Faecal Sterol Analysis
Sterols are lipids that have important biological functions, in plants and animals, including maintenance of cell wall structure. The subgroup of "faecal" sterols is found mainly in animal faeces, and the sterol "fingerprint" can be quite distinctive between species, depending on the interaction of three factors :
- Whether animals synthesise sterols (e.g., humans synthesise cholesterol).
- The animal's diet, which determines the relative quantities of sterol precursors, e.g. plant sterols in herbivores.
- The anaerobic bacteria in the animal's gut, which convert sterols to stanols e.g., cholesterol is the precursor to cholestanol and coprostanol.
Although individual sterols are not unique to a particular animal, examination of the entire sterol profile in a faecal sample can be useful in determining the likely pollution source, particularly when combined with other information such as bacterial indicator results.
Faecal sterols analysis is performed, by filtering at least four litres of river water onto glass fibre filters. Filters are stored frozen until they were analysed using the extraction procedure described by Gregor et al. (2002) (external link) . Each sterol and stanol result is expressed as parts per trillion (ppt).
Faecal sterol analysis in waterways relies on sterol ratios rather than on absolute concentrations.
Key analytical features:
- level of coprostanol, which comprises approximately 60% of the human sterol concentration and occurs in ten fold less concentrations in other animals.
- the ratio of coprostanol:cholestanol is used to indicate whether the coprostanol present is of faecal origin. A coprostanol:cholestanol ratio greater than 0.5 in a water sample suggests faecal contamination.
- the ratio of coprostanol:24-ethylcoprostanol. Humans produce more coprostanol whereas herbivores produce higher concentrations of 24-ethylcoprostanol which is derived from their diet of plant sterols. Therefore human faecal pollution typically has a ratio of greater than one for coprostanol: 24-ethylcoprostanol.
Other sterol ratios point to certain animals and birds. The interpretation of sterol data is complex. A flow diagram outlining the sequential steps followed to establish a faecal source is shown below.
This diagram is based on an expert opinion system and is not presented as a decision tree. An expert opinion system is regularly updated based on continual research input and the integration of complex interrelated factors. For example, it is not necessary for all three coprostanol ratios to indicate human faecal pollution as site specific factors such as climate and/or algal proliferation (resulting in higher levels of cholestanol production) may alter sterol concentrations and thus ratio values.
Interpretation of Faecal Sterol Results
Where a mixed pollution event involving contamination from human and herbivore sources is suspected then the diagram outlines the calulations for an estimation of the faecal contribution from human and herbivore sources.
At ESR, we can analyse water samples for ten different sterols/stanols [PDF, 144 KB], and ratio analysis [PDF, 13 KB] of the results has the potential to identify a wide range of animals and birds. An example of results generated by faecal sterol analysis can be found here [PDF, 201 KB]. In addition, this method may estimate the relative contributions of faecal contamination where a mixed human/herbivore pollution event is suspected. However, faecal sterol analysis requires the collection of at least two litres of water for analysis, and is the most time consuming and expensive of the faecal discrimination tools offered by our laboratory. Analytical costs for faecal sterol analysis can be found at Costs for faecal discrimination testing.
Contamination issues when collecting water for faecal sterol analysis
Generally, four litre water samples are collected for faecal sterol analysis, although turbid or heavily polluted water may only require 2 litre samples. It is important to be aware of contamination issues when collecting water for faecal sterol analysis. It is advisable to wear blue nitrile gloves that are free of cholesterol as human handling may contaminate the water sample. For more information refer to Faecal source ID sample collection.
References for faecal sterol analysis can be found here.