First evidence of terrestrial ambrein formation in human adipocere.

To date, the only known occurrence of ambrein, an important perfumery organic molecule, is in coproliths found in about one in a hundred sperm whales. Jetsam ambergris coproliths from the whale are also found occasionally on beaches worldwide. Here we report on the surprising occurrence of ambrein in human adipocere. Adipocere is a waxy substance formed post-mortem during incomplete anaerobic decomposition of soft tissues. Adipocere samples obtained from grave exhumations were analysed using gas chromatography-mass spectrometry (GC-MS). In addition to the typical fatty acids of adipocere, lesser amounts of ambrein were identified in the samples, in abundances similar to those of the major accompanying faecal steroids. The distribution of these compounds suggests that ambrein was produced post-mortem during the microbial decomposition of faecal residues and tissues. It is assumed that the adipocere matrix of saturated fatty acidsaided the preservation of ambrein over extended periods of time, because adipocere is stable against degradation. The association of ambrein formation in ageing faecal material, under moist, oxygen-depleted conditions, now requires more attention in studies of other mammalian and geological samples. Indeed, ambrein and its transformation products may be useful novel chemical indicators of aged faecal matter and decomposed bodies.

Investigation of sterols as potential biomarkers for the detection of pig (S. s. domesticus) decomposition fluid in soils.

This study was carried out to evaluate the potential of using cholesterol and coprostanol, as indicators for the detection of decomposition fluid of buried pigs (S. s. domesticus) in soils. In May 2007, four pig carcasses (∼35kg) were buried in shallow graves (∼40 cm depth) at the University of Ontario Institute of Technology in Canada. Two pigs were exhumed after three months (Pig 1, Pig 2) and six months (Pig 3, Pig 4) post burial. Soil samples were collected beneath the pig carcasses (∼40cm depth) and from grave walls (∼15-20 cm depth) as well as from a parallel control site. Coprostanol and cholesterol were extracted from soils, purified with solid phase extraction (SPE) and analysed with gas chromatography/mass spectrometry (GC/MS). A significant increase in cholesterol concentrations (p<0.05) and amounts of coprostanol were detected in soil located beneath the pig carcasses after three months of burial. It is assumed that during the putrefaction and liquefaction stages of decomposition pig fluid which contains cholesterol and coprostanol is released into the underlying soil. Therefore, cholesterol and coprostanol could be used as potential biomarkers to detect the presence of decomposition fluid three months after burial under comparable soil and environmental conditions. Further research is suggested for additional soil sampling before and after three months to investigate the abundance of these and other sterols.

Can gas chromatography combustion isotope ratio mass spectrometry be used to quantify organic compound abundance?

Quantifying the concentrations of organics such as phospholipid fatty acids (PLFAs) and n-alkanes and measuring their corresponding (13)C/(12)C isotope ratios often involves two separate analyses; (1) quantification by gas chromatography flame ionisation detection (GC-FID) or gas chromatography/mass spectrometry (GC/MS), and (2) (13) C-isotope abundance analysis by gas chromatography/combustion/isotope ratio mass spectrometry (GC-C-IRMS). This requirement for two separate analyses has obvious disadvantages in terms of cost and time. However, there is a history of using the data output of isotope ratio mass spectrometers to quantify various components; including the N and C concentrations of solid materials and CO(2) concentrations in gaseous samples. Here we explore the possibility of quantifying n-alkanes extracted from sheeps' faeces and fatty acid methyl esters (FAMEs) derivatised from PLFAs extracted from grassland soil, using GC-C-IRMS. The results were compared with those from GC-FID analysis of the same extracts. For GC-C-IRMS the combined area of the masses for all the ions (m/z 44, 45 and 46) was collected, referred to as 'area all', while for the GC-FID analysis the peak area data were collected. Following normalisation to a common value for added internal standards, the GC-C-IRMS 'area all' values and the GC-FID peak area data were directly compared. Strong linear relationships were found for both n-alkanes and FAMEs. For the n-alkanes the relationships were 1:1 while, for the FAMEs, GC-C-IRMS overestimated the areas relative to the GC-FID results. However, with suitable reference material 1:1 relationships were established. The output of a GC-C-IRMS system can form the basis for the quantification of certain organics including FAMEs and n-alkanes.

Protocol for the analysis of n-alkanes and other plant-wax compounds and for their use as markers for quantifying the nutrient supply of large mammalian herbivores.

Plant-wax markers can be used for estimating forage intake, diet composition and supplement intake in grazing livestock, wild ruminants and other mammals. We describe protocols for using the saturated hydrocarbons (alkanes) of plant wax as markers for estimating fecal output, intake and digestibility. Procedures for investigating digestion kinetics are also discussed. Alkanes can also be used to estimate diet composition and the procedures required to do this are also described, including the special case where supplementary feed is treated as a component of the diet composition estimate. The long-chain alcohols (LCOHs) and very long-chain fatty acids (VLCFAs) of plant wax show particular promise for discriminating a greater number of species in the diet. The use of all these plant-wax markers in nutrition studies depends on having quantitative, repeatable and mutually compatible assay procedures for alkanes, LCOHs and VLCFAs; we present protocols for these assays in detail. Analysis of a single sample of feces or plant material for all these plant-wax markers can be completed within 2 days; however, it is possible to process up to 50 samples (analyzed in duplicate) per week.

Phthalate and alkyl phenol concentrations in soil following applications of inorganic fertiliser or sewage sludge to pasture and potential rates of ingestion by grazing ruminants.

Soil concentrations of dioctyl phthalate (DOP) and the alkyl phenols, octyl phenol (OP) and nonyl phenol (NP), after repeated surface applications of sewage sludge to pastures, were investigated. Liquid sludge was applied at a rate of 2.25 tonnes dry matter (DM) per hectare to each of three treated (T) plots on three occasions during the summer and two occasions in the early spring over a period of 2.5 years. Control (C) plots were treated with inorganic fertiliser containing amounts of nitrogen equivalent to those applied to the treated plots. At between 69 and 81 days after the application of sludge, 15 separate soil samples were collected from one half of each of the plots (Experiment 1). Concentrations (microg g(-1)) of DOP were higher (P < 0.001) than those of NP, while those of OP were generally below detectable levels. Mean soil concentrations of DOP were not significantly different in T and C plots [0.233 vs. 0.155 microg g(-1); standard error of the difference (SED) = 0.046; not significant (NS)], partly because there was already a relatively large amount of DOP present. NP concentrations were, however, significantly higher in T than in C plots (0.021 vs. 0.013 microg g(-1) SED = 0.002; P < 0.05). There was no consistent change over time in the mean soil concentrations of these compounds when sampled at intervals of 3-6 months. Concentrations in soil samples collected at monthly intervals following sludge application indicated that the variation in concentrations of these endocrine-disrupting compounds (EDC) was unrelated to time since sludge application. Rates of soil ingestion, expressed as the percentage of DM intake represented by soil, were higher during the winter than the summer (5.40 vs. 1.17; SED = 0.360; P < 0.001) and estimated daily intakes of DOP and NP were up to 150 microg and 8 microg, respectively. It is concluded that the application of sewage sludge to pasture does not increase soil concentrations of phthalate (as DOP) or alkyl phenols. Thus, the risk of increased exposure to these EDC as a result of sludge application is small. However, the small effect of sludge application on soil concentrations may be largely a reflection of the relatively high concentrations of DOP already present in the soil, which may be biologically significant.

The flexibility of an intermediate feeder: dietary selection by mountain hares measured using faecal n-alkanes.

Herbivores with an intermediate feeding strategy either vary their diet between a grazing (bulk roughage feeders) or browsing (concentrate selectors) strategy on a seasonal basis or select a mixed diet at any one time. The underlying ecological causes of the seasonal dietary shift in a small non-ruminant intermediate feeder - the mountain or arctic hare (Lepus timidus L.) were determined. Diet composition and selection relative to availability were investigated for 41 individual free-ranging mountain hares (of which 18 female hares were radio-collared) occupying an upland mosaic landscape in north-east Scotland. Diet composition was determined using faecal n-alkane analysis. Radio-collared hares were designated as pasture, woodland or moorland hares according to the habitat that predominated their home-range. In common with previous studies, mountain hares switched from a browse-dominated diet during winter to a Gramineae-dominated diet in summer, although it was only significant for reproductively active females during the peak breeding season. Diet composition remained consistent regardless of habitat occupied. However, the diet of radio-tracked hares differed significantly from the biomass available in the individual home-ranges; Gramineae were preferentially selected over browse species throughout the year. During winter and in particular during the early breeding season, intermediate feeders, such as mountain hares, ate browse material when the availability of higher quality was restricted. The ability to browse or graze represents a flexible foraging strategy permitting survival and production through periods of changing or unpredictable forage quality and availability.