indicators of faecal contamination

Recreational waters may be contaminated with faecal material from such sources as discharged sewage, stormwater runoff from agricultural or urban areas, wild or domesticated animals, and even through faecal shedding by swimmers themselves. Many epidemiological studies have identified gastrointestinal and upper respiratory illnesses in swimmers as a result of such contamination. Historically, the bacteria in the coliform group and its subgroups (total coliforms, thermotolerant [faecal] coliforms, E. coli) and the enterococci--the more faecal-specific portion of the faecal streptococci group--have been used to monitor recreational waters for the presence of faecal contamination. As such, they have also been used to indicate the possible presence of pathogenic microorganisms responsible for these illnesses. Routine testing of recreational waters for pathogenic organisms is impractical and is not recommended for the following reasons:

• Testing for every possible waterborne disease-causing microorganism would be prohibitive in terms of both the financial resources necessary and the time required to perform the analyses. These organisms are difficult to isolate and quantify, and testing requires proper laboratory containment facilities, specialized equipment and highly trained and experienced microbiologists. Detection methods for some pathogens do not exist at all.
• Pathogens are usually present at low levels and are unevenly distributed in recreational waters, even during disease outbreaks.
• The absence of one pathogen does not necessarily ensure that other enteric pathogens are also absent.

Consequently, authorities monitor for non-pathogenic faecal indicator bacteria that are present in high numbers in both human and animal faeces. Elevated numbers of these indicator bacteria in the aquatic environment are indicative of faecal contamination and therefore suggest the possible presence of enteric pathogens.

The ideal faecal indicator organism would meet the following requirements (Cabelli et al., 1983; Elliot and Colwell, 1985):

• found within the intestinal tract of humans and warm-blooded animals;
• present in faecally contaminated waters when enteric pathogens are present, but found in greater numbers than pathogens;
• incapable of growth in the aquatic environment, but capable of surviving longer than pathogens;
• applicable to all types of natural recreational waters (fresh, estuarine and marine waters); and
• absent from non-polluted waters and exclusively associated with animal and human faeces.

Other desirable qualities for the indicator organism include:

• Density of the indicator should be directly correlated with the degree of faecal contamination.
• Density of the indicator should be quantitatively related to swimmer-associated illnesses.
• Detection and enumeration test methods should be rapid, easy to perform, inexpensive, specific and sensitive.

No single microorganism unequivocally meets all of these criteria. E. coli and enterococci are currently considered the best indicators of faecal contamination in recreational waters, as they most closely fit the above characteristics. There are limitations associated with the use of indicators in assessing the quality of recreational waters. Judicious use of these guideline values as part of a multi-barrier approach to recreational water management represents a sound approach to protecting swimmers against exposure to faecal pathogens in the recreational water environment.

4.1 Indicator organisms for primary contact recreation

4.1.1 Fresh waters: Escherichia coli (E. coli)

Guideline values

For fresh recreational waters used for primary contact activities, the guideline values are as follows:

• Geometric mean concentration (minimum of five samples): ≤ 200 E. coli/100 mL
• Single-sample maximum concentration: ≤ 400 E. coli/100 mL

Calculation of the geometric mean concentration should be based on a minimum of five samples, collected at times and sites so as to provide representative information on the water quality likely to be encountered by users. Further action should be initiated if either of these guideline values is exceeded. Minimum action should consist of immediate resampling of the site(s). In addition, a swimming advisory may be issued should the responsible authority identify that the area is not suitable for recreational water use.

It is further advised that recreational water areas routinely used for primary contact recreation be monitored at a minimum of once per week, with increased monitoring recommended for those beaches that are highly frequented or are known to experience high user densities. Similarly, under certain scenarios, a reduction in the recommended sampling frequency may be justified. Further guidance on sampling frequency recommendations and the posting of recreational waters can be found in Part I (Management of Recreational Waters).

Enterococci (Section 4.1.2) is also recognized as a suitable indicator of faecal contamination in fresh recreational waters (Cabelli, 1983; Pruss, 1998; Wade et al., 2003, 2006). If it can be shown that enterococci can adequately demonstrate the presence of faecal contamination in fresh waters, then the enterococci maximum limits for marine waters may be adopted. If there is any doubt, samples should be examined for both sets of indicators for extended periods to determine whether a positive relationship exists.

Guideline rationale

The guideline values have been developed based on epidemiological evidence relating E. coli concentrations in fresh recreational waters to the incidence of swimming-associated gastrointestinal illness observed among swimmers. The existing epidemiological data are not sufficient to permit the estimation of the level of risk for individual exposures. Based on the U.S. EPA's regression analysis of epidemiological data (Dufour, 1984), Health Canada has estimated that using the guideline values for the recommended indicators of faecal contamination for fresh and marine waters will correspond to a seasonal gastrointestinal illness rate of approximately 1-2% (10-20 illnesses per 1000 swimmers).

In determining the value for the maximum faecal indicator concentration permitted in a single sample, the U.S. EPA equations pertaining to the calculation of a single-sample limit were reviewed (U.S. EPA, 1986). The data regarding the maximum allowable indicator density at designated beach areas is consistent with applying a factor of 2 times the recommended geometric mean value results. Thus, the single-sample maximum concentration of 400 E. coli/100 mL is reaffirmed.

These values represent risk management decisions that have been based on a thorough assessment of the potential risks for the recreational water user. In considering both the potential health risks and the benefits of recreational water use in terms of physical activity and enjoyment, it was concluded that this is a tolerable and reasonable estimate of the risk of illness likely to be experienced by users engaged in a voluntary activity.

An evaluation of the epidemiological information published since the Guidelines were last issued concluded that the current body of evidence supports the existing recommendations regarding the use of E. coli as the indicator of faecal contamination in fresh recreational waters. There has not been substantial evidence to suggest that revision of the existing guideline values is necessary at this time.

Description

E. coli most closely fits the requirements of an ideal indicator of faecal contamination for fresh waters. The organisms are found in high numbers in the intestinal tract and faeces of humans and warm-blooded animals. The vast majority of the E. coli types are harmless. There are several types (serotypes or strains) that possess virulence factors enabling them to act as human pathogens; however it should be noted that faecal concentrations of the typical non-pathogenic E. coli will always be greater than those of the pathogenic strains, even during outbreaks. E. coli is considered a more specific indicator of faecal contamination than either total coliforms or thermotolerant (faecal) coliforms and can be rapidly and easily enumerated in recreational waters.. In addition, a strong correlation has been demonstrated between the concentration of E. coli in fresh waters and the risk of gastrointestinal illness among swimmers (Dufour, 1984; Wade et al., 2003).

For several decades now, recreational water quality experts in Canada have recognized E. coli as the indicator of choice for faecal contamination. The use of E. coli as an indicator of recreational water quality was of limited use until the 1980s, when standardized laboratory methods permitting detection of the organism within 24-48 hours became available. Prior to this, the thermotolerant coliform group was used as the primary indicator of faecal contamination in recreational waters. However, subsequent findings that some thermotolerant coliform species had non-faecal or environmental origins and could be isolated in high numbers from waters receiving waste effluents from such sources as pulp and paper and textile manufacturing facilities (Dufour and Cabelli, 1976; Huntley et al., 1976; Rokosh et al., 1977; Vlassoff, 1977) raised concerns about the reliability of using this group as an indicator of faecal contamination in recreational waters. Despite the availability of methods specific for the detection of E. coli, testing laboratories were already set up to test for thermotolerant coliforms, and the requirements to perform surveillance of recreational waters for these microorganisms were embedded in long-standing regulatory and legislative documents. As a result, it has taken many years and considerable time to amend the existing guidelines and standards, updating them to reflect the current state of knowledge that E. coli is the preferred indicator of faecal pollution for fresh recreational waters.

In the 1992 edition of the Guidelines, the introduction of E. coli as the recommended indicator for freshwater quality represented a new direction for recreational water monitoring, moving away from the 1983 recommendation regarding the use of thermotolerant coliforms. As a result, a provision was made that allowed thermotolerant coliforms to continue to be used if it could be determined that greater than 90% of the thermotolerant coliforms were, in fact, E. coli. This was done to permit an adjustment period for jurisdictions in making the changeover to the new recommendations. Significant time has now passed to allow jurisdictions to make the change from thermotolerant coliforms to the more faecal-specific E. coli. As a result, this third edition does not recommend the use of thermotolerant coliforms as an indicator of the quality of recreational waters. Instead, it reaffirms that E. coli is the preferred indicator for monitoring fresh recreational waters in Canada.

Occurrence in the aquatic environment

Within human and animal faeces, E. coli is present at a concentration of approximately 10⁹ cells per gram (Edberg et al., 2000) and comprises about 1% of the total biomass in the large intestine (Leclerc et al., 2001; Health Canada, 2012a). Human faecal flora characterization studies have reported that E. coli was detected in 94% and 100% of the subjects tested (Finegold et al., 1983; Leclerc et al., 2001). These values were significantly higher than those reported for other members of the coliform group and were matched or exceeded by only enterococci and certain species of anaerobic bacteria (Bacteroides, Eubacterium).

E. coli comprises about 97% of the coliform organisms in human faeces, with Klebsiella spp. comprising 1.5% and Enterobacter and Citrobacter spp. together comprising another 1.7%. E. coli has been shown to represent between 90% and 100% of all coliforms in faeces from eight species of domestic animals, including chickens (Dufour, 1977).

Once shed from a human/animal host, faecal bacteria are not expected to survive for long periods in the aquatic environment (Winfield and Groisman, 2003). Survival of E. coli in the recreational water environment is dependent on many factors, including temperature, exposure to sunlight, available nutrients, water conditions such as pH and salinity, and competition from and predation by other microorganisms.

Numerous authors have reported on the ability of beach sand and sediments to prolong the survival of faecal microorganisms (Whitman and Nevers, 2003; Ishii et al., 2006a; Kon et al., 2007a). This environment is thought to provide more favourable conditions of temperature and nutrients than the adjacent waters, as well as to offer protection from certain environmental stressors such as sunlight. Others have reported on the ability of E. coli to survive in organic-rich environments not known to be associated with faecal contamination, such as industrial process wastes and wastes from pulp and paper manufacturing (Megraw and Farkas, 1993; Gauthier and Archibald, 2001). Recently, researchers have reported on the ability of E. coli and other faecal bacteria to survive within mats of the green algal species Cladophora (Whitman et al., 2003; Olapade et al., 2006).

Historically it was believed that E. coli could only originate from faecal sources and was incapable of growth in the aquatic environment. However, recent studies are raising questions regarding these assumptions (Kon et al., 2007b; Hartz et al., 2008, Vanden Heuvel et al., 2009). Research in this area is ongoing. These recent findings do not invalidate the use of E. coli as the best available indicator for recreational water quality.

E. coli is considered to be a good surrogate of the survival of enteric bacterial pathogens in recreational waters. Several authors have reported similar survival rates for E. coli and enteric bacterial pathogens (Rhodes and Kator, 1988; Korhonen and Martikainen, 1991; Chandran and Mohamed Hatha, 2005). The indicator is regarded to be more sensitive to environmental stresses than human enteric viruses and protozoa and thus does not survive as long in the environment as these organisms.

In many parts of Canada, freshwater beaches are routinely monitored for levels of E. coli as an indication of faecal contamination. Many Canadian recreational waters are of good microbiological quality; however, certain waters are contaminated throughout part, or all, of the swimming season. An examination of beach monitoring data over a 10-year period (1993-2003) at 10 Lake Huron recreational beaches in Ontario demonstrated that levels of E. coli can vary widely at a single location from year to year and between beach locations (Ontario Ministry of the Environment, 2005). E. coli values can range from 0/100 mL in isolated areas to several thousand per 100 mL in areas directly impacted by faecal contamination (Payment et al., 1982; Sekla et al., 1987; Williamson, 1988; Ontario Ministry of the Environment, 2005).

Association with pathogens

E. coli is considered a good indicator for enteric bacterial pathogens such as Salmonella, Shigella, Campylobacter and E. coli O157:H7 (Health Canada, 2012a). Investigations conducted by the Water Environment Research Foundation (Yanko et al., 2004) examined the relationship between E. coli concentrations in surface water samples collected from various watersheds in southern California and the probability of detecting Salmonella and Shiga toxin-producing E. coli (STEC). The results demonstrated that the probability of detecting Salmonella using culture-based methods steadily increased up to a concentration of approximately 1000 E. coli/100 mL. At this point, a 100% probability of detection was reported. Similar results were reported for the detection of STEC strains using polymerase chain reaction (PCR)-based methods. Although there was clearly an association between E. coli concentrations and the probability of detection of Salmonella and STEC strains, the authors reported that no single sample could be used to provide absolute assurance of the presence or absence of these pathogens.

E. coli is a less effective indicator of enteric pathogenic viruses and protozoa. Numerous studies have reported on the lack of a correlation between E. coli concentrations and the presence of enteric viruses and protozoa in surface waters (Griffin et al., 1999; Denis-Mize et al., 2004; Hörman et al., 2004; Dorner et al., 2007).

E. coli are always present in faecal contamination from human and animal sources. Detection indicates faecal contamination of water and thus the possible presence of faecal pathogenic bacteria, viruses and protozoa. The occurrence of faecal pathogens in recreational waters is strongly dependent on the nature of the contamination sources impacting the swimming area. Their presence and numbers in the environment can be sporadic and highly variable. As well, some enteric pathogens can survive longer than the faecal indicators. The absence of E. coli should not be interpreted to mean that enteric pathogenic microorganisms are also absent.

The combination of routine E. coli monitoring alongside actions, procedures and tools to collectively reduce the risk of swimmer exposure to faecal contamination in the recreational water environment represents the most effective approach to protecting the health of recreational water users.

Guidelines used by other countries/organizations

The guideline formats and values established by other government and multinational organizations worldwide for faecal indicator organisms in fresh waters were reviewed in developing the revised edition of this document.

Related epidemiological studies

The U.S. EPA's original epidemiological study in fresh recreational waters. measured the concentrations of faecal indicator organisms (thermotolerant (faecal) coliforms, E. coli, enterococci) in swimming waters and compared them with the rates of swimming-associated illness reported on the same days on which the samples were collected (Dufour, 1984). Statistically significant rates of gastrointestinal illness were observed among swimmers in waters considered to be more faecally contaminated. For symptoms unrelated to gastrointestinal illness, no statistically significant differences were observed. For the data analysis, the mean seasonal faecal indicator concentration per 100 mL was plotted against the seasonal swimming associated rate of gastrointestinal symptoms per 1000 persons for each indicator. Correlation and regression analysis were then used to determine the correlation coefficients and the slope of the linear regression equation for each indicator. The best correlation coefficient (r) was obtained with E. coli (r = 0.80) and an almost equal correlation coefficient was observed with enterococci (r = 0.74). The E. coli data were used to produce a regression equation:

The equation used to calculate the rate of gastrointestinal illness from E. coli concentrations

Several freshwater epidemiological studies have been conducted since the previous version of the Guidelines for Canadian Recreational Water Quality was developed (Lightfoot, 1988; Ferley et al., 1989; Calderon et al., 1991; van Asperen et al., 1998). All have confirmed the existence of a strong relationship between exposure to recreational waters and swimming-associated illness; however, few have been able to demonstrate evidence of a mathematical relationship between faecal indicator counts and illness among swimmers. Van Asperen et al. (1998) reported that the risk of gastroenteritis was significantly higher among triathletes who swam in water with a geometric mean concentration of > 355 E. coli colony-forming units (cfu)/100 mL (equivalent to E. coli/100 mL). Ferley et al. (1989) proposed that faecal streptococci were a better indicator of gastrointestinal illness at freshwater beaches in France. Calderon et al. (1991) observed that total staphylococci counts were most closely associated with gastrointestinal illness among swimmers at a recreational pond not affected by point source discharges.

Only a few epidemiological studies have been conducted that have investigated the health effects of recreational activities other than swimming, such as whitewater canoeing and rafting (Fewtrell et al., 1992; Lee et al., 1997). The data linking water quality and illness through these activities have been less strong. Still, conclusions that have been reported are that gastrointestinal illness similarly constitutes the most frequently reported health outcome during these types of activities; and that factors related to the risk of illness include the quality of the water and the frequencies of immersion and water ingestion.

Several reviews of the epidemiological findings have also been published. In 1998, WHO (Pruss, 1998) published a comprehensive review of the epidemiological research that had been conducted over the period from 1953 to 1996. This was the first extensive review of the existing epidemiological literature. Pruss (1998) concluded that gastrointestinal illness was the most frequent health outcome for which dose-response relationships were reported and that the indicators that best correlated with health outcomes were enterococci for marine waters and E. coli and enterococci for fresh waters.

The U.S. EPA published two reviews of the existing epidemiological literature for recreational waters. The first, published in its Implementation Guidance for Ambient Water Quality Criteria for Bacteria (U.S. EPA, 2002), was a minireview of the epidemiological studies conducted since the previous guidance had been issued, in 1986. The U.S. EPA concluded that the epidemiological methods used to derive its 1986 Water Quality Criteria remained scientifically valid and that no new scientific principles had been established that would require a revision of the current guidelines. Subsequent to this, the U.S. EPA (Wade et al., 2003) conducted a meta-analysis of the existing epidemiological data available in the literature to determine if its current regulatory standards were sufficiently protective against the risk of gastrointestinal illness in recreational waters. The authors demonstrated that in the freshwater studies, E. coli was shown to be the most suitable indicator of recreational water illness. It was further reported that when comparing the summary relative risk values with the U.S. EPA guidelines for fresh water, E. coli densities above the guideline values were associated with an increased risk of illness, whereas exposures below the guideline values did not show an association.

Wiedenmann et al. (2006) reported on the results of a randomized controlled prospective cohort study conducted at freshwater swimming sites in Germany. Earlier randomized controlled trials had been conducted in coastal waters of the United Kingdom (Kay et al., 1994; Fleisher et al., 1996); however, this study was the first of its type to be conducted in fresh waters. The study design used by the authors was similar to that originally used in the UK trials. The authors observed evidence of a relationship between the observed rates of illness and measured concentrations of E. coli, enterococci, Clostridium perfringens and somatic coliphages. No-observed-adverse-effect-levels (NOAELs) were reported for several definitions of gastroenteritis, ranging from 78 to 180 E. coli/100 mL and from 21 to 24 enterococci/100 mL. The authors proposed possible guidelines by combining all of the data derived from the different definitions of gastrointestinal illness investigated, suggesting values of 100 E. coli/100 mL, 25 enterococci/100 mL, 10 somatic coliphages/100 mL and 10 C. perfringens/100 mL. Although the authors propose a value of 100 E.coli/100 mL, it is important to note that the NOAEL reported for gastrointestinal illness that most closely fits the criteria of "highly credible gastrointestinal illness" (as defined by Cabelli et al , 1983) was 180 E. coli/100 mL. In addition, the quartile and quintile breakdown of the data for the UK definition of gastrointestinal illness indicated that the rates of swimmer illness compared to those of the control group were not statistically significant until E. coli concentration ranges approached or exceeded 200 E. coli/100 mL. Even using the least stringent definition of gastrointestinal illness resulted in a NOAEL (NL-2^{Footnote 1}: 164 E. coli/100 mL) well above 100 E. coli/100 mL.

The U.S. EPA and Centers for Disease Control and Prevention (CDC) have also been conducting epidemiological studies at freshwater and marine beaches under the National Epidemiologic and Environmental Assessment of Recreational (NEEAR) Water Study. The studies are intended to support the development of new recreational water quality guidelines (U.S. EPA, 2002; Dufour et al., 2003) as well as to investigate new water quality indicators and rapid methods for water quality monitoring. Data collection for these studies was completed in 2010.

Summary

It has been concluded, based on all of the existing evidence, that E. coli remains the most suitable indicator of faecal contamination in fresh recreational waters. In summary:

1. The guideline values have been developed based on the analysis of epidemiological evidence relating E. coli concentrations in fresh recreational waters to the incidence of swimming-associated gastrointestinal illness observed among swimmers. The values represent risk management decisions based on the assessment of possible health risks for the recreational water user and the recognition of the significant benefits that recreational water activities provide in terms of health and enjoyment.
2. E. coli most closely fits the requirements of an ideal indicator of faecal contamination for fresh waters. E. coli are always present in faecal contamination from human and animal sources. Detection suggests faecal contamination of water and thus the possible presence of faecal pathogenic bacteria, viruses and protozoa.
3. There are limitations associated with the use of indicators in assessing the quality of recreational waters. The occurrence of faecal pathogens in recreational waters is dependent on many factors and can be variable and sporadic. The absence of E. coli should not be interpreted to mean that enteric pathogenic microorganisms are also absent.
4. Combining routine E. coli monitoring alongside actions, procedures and tools to collectively reduce the risk of swimmer exposure to faecal contamination in the recreational water environment represents the most effective approach to protecting the health of recreational water users.

4.1.2 Marine waters: Enterococci

Guideline values

For marine recreational waters used for primary contact activities, the guideline values are as follows:

• Geometric mean concentration (minimum of five samples): ≤ 35 enterococci/100 mL
• Single-sample maximum concentration: ≤ 70 enterococci/100 mL

Calculation of the geometric mean concentration should be based on a minimum of five samples, collected at appropriate times and sites to provide representative information on the water quality likely to be encountered by users. Further action should be initiated if either of these guideline values is exceeded. Minimum action should consist of immediate resampling of the site (or sites). In addition, a swimming advisory may be issued should the responsible authority identify that the area is not suitable for recreational water use.

It is further advised that recreational water areas routinely used for primary contact recreation be monitored at a minimum of once per week, with increased monitoring recommended for those beaches that are highly frequented or are known to experience high user densities. Similarly, under certain scenarios, a reduction in the recommended sampling frequency may be justified. Further guidance on sampling frequency recommendations and the posting of recreational waters can be found in Part I (Management of Recreational Waters).

E. coli (Section 4.1.1) is also recognized as a useful predictor of the risk of gastrointestinal illness in marine recreational waters (Wade et al., 2003). If it can be shown that E. coli can adequately demonstrate the presence of faecal contamination in marine waters, then the E. coli maximum limit for fresh waters may be adopted. If there is any doubt, samples should be examined for both sets of indicators for extended periods to determine whether a positive relationship exists.

Guideline rationale

The guideline values have been developed based on epidemiological evidence relating enterococci concentrations in marine recreational waters to the incidence of swimming-associated gastrointestinal illness observed among swimmers. The existing epidemiological data are not sufficient to permit the estimation of the level of risk for individual exposures. Based on the U.S. EPA's regression analysis of epidemiological data (Cabelli, 1983), Health Canada has estimated that using the guideline values for the recommended indicators of faecal contamination for fresh and marine waters will correspond to a seasonal gastrointestinal illness rate of approximately 1-2% (10-20 illnesses per 1000 swimmers). In Canada, owing to geography and climate, a significantly smaller percentage of the population engages in marine recreational water activities compared with those engaging in freshwater recreation.

In determining the value for the maximum faecal indicator concentration permitted in a single sample, the U.S. EPA equations pertaining to the calculation of a single-sample limit were reviewed (U.S. EPA, 1986). The data regarding the maximum allowable indicator density at designated beach areas are consistent with applying a factor of 2 times the recommended geometric mean value results. Subsequently, a single-sample maximum concentration of 70 enterococci/100 mL is reaffirmed.

These values represent risk management decisions that have been based on a thorough assessment of the potential risks for the recreational water user. In considering both the potential health risks and the benefits of recreational water use in terms of physical activity and enjoyment, it was concluded that this is a tolerable and reasonable estimate of the risk of illness likely to be experienced by users engaged in a voluntary activity.

Evaluation of the epidemiological information published since the Guidelines were last issued concluded that the current body of evidence supports the existing recommendations regarding the use of enterococci as the indicator of faecal contamination in marine recreational waters. There has not been substantial evidence to suggest that revision of the existing guideline values is necessary at this time.

Description

Enterococci are members of the genus Enterococcus. The genus was created to include the more faecal-specific species of genus Streptococcus, formerly considered as group D streptococci. In practice, the terms enterococci, faecal streptococci, Enterococcus and intestinal enterococci have been used interchangeably (Bartram and Rees, 2000). Enterococci are characterized by their ability to fulfil the following criteria: growth at temperatures of 10°C and 45°C, resistance to 60°C for 30 minutes, growth in the presence of 6.5% sodium chloride and at pH 9.6, and the ability to reduce 0.1% methylene blue (Bartram and Rees, 2000; APHA et al., 2005). Species of the genus include E. faecalis, E. faecium, E. durans, E. hirae, E. gallinarum and E. avium.

E. faecalis and E. faecium occur in significant quantities in both human and animal faeces and, along with E. durans, have been reported to be the species most frequently encountered in polluted aquatic environments (Bartram and Rees, 2000). E. gallinarum and E. avium occur at high concentrations in animal faeces, but are not exclusively associated with animal sources.

Enterococci closely satisfy many characteristics of a suitable indicator of faecal contamination in recreational waters. Many species within the group enterococci are found in high numbers in human and animal faeces. They are not commonly found in unpolluted waters and are generally regarded to be incapable of growth in recreational waters (Ashbolt et al., 2001). Compared with other indicator organisms (e.g., E. coli, thermotolerant coliforms), enterococci have demonstrated greater resistance to certain environmental stresses in recreational waters, such as conditions of sunlight and salinity. Enterococci have also demonstrated greater resistance to wastewater treatment practices, including chlorination. A strong correlation has also been demonstrated between the concentration of enterococci in marine waters and the risk of gastrointestinal illness among swimmers (Cabelli, 1983; Kay et al., 1994).

In the past, a ratio of thermotolerant coliforms to faecal streptococci concentrations was used in attempts to indicate the origin of bacterial contamination (Geldreich, 1976; Clausen et al., 1977). A thermotolerant coliform/faecal streptococcus ratio of 4 or higher was said to indicate a human source of contamination, whereas a lower ratio would represent an animal source. However, because of the noted differences in the survival times between these two groups in the environment and the variability of the different methods used for their enumeration, the use of the thermotolerant coliform/faecal streptococcus ratio is now considered inaccurate (Ashbolt et al., 2001; APHA et al., 2005). As a result, the use of this ratio is not recommended. Further information on faecal pollution source tracking can be found in Section 10.0 (Faecal pollution source tracking).

Occurrence in the aquatic environment

Enterococci can be routinely isolated from marine and fresh recreational waters known to be impacted by human and animal faecal pollution sources. These organisms are present in high concentrations in human and animal faeces, with concentrations reported on the order of 10⁶-10⁷/g (Sinton, 1993; Edberg et al., 2000). Overall, it is thought that enterococci are present at concentrations approximately 1- to 3-fold lower than those of E. coli in faeces and municipal wastes (Sinton, 1993; Edberg et al., 2000). Human faecal flora studies reported by Leclerc et al. (2001) demonstrated that Enterococcus species could be detected in the faeces of 100% of the subjects tested.

Several publications have reported that prolonged survival of enterococci is possible in marine and freshwater sediments (Davies et al., 1995; Desmarais et al., 2002; Ferguson et al., 2005). These are thought to provide more favourable conditions of temperature and nutrients than the adjacent recreational waters. Others have reported on the ability of enterococci to survive in organic-rich environments not known to be associated with faecal contamination, such as on mats of the green algae species Cladophora (Whitman et al., 2003).

In Canada, there have been few published investigations on the distribution of enterococci in the marine environment. Gibson and Smith (1988) conducted a study to investigate the distribution of enterococci at 26 marine beaches in the Vancouver region. The findings of this study demonstrated that 1.6% of the results would have exceeded the enterococci geometric mean concentration guideline value of 35/100 mL. In 1988, the New Brunswick Department of Health and Community Services (1989) monitored eight marine beaches along the Northumberland Strait in New Brunswick. The overall enterococci levels were low, showing a geometric mean concentration of 3.5/100 mL. The results of the study indicated that enterococci were absent in 60% of the samples.

Association with pathogens

Enterococci are considered a good indicator for enteric bacterial pathogens. In a survey of surface waters collected from various watersheds in southern California, enterococci were shown to demonstrate good predictive ability with PCR detection of STEC (Yanko et al., 2004). It was reported that above an enterococci concentration of 100 most probable number (MPN)/100 mL, the probability of detection of STEC was approximately 60-70%.

Enterococci are somewhat less effective as an indicator of the presence of enteric pathogenic viruses and protozoa. A number of researchers have reported on the lack of a relationship between enterococci concentrations and the presence of human viruses in surface waters (Griffin et al., 1999; Schvoerer et al., 2000, 2001; Jiang et al., 2001, Jiang and Chu, 2004)

Enterococci are considered the best available indicator of water quality for marine recreational waters (Pruss, 1998; WHO, 1999; Wade et al., 2003). Detection indicates faecal contamination of water and thus the possible presence of faecal pathogenic bacteria, viruses and protozoa. Human enteric viral and protozoan pathogens of faecal origin can survive for prolonged periods in marine waters. Although the presence of high enterococci counts may indicate the possible presence of viral and protozoan pathogens, the opposite--that the absence of enterococci indicates that these pathogens are also absent--cannot be assured.

The combination of routine monitoring for enterococci alongside actions, procedures and tools to collectively reduce the risk of swimmer exposure to faecal contamination in the recreational water environment represents the most effective approach to protecting the health of recreational water users.

Guidelines used by other countries/organizations

The guideline formats and values established by other government and multinational organizations worldwide for faecal indicator organisms in marine waters were reviewed in developing the revised edition of this document.

Related epidemiological studies

The U.S. EPA's original epidemiological studies in marine recreational waters (Cabelli, 1983) measured the concentrations of faecal indicator organisms - total coliforms, thermotolerant (faecal) coliforms, E. coli, enterococci - in swimming waters and compared them with the rates of swimming-associated illness reported on the same days on which the samples were collected. Statistically significant rates of gastrointestinal illness were observed among swimmers in waters considered to be more faecally contaminated. For symptoms unrelated to gastrointestinal illness, no statistically significant differences were observed. For the data analysis, the mean seasonal faecal indicator concentration per 100 mL was plotted against the seasonal swimming associated rate of gastrointestinal symptoms per 1000 persons. Correlation and regression analysis were then used to determine the correlation coefficients and the slope of the linear regression equation for each indicator. The best correlation coefficient (r) was obtained with enterococci (r = 0.75). The following regression equation was produced for the enterococci data :

The equation used to calculate the rate of gastrointestinal illness from enterococci concentrations

Several marine water epidemiological studies have been conducted since the Guidelines for Canadian Recreational Water Quality were last developed (Cheung et al., 1990; Alexander et al., 1992; von Schirnding et al., 1992; Corbett et al., 1993; Harrington et al., 1993; Kay et al., 1994; Kueh et al., 1995; Marino et al., 1995, Fleisher et al., 1996; van Dijk et al., 1996; McBride et al., 1998; Haile et al., 1999; Prieto et al., 2001). All of the studies have confirmed the existence of a relationship between exposure to marine recreational waters of varying quality and symptoms of water-related illness among swimmers. The most significant findings came from the results of the randomized controlled program of epidemiological studies conducted at coastal beaches in the United Kingdom (Kay et al., 1994; Fleisher et al., 1996). These studies were designed to address some of the perceived shortcomings of the traditional beach survey design used in many of the earlier studies. The key features of the randomized controlled design were efforts to ensure a more random distribution of subjects in the swimming and non-swimming groups and tighter monitoring of the water quality experienced by the individual swimmers. Of the faecal indicators monitored, only faecal streptococci levels measured at chest depth showed a significant relationship with the incidence of both gastrointestinal illness and respiratory illness among swimmers. The authors further reported the existence of possible thresholds for an increased risk of gastroenteritis at a concentration of 32 faecal streptococci/100 mL and an increased risk of respiratory illness at a concentration of 60 faecal streptococci/100 mL. In other studies, McBride et al. (1998) reported an increasing risk of respiratory illness with increasing enterococci levels among swimmers at New Zealand beaches. Cheung et al. (1990) observed a moderate correlation (r = 0.63) between enterococci levels and rates of highly credible gastrointestinal illness (HCGI) and skin symptoms combined at coastal beaches in Hong Kong, although a stronger correlation was observed with E. coli (r = 0.73).

A few epidemiological studies have been conducted that have investigated the health effects of recreational activities other than swimming, such as surfing (Gammie and Wyn-Jones, 1997; Dwight et al., 2004). The data linking water quality and illness through these activities have been less strong. Still, conclusions that have been reported are that gastrointestinal illness similarly constitutes the most frequently reported health outcome during these types of activities and that factors related to the risk of illness include the quality of the water and the frequencies of immersion and water ingestion.

Several reviews of the epidemiological findings have also been published. WHO (Pruss, 1998) published a comprehensive review of all of the recreational water epidemiological studies conducted over the period from 1953 to 1996. From the review, it was concluded that gastrointestinal symptoms were the most frequently reported outcome for which water quality dose-response relationships were reported, and that the indicator organisms that best correlated with the health outcomes were enterococci for marine waters and E. coli and enterococci for fresh waters. The U.S. EPA has also published two reviews of the existing epidemiological literature for recreational waters. The first, published in its Implementation Guidance for Ambient Water Quality Criteria for Bacteria (U.S. EPA, 2002), was a minireview of the epidemiological studies conducted since the previous guidance had been issued, in 1986. The U.S. EPA concluded that the epidemiological methods used to derive its 1986 Water Quality Criteria remained scientifically valid and that no new scientific principles had been established that would require a revision of the current guidelines. More recently, Wade et al. (2003) conducted a meta-analysis of all of the existing epidemiological data published since 1950 linking microbiological indicators of recreational water quality to gastrointestinal illness in swimmers. The authors concluded that in the marine water studies, enterococci and, to a lesser extent, E. coli were the most reliable predictors of gastrointestinal illness. Moreover, the authors observed that the reported risks of gastrointestinal illness at enterococci concentrations below the current U.S. EPA standards were not statistically significant, whereas values above the standards were elevated and statistically significant.

The U.S. EPA and CDC are currently conducting epidemiological studies at freshwater and marine beaches under the NEEAR Water Study. The studies are intended to support the development of new recreational water quality guidelines (U.S. EPA, 2002; Dufour et al., 2003), as well as to investigate new water quality indicators and rapid methods for water quality monitoring. Data collection for these studies was completed in 2010.

Summary

Based on all of the existing evidence, the enterococci group remains the most suitable indicator of faecal contamination in marine recreational waters. In summary:

1. The guideline values have been developed based on the analysis of epidemiological evidence relating enterococci concentrations in marine recreational waters to the incidence of swimming-associated gastrointestinal illness observed among swimmers. The values represent risk management decisions based on the assessment of possible health risks for the recreational water user and the recognition of the tremendous benefits that recreational water activities provide in terms of health and enjoyment.
2. Enterococci most closely fit the requirements of an ideal indicator of faecal contamination for marine recreational waters. Detection suggests faecal contamination of water and thus the possible presence of faecal pathogenic bacteria, viruses and protozoa.
3. There are limitations associated with the use of indicators in assessing the quality of recreational waters. The occurrence of faecal pathogens in recreational waters is dependent on many factors and can be variable and sporadic. The absence of enterococci should not be interpreted to mean that enteric pathogenic microorganisms are also absent.
4. Combining routine enterococci monitoring alongside actions, procedures and tools to collectively reduce the risk of swimmer exposure to faecal contamination in the recreational water environment represents the most effective approach to protecting the health of recreational water users.

4.2 Advice regarding water intended for secondary contact recreational activities

The Guidelines for Canadian Recreational Water Quality are intended to be protective for those activities that involve intentional or incidental immersion in natural waters. Due to increased interest from jurisdictions in distinguishing between primary contact activities and secondary contact activities, this current edition of the Guidelines takes an initial step at providing advice for secondary contact activities separately from that of primary contact with respect to faecal indicator concentration.

Recreational activities that have been traditionally considered as secondary contact activities (e.g. canoeing, fishing) involve exposures much different from those associated with primary contact uses. Ingestion of water and, subsequently, the risk of gastrointestinal illness are presumed to be lower during secondary contact recreation. Still, it is expected that there is some degree of risk of acquiring illness through these activities. Inadvertent immersion can result in whole body contact, and splashing can lead to a variety of water exposure scenarios. Illnesses affecting the skin and perhaps the mucous membranes of the eyes and ears may be of relatively greater importance for secondary contact uses (U.S. EPA, 2002). Inhalation may also be an important route of exposure during secondary contact activities in areas where sprays or aerosols are generated.

Limited research has been conducted on the potential risks of acquiring illness during secondary contact activities in recreational waters. In one study which investigated the relationship between water quality and illness acquired during canoeing or rowing, Fewtrell et al. (1994) noted no significant differences between the exposed group and the unexposed group. The bulk of the epidemiological research on recreational water uses and the risk of acquiring illness have been generated for primary contact activities. As a result, insufficient data are available to derive precise health-based faecal indicator limit values intended to protect users engaged in secondary contact recreational activities from exposure to faecal contamination. However, it is recognized that because a lower degree of water exposure can be expected at most times during the majority of secondary contact recreational activities, there may be some water areas where a secondary contact use designation with separate water quality values is desired and considered reasonable and acceptable to local and regional authorities.

When contemplating the establishment of separate faecal indicator values for water areas used entirely for secondary contact recreational uses, a clear understanding of the types of activities that would be considered to fit under this description is required. WHO, in its Guidelines for Safe Recreational Water Environments: Volume 1--Coastal and Fresh Waters (WHO, 2003a), has proposed a scheme for the classification of recreational water activities according to their degree of water exposure. The following descriptions (adapted from WHO, 2003a), may be used as an initial guide when determining whether a specific recreational activity would be considered as either primary or secondary contact:

• Primary contact: Recreational activity in which the whole body or the face and trunk are frequently immersed or the face is frequently wetted by spray, and where it is likely that some water will be swallowed. Inadvertent immersion, through being swept into the water by a wave or slipping, would also result in whole body contact. Examples include swimming, surfing, waterskiing, whitewater canoeing/rafting/kayaking, windsurfing or subsurface diving.
• Secondary contact: Recreational activity in which only the limbs are regularly wetted and in which greater contact (including swallowing water) is unusual. Examples include rowing, sailing, canoe touring, or fishing.

Even if these classification criteria are used, it remains a significant challenge to discern which activities constitute primary contact and which constitute secondary contact. The classification of certain recreational water activities will be clear, whereas that of others may be less obvious and more open to interpretation. Activities considered as potential candidates under a secondary contact use designation should be evaluated on a case-by-case basis.

Other factors to consider before assigning a secondary contact use designation to a recreational water area include:

• The water area should first be subject to an assessment of existing uses, water quality and the potential for improvement as well as any other relevant factors, such as health or environmental considerations.
• The secondary contact designation should not be applied where an assessment has shown primary contact recreation to be a significant use.
• Where the water area has a shared use (e.g. swimming and canoeing), it is the primary contact values that should apply.
• When an area is posted as suitable only for secondary contact recreational uses, communication material should clearly convey that accidental immersion (through falls, canoe spills, etc.) can lead to whole body exposure; under these circumstance, water ingestion may result in illness.
• Users should be reminded to take the precautions necessary to ensure that these types of exposures are avoided as much as possible; the skill of the person performing the activity may strongly influence the degree of water exposure.
• Further guidance on the posting of information at recreational water areas can be found in Part I (Management of Recreational Waters).

Responsible authorities have a duty to take precautions, protect the health and safety of all recreational water users and maintain the best water quality possible. The existence of the secondary contact values should not be used as a mechanism for downgrading the status of an area in response to poor water quality issues. This is particularly important where an assessment has shown that the primary contact guideline values could be achieved.

Based on an assessment of the available information, where a water area is intended to be used specifically for secondary contact recreation (i.e. where primary contact is not an existing use), the application of a factor of 5 to the geometric mean faecal indicator concentration used to protect primary contact recreation users may be used as an approach to establish faecal indicator limits. The corresponding values for E. coli and enterococci concentrations would therefore be as follows:

The operation used to calculate the secondary-contact guideline values

These values represent a risk management decision based on the assessment of the expected exposure scenarios and potential health risks for the recreational water user. They are intended to allow specified water areas to have a secondary contact use designation where this has been considered appropriate by the responsible local or regional authorities, while still providing some level of protection for secondary contact recreational users until epidemiologically based guideline values can be derived. In considering both the potential health risks and the benefits of recreational water use, it was concluded that this is a tolerable and reasonable approach to protect users engaged in a voluntary activity. These values are also consistent with advice provided by other jurisdictions (Saskatchewan Environment, 1997; Alberta Environment, 1999; U.S. EPA, 2002; British Columbia Ministry of Health Services, 2007; MDDEP, 2007). The values will be periodically reviewed or adjusted, however, as new or more significant data become available.

Insufficient information is availableto develop separate values with respect to water areas used solely for secondary-contact for other parameters in the Guidelines for Canadian Recreational Water Quality. Area operators, service providers and responsible authorities should remain aware that these parameters may also affect water areas intended only for secondary contact uses. Where separate guidance for secondary contact does not exist, it is advised that the values and associated guidance provided in the Guidelines apply to all recreational waters, regardless of the types of activities practised.

4.3 Other organisms as potential indicators

Many different waterborne pathogens can be encountered in Canadian recreational waters. As discussed in the previous section, recreational water quality is most frequently determined by the presence of faecal indicator bacteria which, by extension, suggests the possible existence of faecally transmitted waterborne pathogens. Currently, E. coli (fresh waters) and enterococci (marine waters) remain the best available indicators of recreational water quality, as they, above all other organisms, have been shown to be the most successful in meeting the desired indicator criteria.

Nevertheless, the current indicators do not unequivocally meet all of the requirements of an ideal indicator, and the limitations of these two organisms as pathogen indicators are well known. It is understood that no single organism would be able to fill all of the roles of what might be considered a perfect indicator of recreational water quality--one that models all of the known pathogens, provides information on the degree and source of faecal contamination and communicates the potential risk of illness for recreational water users. It has been proposed that this task would require multiple indicators, each with unique characteristics that would enable them to satisfy specific roles (Ashbolt et al., 2001).

The term indicator can be further specified to reflect these different functions. Indicators may be considered as faecal indicators (indicative of the presence of faecal contamination, but not necessarily of specific pathogens) or pathogen indicators (indicative of the presence and behaviour of specific pathogens). Moreover, faecal indicators may be further categorized into what can be considered primary indicators--those that provide information on the magnitude or extent of faecal contamination-- and secondary indicators--those that provide information on the source of faecal contamination.

The objective of this section is to provide a summary of what is known regarding other microorganisms that have been widely discussed among recognized experts, academics and policymakers as potential indicators for recreational water. The organisms covered are Bacteroides spp., Clostridium perfringens, F⁺ RNA coliphages and bacteriophages infecting Bacteroides fragilis. A summary of the characteristics of the recommended indicators and the other potential indicator organisms discussed is presented in Table 4.

Potential indicators

Bacteroides spp.

Bacteroides spp. are Gram-negative, rod-shaped, obligate anaerobic bacteria. The genus is considered to represent the most abundant bacterial genus in human faeces (Fiksdal et al., 1985). The four dominant species--B. fragilis, B. vulgatus, B. distasonis and B. thetaiotaomicron--can reach concentrations on the order of 10¹⁰ cells/g faeces (Kator and Rhodes, 1994), outnumbering E. coli concentrations by as much as 100- to 1000-fold (Slanetz and Bartley, 1957; Holdeman et al., 1976; Fiksdal et al., 1985). Bacteroides species can occur in much lower densities (10⁵- fold to 10¹⁰-fold lower) in animals (Allsop and Stickler, 1985; Kator and Rhodes, 1994), although higher densities have been observed in some species, such as domestic pets and gulls (10⁷-10⁸ cfu/g) (Allsop and Stickler, 1985).

Because of their high numbers in human faeces, Bacteroides species have long been considered a candidate indicator of faecal pollution; however, the difficulties associated with culturing anaerobic bacteria discouraged their use in investigations (Kreader, 1995; Bernhard and Field, 2000a). Recent advances in molecular biology have overcome this problem. Researchers have developed PCR assays for the detection of both generic and species-specific (human, bovine) Bacteroides genetic markers in faeces. In this case, the presence of the genetic markers is considered to infer the presence of Bacteroides cells.

Bacteroides PCR methods have been successfully used for the detection of faecal pollution in contaminated water samples (Kreader, 1998; Bernhard and Field, 2000b; Field et al., 2003). As well, quantitative PCR (QPCR) methods have been developed that have enabled the near real-time enumeration of Bacteroides spp. in recreational waters (Fung, 2004; Seurinck et al., 2005; Wade et al., 2006). The U.S. EPA included QPCR monitoring for Bacteroides as part of its NEEAR Waters Study (Wade et al., 2006).

Detection of Bacteroides genetic markers in recreational waters is a relatively new area of research. Few studies have been conducted to date that provide an analysis of Bacteroides markers relative to faecal indicator organisms, faecal pathogens or rates of illness among swimmers. Wade et al. (2006) reported a positive but weak association between Bacteroides and gastrointestinal illness among swimmers at one of two freshwater beaches investigated during the NEEAR Waters Study. A problem with the sensitivity of the QPCR method was cited by the authors (Wade et al., 2006).

Noted strengths of Bacteroides as a possible primary indicator of faecal contamination include their high occurrence in human faeces and sewage, the inability of the bacterium to grow in the environment and the long environmental persistence of the DNA markers. Limitations include the lower concentrations observed in non-human faecal sources, current data gaps regarding its capabilities as an indicator (primary indicator, pathogen indicator and indicator of swimming-associated illness) and challenges relating to the analytical methods (expensive, technically demanding, issues of sensitivity).

Information generated to date suggests that Bacteroides markers may have a stronger role as a secondary indicator of faecal contamination, providing information on the potential sources of faecal material.

Clostridium perfringens

C. perfringens is a Gram-positive, rod-shaped, spore-forming anaerobic bacterium that is consistently found in both human and non-human faeces (Bisson and Cabelli, 1980). Clostridium species have the ability to enter into a protective spore form that is able to resist environmental stresses and that can allow the organism to persist in the environment for long periods of time.

C. perfringens has long been considered a valuable indicator of the sanitary quality of water, dating back to the late 19th century (Ashbolt et al., 2001). Much of the interest in the use of C. perfringens as an indicator of recreational water quality has stemmed from research conducted in the state of Hawaii. Researchers there had observed that many stream and soil samples not thought to be affected by a known source of faecal pollution contained faecal coliforms and E. coli in excess of the current water quality standards (Fujioka and Shizumura, 1985). It was also observed that C. perfringens concentrations in streams receiving wastewater discharges were consistently higher than in streams that were not affected (Fujioka and Shizumura, 1985). The researchers subsequently asserted that C. perfringens was a more reliable indicator of faecal contamination in Hawaiian waters. It has since been proposed that this situation may be encountered in other tropical locations in the United States (U.S. EPA, 2001b). At present, Hawaii is the only known jurisdiction that includes monitoring for C. perfringens as a recreational water quality indicator (Anon., 1996).

The concentration of C. perfringens in human and animal faeces is considerably less than that of E. coli or enterococci (Wright, 1982). Published data suggest that C. perfringens may be detected in only a small to moderate percentage of human faecal samples (13-35%), with an average faecal concentration of approximately 10³ cells/g (Ashbolt et al., 2001). Higher concentrations of C. perfringens have been reported in sewage (Fujioka and Shizumura, 1985). C. perfringens has been detected in the faeces of a wide range of animals, including birds, mammals, reptiles and amphibians (Conboy and Goss, 2003). Significant numbers of the organism have been encountered in the faeces of a few animal species, including dogs (10⁸ cells/g), cats (10⁷ cells/g) and sheep (10⁵ cells/g) (Ashbolt et al., 2001). C. perfringens is not exclusively associated with faecal wastes and is a common inhabitant of soils (Toranzos, 1991).

Water quality investigations conducted in coastal waters in Florida have indicated that concentrations of C. perfringens do not correlate well with levels of faecal indicator bacteria (Griffin et al., 1999; Lipp et al., 2001) or with the presence of enteric viruses (Griffin et al., 1999). Furthermore, the levels of C. perfringens were generally lower than those of enterococci or the faecal coliform group in the water column, but were substantially greater than the levels of either indicator in the underlying sediment (Lipp et al., 2001). In a study of indicator and pathogen presence in selected lakes and rivers in southwestern Finland, Hörman et al. (2004) reported a positive correlation between the presence of C. perfringens and the detection of one or more of the pathogens being tested for (Cryptosporidium, Giardia, Campylobacter, noroviruses). However, the absence of C. perfringens demonstrated only a weak predictive value for a negative pathogen sample.

Several epidemiological studies have included C. perfringens as an indicator when investigating the relationships between water quality and illness in swimmers (Cabelli, 1983; Harrington et al., 1993; Kueh et al., 1995; Lee et al., 1997; Wiedenmann et al., 2006). Cabelli (1983) observed a weak correlation between C. perfringens densities and acute gastrointestinal illness in swimmers during the U.S. EPA's original epidemiological studies at marine beaches in the 1970s. Kueh et al. (1995) noted a positive, but not strong, correlation with the incidence of gastroenteritis among swimmers and C. perfringens in two marine beaches in Hong Kong. Wiedenmann et al. (2006) did report a relationship between C. perfringens and the incidence of gastroenteritis among swimmers during a randomized controlled epidemiological study at German freshwater beaches. A NOAEL of 13 C. perfringens/100 mL was reported for several definitions of gastrointestinal illness.

Strengths of C. perfringens include its inability to grow in the environment and its capability for surviving longer than faecal waterborne pathogens. Advances in culture methods (Adcock and Saint, 2001) have helped to improve the ease with which C. perfringens can be detected--previously an impediment surrounding the use of this organism as a water quality indicator.

Limitations include not being faecal specific, having lower faecal numbers relative to other indicator bacteria; detection being strongly dependent on the source of contamination; presence not necessarily indicative of recent contamination (owing to the long environmental persistence of the spores); and a lack of epidemiological evidence linking C. perfringens concentrations to the potential for acquiring swimming-associated illness.

C. perfringens may be better suited as an indicator of the effectiveness of drinking water treatment processes (Bisson and Cabelli, 1980; Payment and Franco, 1993) or as an indicator of intermittent or cumulative sewage inputs (Sorensen et al., 1989; Hill et al., 1993; Lisle et al., 2004). At present, C. perfringens appears to more closely satisfy the role of a pathogen indicator or perhaps a secondary indicator of faecal contamination.

F⁺ RNA coliphages

Coliphages are bacteriophages (viruses infecting only bacteria) that specifically infect E. coli cells. The reasons cited for investigating coliphages as potential indicators of faecal contamination are that coliphages are thought to more closely resemble enteric viruses in terms of their physical characteristics, environmental persistence and resistance to disinfection, compared with the traditional bacterial indicators of faecal contamination. They are also less costly and easier to enumerate than human viruses. Also, because they theoretically infect only E. coli cells, it is thought that their detection should be sufficiently indicative of the presence of faecal contamination.

There are two main types of coliphages: somatic coliphages and male-specific (F⁺) coliphages. Somatic coliphages infect E. coli cells by attaching to the lipopolysaccharide component of the cells' outer membranes. They have been investigated as potential indicators of swimming water quality (Contreras-Coll et al., 2002; Vantarakis et al., 2005; Wiedenmann et al., 2006); however, they are thought to represent a less specific group than the F⁺ coliphages, and knowledge of their sources and behaviour is currently lacking. By comparison, F⁺ coliphages have been more extensively studied (Duran et al., 2002).

F⁺ coliphages demonstrate a greater specificity than somatic coliphages, infecting E. coli cells possessing F-pili--tube-like structures coded for by an F-plasmid and that allow connections to form between cells for the transfer of genetic material (Singleton and Sainsbury, 1997; Scott et al., 2002). It is these F-pili that serve as the site of phage attachment.

F⁺ coliphages include F⁺ RNA phages and F⁺ DNA phages. F⁺ RNA phages more closely resemble the human viruses of waterborne significance and have thus been preferentially explored (Sobsey, 2002). Using immunological or genetic methods, the group can be further categorized into four distinct serogroups or genogroups, and subsequent source tracking investigations have identified that the presence of a particular subgroup has some merit in distinguishing the source of faecal contamination (Havelaar et al., 1990; Brion et al., 2002; Schaper et al., 2002b; Cole et al., 2003). In general, Groups II and III have been shown to be highly associated with human faecal contamination (e.g., domestic or municipal sewage), Group IV has been shown to be predominantly linked to animal faecal contamination and animal waste materials, Group I has been isolated from both human and animal faecal material and wastes (Scott et al., 2002; Sobsey, 2002).

F⁺ RNA phages are not always present in human faeces and, when detected, are often present in low numbers (Havelaar and Pot-Hogeboom, 1988; Havelaar et al., 1990; Luther and Fujioka, 2004). Researchers have similarly reported low isolation frequencies among septage samples or waters known to be affected by septic system wastes (Calci et al., 1998; Griffin et al., 1999). A low isolation frequency among animal faecal samples has also been reported (Calci et al., 1998; Luther and Fujioka, 2004). Significantly higher numbers of F⁺ RNA coliphages have been detected in sewage and wastewater (Contreras-Coll et al., 2002; Lucena et al., 2003).

Sinton et al. (1999) reported that the degree of survival for various indicator organisms in sewage-polluted seawater during sunlight inactivation experiments (simulated summer conditions) was (from greatest to least): somatic coliphages > F⁺ RNA phages > enterococci > E. coli. The F⁺ RNA coliphage groups have been shown to vary markedly in their ability to persist in the environment (Brion et al., 2002; Schaper et al., 2002a; Sobsey, 2002; Long and Sobsey, 2004). In general, it has been observed that Group I is capable of the longest environmental persistence, followed by Groups II and III, with Group IV demonstrating the shortest period of survival (Brion et al., 2002; Schaper et al., 2002a; Long and Sobsey, 2004).

Evidence as to whether F⁺ RNA coliphages are a reliable indicator of faecal pollution in natural waters has been conflicting. Havelaar et al. (1993) and Ballester et al. (2005) reported that F⁺ RNA coliphage concentrations were more strongly correlated with concentrations of infectious enteroviruses and enteric viruses than either faecal coliforms or enterococci in environmental waters. The authors did report, however, that in a few instances, viruses were isolated in the absence of the coliphages, and that the reverse was also true (Havelaar et al., 1993). Griffin et al. (1999) demonstrated a lack of predictive ability when using coliphages (somatic and F⁺ RNA) as indicators of the presence of enteric viruses in canal waters in the Florida Keys. Jiang and Chu (2004) reported no apparent relationship between the detection of adenovirus, enterovirus and hepatitis A virus (HAV) genomes and F-specific coliphage concentrations in a study of human viral contamination in river and coastal waters in southern California.

Grabow (2001) suggested that a direct correlation between the number of coliphages and enteric viruses in water environments cannot be expected, as coliphages are excreted at all times by a certain percentage of the human population, whereas enteric viruses are largely excreted during infection, which can be intermittent and seasonal.

There has been some information collected regarding the relationship between coliphage concentrations and the rate of swimming-associated illness. Lee et al. (1997) reported a significant association between the concentration of F⁺ RNA coliphages and the reporting of gastrointestinal symptoms among canoeists and rafters at an artificial freshwater canoe course in the United Kingdom. Other researchers have included coliphages among the panel of prospective indicators monitored during epidemiological investigations (von Schirnding et al., 1992; Marino et al., 1995; McBride et al., 1998; van Asperen et al., 1998); however, no significant correlations were identified.

Strengths of F⁺ RNA phages as a possible faecal indicator include the noted similarities to human enteric viruses, strong evidence of being exclusively associated with human and animal faecal material, an apparent inability to replicate in the environment and potential applications in faecal source identification. Limitations include presence being dependent on the source of contamination, different survival rates of the individual phage groups and a lack of a demonstrated relationship with either enteric virus presence or rates of swimming-associated illness.

F⁺ RNA coliphages appear to be more useful as an indicator of sewage, rather than of faecal contamination in general. At present, they appear to be better positioned as potential pathogen indicators or secondary indicators, as opposed to primary indicators of faecal contamination (Chapron et al., 2000).

Bacteriophages of Bacteroides fragilis

Bacteriophages of B. fragilis have been investigated as a possible indicator of faecal contamination on the theory that the organism might combine some of the desirable properties reported for both the coliphage group and Bacteroides spp.--the potential to be present in high numbers in faecal material, while demonstrating survival traits more representative of enteric viruses.

Although concentrations of B. fragilis in human faeces are high, B. fragilis phages have been shown to be isolated somewhat infrequently in faeces, and in lower numbers (Tartera and Jofre, 1987; Grabow et al., 1995). Published accounts have placed the percentage of human faecal samples from which B. fragilis phages have been isolated in the range from 10% to 28% (Tartera and Jofre, 1987; Grabow et al., 1995; Puig et al., 1999; Gantzer et al., 2002). Phage isolation from human and animal faeces has been shown to be largely dependent upon the B. fragilis host strain used for recovery (Tartera and Jofre, 1987; Puig et al., 1999). Molecular methods for the detection of B. fragilis bacteriophages are under investigation, and these are expected to eliminate many of the difficulties associated with the current host recovery methods (Puig et al., 2000).

B. fragilis phages have been shown to be routinely isolated from sewage; however, the concentrations encountered (< 10-10⁵ phages/100 mL) are frequently less than those reported for somatic and F⁺ RNA coliphages (Puig et al., 1999; Contreras-Coll et al., 2002; Lucena et al., 2003). Tartera and Jofre (1987) reported that B. fragilis phages could be detected in all water and sediment samples from highly polluted rivers (10¹-10⁵ plaque-forming units [pfu]/100 mL), but not in samples collected from areas not known to receive sewage pollution. In a more recent investigation of the occurrence and levels of phages of B. fragilis in swimming waters throughout Europe, Contreras-Coll et al. (2002) documented a median phage concentration well below 10 pfu/100 mL, with 95% of the samples below 10² pfu/100 mL. Lucena et al. (2003) reported similar numbers during a survey of water samples collected from selected rivers in Europe and South America.

No correlation has been shown between the concentration of faecal indicator bacteria and levels of B. fragilis phages in recreational waters (Tartera et al., 1989; Contreras-Coll et al., 2002; Lucena et al., 2003). Certain studies have suggested that B. fragilis phages are reliable indicators of viral contamination in treated wastewaters (Gantzer et al., 1998) and shellfish (Hernroth et al., 2002; Formiga-Cruz et al., 2003). However, there has been limited information published to date in which the occurrence and concentrations of enteric viruses and phages of B. fragilis in recreational waters have been directly compared. In a study of river water samples where domestic sewage was cited as its primary contamination source, Tartera et al. (1989) reported that B. fragilis phages could be consistently isolated from samples in which enteroviruses were detected.

Of the organisms suggested as possible alternative indicators of faecal pollution, B. fragilis phages have been perhaps the least well investigated. Potentially useful properties include the absence of significant non-faecal sources, the inability to replicate in the environment and structural similarities to the enteric viruses. Limitations include low faecal concentrations, variable isolation depending on the contamination source and difficulties associated with recovery of the organisms.

Currently, it is speculated that, similar to the F⁺ coliphages, B. fragilis phages may be more suitable as indicators of sewage contamination--and thus as possible secondary indicators of faecal contamination.

Summary

1. The faecal contamination of recreational waters and the associated risk for water users are a complex issue. E. coli and enterococci are considered the best available indicators of faecal contamination in recreational waters; however, no single organism is capable of satisfying all of the roles of an ideal indicator. Multiple indicators may be required for a more complete understanding of this subject.
2. The organisms most widely discussed as other potential recreational water indicators include Bacteroides spp., C. perfringens, F⁺ RNA coliphages and bacteriophages infecting B. fragilis (see Table 4).
3. At present, none of the proposed indicators investigated meet a sufficient number of the requirements necessary to be successful as a routine indicator of recreational water quality. None of these organisms has demonstrated a consistent correlation with the presence of waterborne pathogens in recreational waters, nor is there evidence of a strong epidemiological link between the occurrence of these organisms and the incidence of illness among recreational water users.
4. Nevertheless, these organisms do possess certain unique properties that might enable them to fulfil other roles as recreational water indicators. Currently, these organisms appear to be better suited as possible pathogen indicators or as faecal source indicators. Advances in detection and enumeration methods may help improve our understanding of these organisms and the roles they may play in recreational water monitoring programs in the future.

Footnotes

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