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Workshop Report: Developing Pollution Source Tracking for Recreational and Shellfish Waters a
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K. R. Pond , R. Rangdale , W. G. Meijer , J. Brandao , L. Falcāo , A. Rince , B. f
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Masterson , J. Greaves , A. Gawler , E. McDonnell , A. A. Cronin & S. Pedley
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Robens Centre for Public and Environmental Health, University of Surrey, Guildford, UK
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CEFAS, Weymouth Laboratory, Barrack Road, The Nothe, Weymouth, UK
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Department of Industrial Microbiology, University College Dublin, Belfield, Dublin, Ireland
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Instituto Nacional de Saude, Lisbon, Portugal
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USC INRA Microbiologie de l'Environnement, IRBA, Université de Caen, Caen cedex, France
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Biochemistry Department, The Conway Institute, University College, Belfield, Dublin, Ireland g
Strategic Environmental Planning, North West Region, Environment Agency, UK
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National Laboratory Service of the Environment Agency at NLS, Starcross Laboratory, Staplake Mount, Starcross Nr. Exeter, UK i
Water Quality Division, Department for Environment, Food and Rural Affairs, Ashdown House, London, UK Available online: 11 Aug 2010
To cite this article: K. R. Pond, R. Rangdale, W. G. Meijer, J. Brandao, L. Falcāo, A. Rince, B. Masterson, J. Greaves, A. Gawler, E. McDonnell, A. A. Cronin & S. Pedley (2004): Workshop Report: Developing Pollution Source Tracking for Recreational and Shellfish Waters, Environmental Forensics, 5:4, 237-247 To link to this article: http://dx.doi.org/10.1080/15275920490887968
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Environmental Forensics, 5:237–247, 2004 C AEHS Copyright ISSN: 1527–5922 print / 1527–5930 online DOI: 10.1080/15275920490887968
Workshop Report: Developing Pollution Source Tracking for Recreational and Shellfish Waters K. R. Pond,1 R. Rangdale,2 W. G. Meijer,3 J. Brandao,4 L. Falc¯ao,4 A. Rince,5 B. Masterson,6 J. Greaves,7 A. Gawler,8 E. McDonnell,9 A. A. Cronin,1 and S. Pedley1 Downloaded by [b-on: Biblioteca do conhecimento online IPSantarem] at 07:39 04 April 2012
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Robens Centre for Public and Environmental Health, University of Surrey, Guildford, UK CEFAS, Weymouth Laboratory, Barrack Road, The Nothe, Weymouth, UK 3 Department of Industrial Microbiology, University College Dublin, Belfield, Dublin, Ireland 4 Instituto Nacional de Saude, Lisbon, Portugal 5 USC INRA Microbiologie de l’Environnement, IRBA, Universit´e de Caen, Caen cedex, France 6 Biochemistry Department, The Conway Institute, University College, Belfield, Dublin, Ireland 7 Strategic Environmental Planning, North West Region, Environment Agency, UK 8 National Laboratory Service of the Environment Agency at NLS, Starcross Laboratory, Staplake Mount, Starcross Nr. Exeter, UK 9 Water Quality Division, Department for Environment, Food and Rural Affairs, Ashdown House, London, UK 2
Traditional methods, such as the detection of total and fecal coliforms, and more recently enterococci, that are used to detect fecal pollution levels do not identify the source of the pollution. Despite a significant volume of research in this field, at present there is no common methodology to identify sources of fecal contamination affecting bathing and shellfish waters in Europe. Keywords: recreational water, source tracking, fecal pollution
The Environment Agency of England and Wales obtained European Community Initiative INTERREG IIIB funding for a project called ICReW—Improving Coastal and Recreational Waters. The project consists of seven pilot actions aiming to contribute to the reduction of pollution, to enhance water quality, and to ensure that land-use practices and recreational activities can exist side by side without impacting public health. One of these actions is to identify and develop a common methodology for source-tracking fecal pollution, for regulatory purposes, over a wide geographical area in Europe. In order to do this the Department for Environment, Food and Rural Affairs, UK, sponsored the first international workshop on the subject. Key researchers from around the world were invited to attend to recommend the most appropriate method(s) for development and field trial in Europe. The meeting concluded that for the specific requirements of the ICReW project two methods should be developed and trialled: bacteroides genotyping and F+ RNA coliphage genotyping. This article summarizes the reasons why these methods were chosen as the most appropriate for the circumstances of this particular project. The inherent challenges of establishing a pilot program to test the methods are outlined and recommendations were provided for the trial. Received 17 June 2004; accepted 13 September 2004. Address correspondence to K. R. Pond, Robens Centre for Public and Environmental Health, University of Surrey, Guildford, GU2 7XH, UK. E-mail:
[email protected]
Background European bathing water quality is regulated by the EU Directive 76/160/EEC (CEC, 1976), which requires regulatory authorities to sample identified bathing waters and analyze the samples for the traditional fecal bacteria indicators Escherichia coli, total coliforms, and fecal streptococci in order to assess pollution status and to monitor deterioration and improvement in bathing water quality. The directive sets two levels of standards: the Imperative standards, which must be met at all identified sites, and the Guideline standards, which member states are required to endeavor to meet at all identified sites. The UK has over 550 identified bathing waters, the vast majority of which are located on the coast. Compliance with the directive requirements has improved steadily since its implementation, and in 2003 98.4% of sites in the UK met the Imperative standards and 74% met the Guideline standards. However, the directive is under revision, and the standards are likely to be considerably tightened. Although indicator species are found in both human and animal feces, existing analytical methods do not distinguish between bacteria of human and nonhuman sources. It is likely that bathing water quality will need to be further improved, but these improvements will be difficult to obtain if there is not a better understanding of the sources of pollution, and in particular the balance of impact between point source and diffuse pollution. The ability to identify the source 237
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of indicator organisms forms an important part of water quality management, enabling targeted risk management and remediation to improve water quality and protect public health.
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Introduction Fecal pollution from nonhuman (pets, livestock, or wildlife) and human sources is one of the major factors that contribute to the degradation of water quality (Bernhard and Field, 2000) and that restricts its use. Currently, waters used for bathing and shellfish harvesting have particular requirements regarding quality. There is a proven cause–effect relationship between fecally polluted recreational water and acute febrile respiratory illness and enteric illness in bathers (WHO, 2003). Shellfish contamination from sewage-polluted waters leading to illness and sometimes death in consumers of raw shellfish is also a well-known problem (Ripabelli et al., 1999). Methods to differentiate animal from human sources of fecal coliform contamination will assist water resource managers in developing strategies to protect shellfish harvesting areas and recreational waters and thus reduce the public health risk from these waters. In 2003 the Environment Agency of England and Wales secured EU INTERREG IIIB funding for a project called ICReW—Improving Coastal and Recreational Waters. The project is intended to help five EU member states—the UK, Ireland, France, Portugal, and Spain—improve the quality of such waters and prepare for the implementation of new European Commission bathing waters legislation. The ICReW project consists of seven projects or pilot actions occurring between 2003 and 2006. One of these pilot actions addresses the issue of microbial source tracking of fecally derived pollution from a regulatory perspective. Its objective is “to produce a working tool which can be used to discriminate the sources of pollution contributing to an environmental sample.” The project aspires to bring source tracking into practical use as a catchment management tool in the EU, specifically to help achieve the stringent standards for E. coli and intestinal enterococci that are proposed by the European Commission. However, the method of choice for the project is required to be applicable to all the selected pilot test sites.
The issue is also of global interest. In the late 1980s the contribution of storm water runoff to contamination of recreational waters became an important issue in the United States and the Southern Hemisphere. It has been shown that animal loads significantly contribute to the total maximum daily load, and this has driven the work in the United States and other countries towards verifying sources of contamination as well as identifying quantities of pollution (Leeming et al., 1996; Parveen et al., 1999; Carson et al., 2003; Scott et al., 2002; McLellan et al., 2003). Previous work has identified a number of unresolved issues associated with microbial source identification, which include temporal issues, geographic stability, and which microbe to use for identifying sources (Leung et al., 2004). Although reviews of methods of source-tracking microbial pollution have been undertaken previously (Parveen et al., 1999; Carson et al., 2003; Scott et al., 2002; Meays et al., 2004), Rangdale et al. (2003) under the ICReW project have undertaken a systematic review of the relevant literature specifically to identify microbial source-typing methods with the best potential for development for regulatory use across Europe. An initial review by the ICReW project team was undertaken of 22 microbiological, chemical, phenotypic, and genotypic library-dependent and -independent methods that have been applied in source tracing (Rangdale et al., 2003). From this review 12 techniques were identified as having potential for development for use in the ICReW project (Table 1). These were selected based on their performance against 16 criteria, detailed in Table 2. The other methods reviewed—ribotyping, multiple antibiotic resistance, E. coli serotyping, carbon source profiling, 23S rDNA sequencing, sequence homology comparison, temperature gradient gel electrophoresis, amplification of DNA fragments surrounding rare restriction sites, plasma typing, and amplified fragment length polymorphism—were rejected for the purposes of the ICReW project for a variety of reasons. Ribotyping, for instance, has been used successfully by Parveen et al. (1999), Carson et al. (2003), Scott et al. (2002), and Jenkins et al. (2003) to analyze E. coli strain isolates from human and nonhuman sources. It is currently being applied commercially in Florida. However, ribotyping has been shown to lose its effectiveness when isolates are
Table 1. Methods and techniques identified as having potential for development for use in the ICReW project for source tracking microbial pollution in bathing and shellfish waters Methods/techniques 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.
Rep PCR (repetitive element PCR fingerprinting) RAPD (random amplified polymorphism DNA) RFLP (restriction fragment length polymorphism) Multi locus sequence typing (MLST) Pulsed field gel electrophoresis (PFGE) Detection of adenoviruses Infrared vibrational spectroscopy (IR-VS) Bacteroides genotyping—PCR-based method Sorbitol-fermenting bifidobacteria Enterotoxin markers—PCR-based method Detection of fluorescent whitening agents and fecal sterols F+RNA coliphage typing
Type Genotypic library dependent Genotypic library dependent Genotypic library dependent Genotypic library dependent Genotypic library dependent Genotypic library dependent Library dependent Genotypic library independent Phenotypic/genotypic library independent Genotypic library independent Chemical library independent Genotyping library independent
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Table 2. Performance of twelve methods and techniques of sourcing microbial pollution against selected criteria Performance Criterion Is the technique reliant upon the use of a comparative library? Has the method been used extensively?
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Is the technique applicable to a range of environmental media?
Are spatial and temporal issues taken into account? Is it reproducible? Does it provide adequate discrimination? Is it quantitative?
Yes rep PCR RAPD RFLP rep PCR PFGE
MLST PFGE IR-VS IR-VS
rep PCR RAPD RFLP MLST PFGE MLST PFGE IR-VS Rep PCR RAPD RFLP MLST Adenovirus FWAs
Adenovirus IR-VS Enterotoxin biomarkers F+ RNA phage
Partially RFLP
Adenovirus Bacteroides Bifidobacteria Adenovirus Bacteroides Bifidobacteria FWAs
Enterotoxin biomarkers FWAs F+ RNA phage Enterotoxin biomarkers FWAs
RAPD Adenovirus Bifidobacteria
FWAs
PFGE IR-VS Bifidobacteria
Bacteroides
Enterotoxin biomarkers
Bacteroides FWAs F+ RNA phage
Bifidobacteria
RAPD FWAs RFLP F+ RNAphage MLST Bacteroides Bifidobacteria
Enterotoxin biomarkers F+ RNA phage
rep PCR RFLP
PFGE IR-VS Enterotoxin biomarkers FWAs F+RNA phage
Adenovirus Bifidobacteria Bacteroides F+ RNA phage rep PCR RFLP RAPD MLST rep PCR RAPD MLST Adenovirus
Is the method amenable to routine laboratory use?
RFLP PFGE IR-VS
Bacteroides Enterotoxin biomarkers
Are widely accepted standard methods available?
MLST
F+ RNA phage
Is it amenable to accreditation?
IR-VS Bacteroides
Enterotoxin biomarkers F+ RNA phage
MLST Adenovirus
FWAs
Are interrelationships with existing fecal indicator organisms considered? Has it been validated with regard to source assignment?
rep PCR IR-VS Bacteroides rep PCR
Enterotoxin biomarkers F+RNA phage
RAPD RFLP MLST RAPD RFLP Adenovirus
PFGE Adenovirus
Enterotoxin
Has it been used in a regulatory or legislative capacity?
Are there significant cost implications MLST to set up the method and per PFGE sample? IR-VS (to set up) Can results be generated rapidly?
Is considerable research and development required? Is further research recommended?
Adapted from Rangdale et al. (2003). Abbreviations are given in Table 1.
MLST adenovirus IR-VS
FWAs (to set up) F+RNA phage
rep PCR RAPD RFLP Adenovirus
Bacteroides rep PCR enterotoxin biomarkers RAPD RFLP FWAs MLST Enterotoxin biomarkers rep PCR Adenovirus FWAs RAPD RFLP Bifidobacteria F+ RNA phage MLST Adenovirus repPCR PFGE IR-VS RAPD RFLP Bacteroides
No
Rep PCR RAPD RFLP PFGE Adenovirus Rep PCR RAPD RFLP Bacteroides Bifidobacteria
IR-VS MLST F+RNA phage PFGE Bacteroides rep PCR RAPD RFLP MLST PFGE Adenovirus IR-VS Bacteroides IR-VS per enterotoxin sample biomarkers FWAs (per sample) PFGE F+ RNA phage Bacteroides Bifidobacteria PFGE IR-VS Bacteroides bifidobacteria FWAs F+ RNA phage
IR-VS Bacteroides Bifidobacteria Enterotoxin biomarkers FWAs PFGE Bifidobacteria FWAs Bacteroides FWAs Bacteroides Bifidobacteria Enterotoxin biomarkers FWAs F+ RNA phage Bifidobacteria
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collected from a wide geographical area. Also, automated ribotyping requires extensive equipment, and manual ribotyping by Southern blot hybridization was considered too time consuming. The ICReW project is being applied over a wide geographical area of Europe from Mediterranean climate to temporal climates in the UK, and therefore methods that require large libraries, or where there are doubts concerning spatial transferability, temporal stability, or methods that are costly to establish and carry out, were not considered suitable for this particular application where reasonable alternative methods were available for preliminary trials. Detailed reasons for the rejection of the methods considered can be found in Rangdale et al. (2003). Based on the results of the review undertaken by Rangdale et al. (2003) key researchers were invited to attend an international workshop sponsored by the Department for the Environment, Food and Rural Affairs, UK on pollution source-tracking approaches in order to assist the ICReW project team in deciding which source-tracking approach would be most appropriate to the needs of the project. The intention is to develop the chosen methods between 2004 and 2006, establishing them in a laboratory in each participating country, and then undertaking field trials to demonstrate their accuracy.
below. Full descriptions of the methods can be found in Rangdale et al. (2003) and indicated references.
Microbial Library-Dependent Methods Repeated Sequences PCR (rep PCR) The rep PCR technique has been described by Rademaker et al. (1998) and Myoda et al. (2003), and it has been successfully used on saline and freshwater to discriminate E. coli isolates from humans, geese, ducks, sheep, pigs, chicken, gulls, horses, and cows (Dombek et al., 2000; Carson et al., 2003; McLellan et al., 2003; Seurinck et al., 2003). The method requires the isolation of bacterial isolates from the species to be analyzed. PCR amplification of the DNA between adjacent repetitive extragenic elements with specific primers results in strain-specific DNA fingerprints. Amplified genomic fragments are then separated using electrophoresis in agarose gels. The gels are stained with ethidium bromide and banding patterns are compared. The genetic fingerprints contain several bands which can then be used to construct a database (Versalovic et al., 1994; Scott et al., 2002; Byoung-Kwon et al., 2003). A disadvantage of this technique is that it requires pure cultures of the bacterial isolate that are time consuming to produce. The reproducibility of this method has also been questioned (Scott et al., 2002).
The Workshop The workshop included presentations by the ICReW project team that described review work undertaken so far and contributions from key researchers currently working with methods that seem to offer the most promise for the project’s application. The workshop was to be used for peer review and challenge the conclusions drawn from that review. Many fecal source-tracking methods are culture-based methods in which the occurrence of phenotypic and genotypic traits in fecal isolates from water is compared with occurrence in a library of isolates from fecal samples. Although these have proved to be successful in a number of individual studies (Gustavo dos Anjos Borges et al., 2003; McLellan et al., 2003), considerable genetic heterogeneity between isolates obtained in natural environments may be observed. This is due to differences between sampling sites, particularly when the sampling site is subject to high contamination from diverse sources and environmental factors such as the disposal of sewage, the presence of domestic animals and livestock, and the erosion of river banks. A considerable increase in the number of unique-pattern strains is ascribed to the addition of strains originating from the human intestine, animal, and sewage to indigenous strains (Rangdale et al., 2003). It is therefore essential to have a large library when sampling from different geographical areas is undertaken. However, discussions in the workshop revealed that as the number of strains in the library increases (>1000 isolates) the reliability of library-dependent methods decreases. Seven library-based methods and five library-independent methods were selected for discussion and consideration of their use in the ICReW project (Table 1). These are briefly described
Randomly Amplified Polymorphic DNA (RAPD) This method produces a spectrum of amplified products characteristic of the template DNA by random priming at multiple locations throughout the genome using arbitrary primers to identify selected polymorphisms (Williams et al., 1990; Hilton and Penn, 1998). It is highly discriminative for typing epidemiologic E. coli strains, but results have proved difficult to reproduce. Restriction Fragment Length Polymorphism (RFLP) RFLP is a strain-specific genetic fingerprinting method. There are four main methods of RFLP that can be used to type bacteria: restriction fragment end labeling (RFEL), RFLP by Southern blotting, RFLP-PCR, and terminal-restriction fragment-length polymorphism (t-RFLP). RFLP-PCR requires the isolation of bacterial isolates. t-RFLP has the advantage of being able to bypass the isolation step and extract the DNA from an environmental sample. RFEL and RFLP by Southern blotting also require purification of genomic DNA. The methods are described by Hermans et al. (1995), Olive and Bean (1999), Nachamkin et al. (1993), and Amman et al. (1995). Field et al. (2003) successfully used t-RFLP of 16SrRNA genes from the fecal bacteria bacteroides to distinguish between ruminant and human feces. The main disadvantage with t-RFLP is that it requires expensive equipment and is technically demanding (Meays et al., 2004). Multilocus Sequence Typing (MLST) MLST is a sequence-based typing system that involves PCR amplification and nucleic acid sequencing. It requires the use of
Pollution Source Tracking Methods
bacterial isolates rather than direct samples. Following DNA extraction, internal fragments of seven housekeeping genes are amplified and sequenced using nested sequencing primers (Jolley, 2000). MLST has been used successfully to discriminate at both the species level and also within species (Feil and Spratt, 2001). However, the method requires the development of a large database library.
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Pulsed Field Gel Electrophoresis (PFGE) PFGE is a genotypic library-dependent method that involves the lysis of cells in situ in agarose plugs to release intact chromosomal DNA. All except the DNA is removed from the plugs, which is then restricted with rare-cutting restriction endonucleases. PFGE is performed on the restricted DNA, followed by staining and analysis of the separated fragments. The method is described by Barrett et al. (1994) and has been recently used by Hahm et al. (2003) to subtype environmental isolates of E. coli. The technique is extremely sensitive to very small genetic differences, requires a long assay time, simultaneous processing is limited, and a large library database is required (Meays et al., 2004). It also requires a specialized gel electrophoresis unit, which increases the costs (Byoung-Kwon, 2003).
Microbial Library-Independent Methods Detection of Adenoviruses Adenoviruses are commonly found in raw sewage and fecally contaminated surface and groundwater, and they have been detected using either cell culture or PCR methods (Jiang, 2002; Noble et al., 2003). Advances in PCR technology allow detection of all adenovirus subgenera from clinical samples (PringAkerblom et al., 1999). Disadvantages of using adenovirus are that the method is not quantitative and to date has not been widely tested. Bacteroides Genotyping, Host-Specific PCR-Based Method The bacteroides group is found in human and animal feces. It has limited survival and reproduction upon release into the environment, but there are strain differences among different animal hosts and a high level of genetic diversity. Species-specific bacteroides strains have been identified, and primers that are specific for the 16S rRNA of these species have been developed. To date, primer sets are available to differentiate between bacteriodes species specific for ruminants and humans. The method has been developed, tested in the field, and compared with other methods in the United States by Field et al. (2003). The detection of Bacteroides fragilis is a highly specific method for tracking the source of human fecal pollution (Scott et al., 2002). The method does not require culturing of the organism, but it is only semiquantitative and the methods need further development. However, the use of bacteriophages of B. fragilis HSP40 has the advantage of being highly specific to human fecal pollution.
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Sorbitol-Forming Bifidobacteria The genus bifidobacteria is commonly found in the human intestine. Certain species of bifidobacteria have been found to be exclusive to the feces of humans and pigs (Resnick and Levin, 1981) and therefore bifidobacteria has been investigated as a potential indictor of human fecal pollution (Jagals et al., 1995; Rhodes and Kator, 1999). Bifidobacteria longum and B. adolescentis are the most frequently isolated and widely distributed of the species and have been found to have the ability to ferment sorbitol, a characteristic not identified in Bifidobacteria spp. found in animal sources. Sorbitol-fermenting bacteria have been used to identify human fecal material, but no information on animal type or species is currently possible using this method. Disadvantages of the methods are that there are low numbers of bifidobacteria present in the environment, they have variable survival rates (Scott et al., 2002), culture of anaerobic cells may be required, and validation of the method is needed. Enterotoxin Markers—PCR-Based Method Pathogenic E. coli strains produce a number of toxins that interact with specific receptors located on mammalian cells. Detection of variable regions of enterotoxin genes of E. coli associated with a particular host allows discrimination between the origins of fecal contamination. The method has been described by Oshiro and Olson (1997), Khatib et al. (2002), Bernhard et al. (2003), and Tsai et al. (2003). The method has been shown to successfully discriminate between fecal contamination from humans, cattle, and pigs. The methods are not quantitative and need standardizing and validating. F+ RNA Coliphage Typing Due to their persistence in the environment and resistance to treatment processes, it has been suggested that coliphages may be better indicators for pathogenic organisms than coliforms (Havelaar, 1993). F+ RNA phage fall into four distinct subgroups, differentiated by serotyping or genotyping. Serogroups II and III predominate in humans, while I and IV predominate in animals. Detection of F+ RNA coliphages is undertaken by infecting susceptible bacterial strains, either Salmonella typhimurium WG49 (Havelaar and Hogeboom, 1984) or E. coli HS(pFamp)R (Debartolomeis and Cabelli, 1991) according to a standard procedure described by Mooijman et al. (2002). Hybridization with specific probes or RT-PCR is then used to detect the F+ RNA coliphage (Rose et al., 1997; Schaper and Jofre, 2000). The method has been used successfully to discriminate between human and nonhuman sources of pollution but not to identify particular species (Brion et al., 2002). F+ RNA coliphages may be found in low numbers in pristine waters, and as yet their relationship with compliance organisms needs to be established. Infrared Vibrational Spectroscopy (IF-VS) Infrared vibrational spectroscopy allows the distinction of intact microbial cells and the production of biochemical
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fingerprint-like spectra that are reproducible and discrete for different microorganisms. Infrared spectroscopy measures molecular vibrations on the basis of the absorption of infrared radiation interacting with a sample (Maquelin, 2003). The technique, which requires cell culture, has been used to distinguish a number of microorganisms, including enterococci, from a variety of origins (Kirschner, 2001). However, there are a number of disadvantages that make the method unsuitable for the purposes of the ICReW project. These include the requirement of a large library database, expensive startup costs, no available standard methods, and the method has not been widely tested and applied.
Chemical Methods Fluorescent Whitening Agents (FWAs) FWAs are found in washing powders and detergents, resulting in considerable loads in effluent and receiving waters. FWAs absorb UV light at 350 nm and emit visible blue light at a maximum of 430 nm, and they may be detected by a high-resolution spectrofluorometer (Cioffi, 2003). A number of studies have been undertaken using FWAs to detect the origin of fecal pollution in rivers (Gilpin et al., 2002, 2003) and marine waters (Cioffi, 2003). However, there is currently no standard method available,
and it is thought that natural fluorescence from organic materials may make interpretation difficult. Fecal Sterols Fecal sterols and stanols have been suggested as indicators of source-specific fecal contamination. Most commonly, coprostanol (5β(H)-cholestanol-3 β-ol) has been used (Leeming et al., 1997). Coprostanol is produced from cholesterol in the intestine of most higher mammals. The relative and absolute amounts of coprostanol is lower in humans than in other mammals such as pigs, cats, and some birds (Leeming et al., 1994, 1996). In addition, very low amounts of coprostanol is found in herbivorous ruminants and higher proportions of 24-ethylcoprostanol and 24-ethyl epi-coprostanol (Leeming et al., 1996). It is therefore possible to compare the ratios of coprostanol and 24-ethyl coprostanol and 24-ethyl epicoprostanol and identify the source. The disadvantages include the natural presence of sterols in sediments, expensive analysis, and low prevalence of sterols, which makes sensitivity an issue (Meays et al., 2004).
Comparison of Techniques The following criteria were deemed to be essential to the suitability of the chosen methods for the requirements of the ICReW project:
Table 3. Advantages and disadvantages of library-dependent and -independent methods of source tracking microbial pollution Advantages Library-dependent methods • Proved to distinguish between animal and human sources • Successful investigations using these methods have been published • May be valuable for targeted investigations • May be useful as a tracer • Library systems that use standard indicator organisms such as E. coli may be useful for regulatory purposes due to familiarity from historical use • Several techniques are well developed and peer reviewed
Library-independent methods • Does not require the development of a library • Possible to distinguish between animal and human sources • Some methods do not require culturing, reducing time and possibly costs • Analysis is generally more rapid than library dependent methods • F+ RNA phage have been used been successfully in coastal areas in several countries • F+ RNA phage have been successfully used where there are hotspots of pollution to differentiate animal and human sources • Level of discrimination of enterotoxin markers is very good: enterotoxin markers have been used successfully as part of a nest of markers, including bacteroides genotyping • Chemical markers have been used successfully to distinguish human and animal sources • Several methods can be grouped together to improve the resolution of the techniques • Most of the methods are applicable on a broad geographical scale
Disadvantages • Requires development of a library • Heterogeneity of the indicator at the molecular level • Uncertainty about the number of isolates required to construct a library—too few isolates can limit the value of library, whereas too many can lead to loss of specificity • Library maintenance is costly and takes time • Specific limitations of all the techniques exist • Analyst variability and interlaboratory variability are key issues (in cases where several laboratories are involved) • Validation of the methods is time consuming • Low throughput and high costs • Methods based on spectroscopy are heavily dependent on the quality and reproducibility of the methods used to prepare the samples • The minimum population size for reliable detection of pathogen markers when present is unknown • Chemical markers often need analysis of large volumes to get detects • Validation and reproducibility problematic with some methods • Some of the chemical and microbial markers are human specific and would detect pollution from animal sources • The use of pathogen-specific markers depend on the organism present in the population • Problems with the fluorescent whitening agents (FWAs) occur as the sampling site moves further away from the source as a result of dilution and dispersion • FWAs are probably not sensitive enough to use in coastal environments; these markers have worked very well close to the source, but the sensitivity of the method declined very rapidly as they moved away from the source
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Table 4. Advantages and disadvantages of selected methods for microbial source tracing
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Technique
Advantages
Disadvantages
Rep-PCR
Traditional method is relatively inexpensive and has high reproducibility Able to distinguish animal types
MLST
Able to distinguish animal types High specificity
PFGE
Able to distinguish animal types
Library-based method Time consuming High variations
RFLP
Able to distinguish animal types
Library-based method
RAPD
Able to distinguish animal types
Library-based method
Vibrational spectoscopy
May allow discrimination between species; gives fast, reproducible results; and may be automated with computerized data analysis Applied to compliance organisms Library independent Able to distinguish between animal and human sources, controllable and non-controllable (avian) sources, and type of animal Well-characterized methods available Glass wool concentration method is effective and cheap No significant startup costs PCR consumables available commercially Method fairly easy to use and robust Library independent Able to distinguish between human and animal sources Able to detect bifidobacteria up to 5 Km from source Method easy to use Can be detected at high dilutions Less variable than E. coli Has been isolated in animals, but the isolates cannot ferment sorbitol, whereas the isolates in humans can Costs per sample low
Library-based method Not sufficiently developed and tested for this purpose
Detection of adenovirus
Sorbitol-fermenting bifido-bacteria
Enterotoxin markers
Library independent Able to distinguish between animal and human sources animal types Level of discrimination is good, but method depends on organism being present in the host population Good method to be used in conjunction with others, e.g., bacteroides genotyping Bacteroides spp. genotyping Library independent Found in human and animal faeces Strain differences among different animal hosts High level of genetic diversity Primers available for cattle, human, elk dog, and horses
Library-based method Modified method (developed by Nakatsu) that needs an expensive automatic sequencer, and this method needs further field testing Library-based method Expensive
Comments/development needs Requires generation of a representative library Work required to quantify the approach
Requires generation of a representative library and extensive validation required Work required to quantify the approach Requires generation of a representative library Extensive validation required Work required to quantify the approach Requires generation of a representative library, and extensive validation required Work required to quantify the approach Requires generation of a representative library, and extensive validation required Work required to quantify the approach Requires generation of a representative library, and extensive validation required Work required to quantify the approach
Costs per sample high May need a minimum population size to ensure detection if present
Extensive validation required Work required to quantify the approach Need to establish the relationships with compliance organisms
Survival in the environment is questionable No markers for specific animals developed No commercially available kits Method is not robust Requires anaerobic conditions for growth
The use of bifidobacteria holds promise, but issues exist with survival and culturing Further work needed on presence in sediments Need more investigation into bifidobacteria in animal species New techniques to develop specificity and sensitivity need to be developed Work required to quantify the approach and to establish relationships with compliance organisms; extensive validation is required Extensive validation required Work required to quantify the approach
Costs per sample high Method is not robust Nonquantitative
Limited survival and Requires some development but can be reproduction in environment made quantifiable Limited species markers Extensive validation required available Need to establish the relationship with Specificity of the markers depend compliance organisms on the stringency of PCR Costs per sample high (Continued on next page)
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Table 4. Advantages and disadvantages of selected methods for microbial source tracing (Continued)
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Technique
Advantages
Can use real-time PCR Inexpensive, quick method Already tested in the field Commercial kits available Method robust and easy to use Method sensitive with low volumes F+ RNA phage Library independent Able to distinguish between animal and human sources animal types Methods are well characterized ISO method exists No significant startup costs Reagents available commercially Simple, rapid and affordable Detects occurrence of fecal contamination and viruses Can adapt methods to detect only animal or only human sources Sufficiently UV resistant Direct PCR demonstrated successfully Costs per sample low Has been tested in coastal areas Chemical markers Fluorescent whitening agents are fairly easy to analyze and are specific to human populations Fecal sterols able to distinguish between controllable animals and noncontrollable (avian) species Capable of whole sample analysis FWAs are very stable and the sample can be stored and analyzed at later date Good indicators of fecal contamination Library independent Faecal sterols require complicated assay but provide a lot of information Good marker in suspended solids FWAs reasonable consistent between manufacturers and countries, therefore useful on a wide geographical scale
Disadvantages
Comments/development needs
May be issues regarding minimum population size
Survival may decrease at higher temperature Differences in survival among groups may alter the perceived relative contribution to receiving water Group I coliphages may have both human and animal origins; not diagnostic of source Mixed sources may create difficulties with data analysis Specificity of human groups is not absolute; some animal sources
Method needs developing but this is possible within reasonable time frame Need to investigate die-off rates Validation required Need to establish the relationship with compliance organisms
Issue of sensitivity since the marker cannot be amplified Method works well close to source but sensitivity declines with distance from the source Large volumes of the sample are needed for analysis Samples with mixed sources are more complicated to analyze Analysis of fecal sterols is time consuming Analysis is costly FWAs only detectable if there is greywater influx to the recreational water area No standard method available
Need to establish the relationship with compliance organisms
r Quantitative—able to describe the relative contributions of different pollution sources to a sample.
r Statistically robust—allowing confidence limits to be placed
r The positive and negative aspects of the 12 methods described above were outlined in the context of suitability for use as a regulatory tool (Table 4).
around the quantitation.
r Cost effective—in the context of alternative approaches, e.g., r r r
catchment investigations. Applicable to water column and sediments—in order that acute and chronic pollution episodes can be commented on. Ideally be based on a compliance parameter—to maintain direct relevance to the legislative drivers. The outcome must be credible and able to be trialled in different catchments.
The relative benefits and drawbacks of the methods and techniques were compared using a two-tier approach:
r The advantages and disadvantages of library dependent and library independent methods were identified (Table 3).
Issues to Consider when Developing the Sampling Program In addition to selecting the most appropriate methods for the ICReW project, issues regarding the design of the fieldwork program were discussed. In all fieldwork programs, case-specific reasons for carrying out the research should be identified prior to carrying out the fieldwork; for example, is the sampling for public health reasons or regulatory purposes? This will dictate the precision of the information required, e.g., is it only necessary to know that the pollution is from nonhuman sources or that the pollution comes from a particular species of livestock. An examination of the land-use in parallel with trialing the chosen technique is important to understand the full range of fecal sources and risks in the area. Other issues identified as being
Pollution Source Tracking Methods
of importance when developing a sampling program in various geographical locations are:
r Laboratory technicians should be trained centrally. r Rigorous validation and quality control procedures such as the
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r
r
r r r
r
use of spiked samples, analysis of simulated samples, testing of positive (i.e., sewage plant or highly contaminated receiving waters) and negative control samples, and interlaboratory comparisons should be undertaken. A quality assurance scheme with each laboratory documenting its internal quality controls should be established. This can be trialled using a round-robin testing of water samples between participating laboratories during the pilot. Comprehensive standard operating procedures should be documented and employed by each of the participating laboratories. The information contained within these can be condensed into bench sheets that can be used by technical staff. The fieldwork in each country should follow a common study design and where possible use the same batch of reagents. The trial should commence on a simple well-characterized watershed. It is advisable to start work close to the known source of contamination and work down the catchment. Sampling plans need to consider seasonality, time of day, and other factors such as migration patterns of birds. Composite samples can be used to overcome the potential problems of temporal changes in the concentration of the target organism. The results of the pilot scheme must be evaluated and the sampling procedure amended as necessary.
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First, there will be a 6-month period in which methods are established and tested for comparability of performance between the partner laboratories. This will be followed by a series of iterative field trials, designed to prove the methods in real situations, chosen as areas where the polluting inputs to watercourses are already well characterized. Multiple methods will be applied to each environmental sample collected so that comparisons can be made both between countries and between methods within each country. This follows the consensus from the workshop that the way forward is the application of a “basket” of methods that should be mutually supporting in their outcomes, proving cross-validation between approaches. The outputs of this project will include tools and guidance to help managers identify sources of pollution in bathing and shellfish waters and will ultimately help to improve their quality.
Acknowledgements The authors thank the following researchers who attended the workshop: Nicholas Ashbolt, Andy Ball, Nicola Cunningham, Alfred P. Dufour, Katherine Field, Brent Gilpin, Valerie J. Harwood, Bodril Hernroth, Joan Jofre, Keith Jones, Gerry Lukasik, Jorge Machado, Sandra McLellan, Steve Mudge, Cindy Nakatsu, Laura Rosado, David Sartory, Joyce Simpson, Mark D. Sobsey, Huw D. Taylor, Tony Warn, and Bruce Wiggins. The workshop was sponsored by the Department for Environment, Food and Rural Affairs, UK.
References Conclusions It was concluded that within the aims and budget of this project it would not be appropriate to choose a library-dependent method, as extensive background work would be needed to populate and test the library. Due to time constraints and the large geographical area that the trial will be carried out on, this is not feasible. Hence, while many of the library-dependent methods are promising and have been used successfully in a number of areas, particularly for geographically restricted studies, this project will employ a library-independent method. When the advantages and disadvantages of each method were examined in detail, it was concluded that the two most appropriate techniques for this project to carry forward to the field phase were Bacteroides spp. genotyping and F+ RNA phage. It should be noted that all of the techniques reviewed can be used under certain circumstances and with varying degrees of success, but not all are appropriate for use on a wide geographical scale (for example, library-dependent methods would require the development of an extremely large library database), or for regulatory purposes, where costs and reliability become significant factors. The ICReW project will develop and apply the chosen methods over a two-year period from March 2004 to March 2006. This development will take place in two phases in each of the ICReW partner countries—the UK, Ireland, France, and Portugal.
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