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The effects of agricultural activities on stream water quality were assessed by nitrogen analysis, further investigated by gas chromatography mass spectrometry (GC-MS) sterol analysis (including chemometric analysis), and characterized by bacterial source tracking (BST). Surface water samples were collected from five sites, throughout the agriculturally-influenced Nathan Creek watershed, British Columbia, Canada and a nearby control site between October 2005 and March 2006. From a total of 48 samples, Canadian Water Quality Guidelines were exceeded nineteen times for nitrate (NO₃ ⁻; guideline value: 2.94 mg/L N) and four times for un-ionized ammonia (NH₃; guideline value 0.019 mg/L N). Gas chromatography mass spectrometry single ion monitoring (GC-MS SIM) analysis of 18 sterols showed that five fecal sterols (coprostanol, episoprostanol, cholesterol, cholestanol, desmosterol) were detected at all sites except the control site (where only cholesterol, cholestanol and desmosterol were detected). Three phytosterols (campesterol, stigmasterol and β-sitosterol) were also detected at all sites while the hormone estrone was present at one site on two occasions at concentrations of 0.01 and 0.04 μg/L. Chemometric analysis (principal component analysis and cluster analysis) grouped sites based on their similarities in sterol composition. Analysis of ten sterol ratios (seven for identifying human fecal contamination and four for differentiating sources of fecal contamination) showed multiple instances of human and animal contamination for every site but the control site. Application of a Bacteroides-BST method confirmed contamination from ruminant animals, pigs and dogs in varying combinations at all impact sites. Together, these results confirmed the impact of agricultural activities on the Nathan Creek watershed and support a need for better land management practices to protect water quality and aquatic life.