ReportNo WSAA 204           

November 1998




Theproject aims were to: (i) estimate the numbers of faecal indicatormicroorganisms (bacteria and bacteriophages of Bacteroides fragilis) and concentrations of sterols present in thefaeces of native and domestic animals and in sewage to validate whether profiledifferences measured in pilot studies could be used to quantify sources offaecal contamination, (ii) determine the relationship between, and relativepersistence of, faecal sterols and indicator microorganisms during microcosmfield experiments and (iii) develop a quantitative method which could distinguishhow much faecal matter is present from various sources in a polluted receivingwater and test the technique in the field.


Theresults for objective (i) indicate that the differences in faecal sterols andbacterial indicators between the animal groups were consistent over a range ofenvironments. The concentrations of faecal sterols and bacterial indicatorsmeasured can be variable, but the ratios of the parameters, on the basis ofpopulations, are consistent within animal groups and compared to referencesamples. Reference samples were collected during field studies from sewer mainsor overflows, livestock saleyards and bird resting sites adjacent to fieldsites and results from these samples provided corroborating in situ ratios to estimate faecal contributionsto specific environments. The independent use of at least two and preferablyfour subgroups of bacterial indicators (thermotolerant coliforms, Escherichia coli, faecal streptococciand enterococci) as a common denominator of all faecal pollution, strengthensthe degree of confidence in the estimates. Estimates from calculationsperformed for field studies, where the wider range of bacterial indicators wasused, generally varied by less than 10%. The use of a range of ratio valuesbased on the variance measured in the faecal and field samples enables a rangeof contributions to be expressed. These are summarised in the legend of Figure4 in the report.


Thebacteriophages to various strains of B.fragilisused to characterise human, pig or poultry, cattle and sheep faeces were oftenabsent from the faeces of individual animals. Nonetheless, when bacteriophageswere present in the faeces of cattle or pigs, they consistently contained onlyphages specific to their respective host strain, and human sewage alwayscontained the bacteriophages to HSP40, the human-specific strain. As observedfor the HSP40 strain in North America, the results indicated that only about10% of individuals excreted their respective bacteriophages, and if faecalcontamination from cattle, pigs or humans was present, then up to 1000 plaqueforming units of their specific phages per millilitre of effluent could beexpected. Hence, given the likely low numbers of phages in environmentalwaters, enrichments for the specific phages probably will be needed to confirmthe presence of cattle, pig or human faecal contamination.


Withthe use of any single or multiple indicator system, the efficacy of the resultor relevance of the interpretation is a function of time and / or distance fromthe source. Results for objective (ii) indicated that faecal sterols degrade atthe same rate as each other and most bacterial indicators die-off more rapidlythan faecal sterols degrade, with C.perfringensspores being the exception. However, significant divergence of the ratiosbetween faecal sterols and bacterial indicators are unlikely to occur within2-3 days. Under the environmental situations of field studies undertaken sofar, this time constraint was generally not considered to have been exceeded. Thiswas because the survey / catchment areas were not large and even low flow ratesoccurring in dry weather were estimated to transport faecal matter along thelength of the catchments inside 2-3 days. During wet weather when faecalcontamination is most prevalent, residence times are even shorter. A settlingexperiment was conducted to examine the particle size distribution for faecalsterols and bacterial indicators. The results indicated that only in the veryfine particle range (equivalent settling velocity to 9 ф quartzparticles) was there any significant increase in bacterial indicators. Thismeans that only in standing waters would the ratio of bacterial indicators tofaecal sterols increase.


Resultsfrom field studies showed the technique conclusively distinguished episodes ofhuman faecal contamination. Faecal contamination from humans and herbivoressuch as cattle, sheep, kangaroos, etc. could be distinguished from each otherwith a high degree of accuracy. Based on the findings of this study, faecalcontamination from dogs might also be evident, if present in significant amount, because of a distinctive faecalprofile of high C. perfringens spores(≈ equal to thermotolerant coliforms) and high cholesterolconcentrations. However, in field studies performed during the period of thisstudy, dogs were not considered to be a significant source of faecalcontamination, hence the efficacy of the technique to distinguish faecalcontamination from dogs remains untested. In the absence currently of aspecific marker for bird faeces, the faecal profile for birds is highabundances of thermotolerant coliforms and faecal streptococci, low abundancesof C. perfringens spores and highconcentrations of  Δ5-sterolswith 5ß-stanols absent or in environmentally insignificant trace amounts. Otherpossible sources of faecal contamination which share a similar faecal profileare invertebrates, reptiles and fish. Because there are presently no specificbiomarkers for faeces from these species, faecal contamination from thesesources are designated “birds, dogs and other diffuse sources”. However, inseveral field studies, birds were proposed to be the dominant contributor tothis grouping because they were ubiquitous and prolific.


Insummary, the technique can, with a high degree of certainty, determine anddistinguish human versus herbivore faecal contamination and these two sourcesversus other sources. The technique can then, with a diminished degree of precision,estimate the contribution from birds, dogs and other diffuse sources from totalfaecal contamination. The results from field studies show the technique is agreat benefit to water managers for making effective management decisions.



Thewater industry has already responded positively to the concept of usingadditional indicators of faecal contamination to characterise and understandcatchments. No one indicator or one approach could represent all the facets andissues associated with contamination of our waterways with faecal matter. Thetechnique described herein has, as of publication, been used in over 20different environments for local councils, environment protection agencies andwater authorities with consistent results. This technique is only a first steptoward devising a set of tools that water managers will have at their disposalto investigate the entry of faecal contamination and other organic matterinputs into aquatic systems.


Inlight of the results of this study, it is recommended that further research beconducted in several complementary areas. Firstly, other classes of lipidbiomarkers (e.g. cholanic bile acids and bile pigments) show distinctiveprofiles between animals. Additional research to discover specific biomarkersfor birds and/or dogs would be highly desirable and appears achievable.Secondly, research on bacterial phages could provide qualitative confirmationsof faecal sources to complement biomarker assays and to promote a diversity ofapproaches to solve current problems. Finally, combined PCR (polymerase chainreaction) and biomarker analyses of environmental samples could also provide anadvantageous and diversified approach to distinguishing faecal pollution.


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