Despite the importance of atmospheric methane as a potent greenhouse gas and the significant contribution from ruminant enteric fermentation on methane emissions at a global scale, little effort has been made to consider the influence that different plant-based natural diets have on methane emissions in grazing systems. Heathland is an ericaceous dwarf-shrub-dominated habitat widespread across the northern hemisphere, in Europe, provides valuable ecosystem services in areas with poor soils, such as water flow regulation, land-based carbon skin, energy reservoir and habitat of key game species. We (i) measured methane emissions from red deer (Cervus elaphus) and sheep (Ovis aries) fed mixed diets of natural grass plus ericaceous species (either Calluna vulgaris or Vaccinium myrtilius) using open-circuit respiration chambers; and (ii) modelled the results to estimate methane emissions from red deer and sheep populations inhabiting heathland habitats across Europe under different scenarios of grass-based mixed diets with varying proportions of ericaceous species. Our results indicated that methane emissions per unit of digestible organic matter intake decreased as the proportion of ericaceous species in diet increased, but this relationship was complex because of the significant interaction between the proportion of ericaceous species in the diet and digestible organic matter intake. According to our estimates red deer and sheep populations across European heathlands produce 129.7 kt.y(-1) methane (se = 1.79) based on a hypothetical grass-ericaceous species mixed diet containing 30% of ericaceous species; this is 0.5% of total methane emissions from human activity across Europe (24,755 kt.y(-1)), and a reduction in methane emissions of 63.8 kt.y(-1) against the same deer and sheep populations, if assumed to consume a grass-only diet. We suggest the implementation of carbon credits as a measure to value the relevance of heathland systems to promote biodiversity and its potential contribution to reduce methane emissions in ruminant grazing systems. (C) 2020 Published by Elsevier B.V.

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.

decomposition of animal and human cadavers can lead to comprehensive chemical and biochemical changes in soil, which can provide helpful information for the analysis of archaeological and crime scenes. The current study focused specifically on the concentration and distribution patterns of steroids in soil with the aim of assessing their suitability for demonstrating the presence of human decomposition products. Soil samples were collected from a forensic site where a human corpse had been lying on the soil surface for 18 days. Total organic carbon (TOC) and steroid concentrations were analysed in soil samples taken from beneath the body at the time the corpse was removed and also one year later. High concentrations of TOC and steroids were detected close to the soil surface at the time the body was removed, and the concentration of cadaver-derived cholesterol was considerably higher than that of the plant-derived sitosterol. The soil beneath the cadaver contained higher concentrations of faeces-derived 5 beta-stanol coprostanol than the control soil. Different concentrations and distribution patterns of steroid compounds in the soil upon removal of the corpse and one year later indicate that significant transformation, degradation and translocation processes had taken place during this period of time. The depth distribution of steroids beneath a decomposing body along with changes in the human steroidal fingerprints in soil over time were found to provide information that could help resolve forensic and archaeological issues. The combined analysis of tissue steroids (cholesterol), the reduction product of cholesterol in soil (5 alpha-cholestanol) as well as faecal steroids (coprostanol, 5 beta-stigmastanol and their conversion products epicoprostanol and epi-5 beta-stigmastanol) represent excellent additions to the forensic toolbox for determining whether a soil of interest has ever been affected by a human cadaver or not. (C) 2018 Elsevier Ltd. All rights reserved.

We studied the changes in concentration and relative abundance of human-derived fatty acids (FAs) in soil over a period of one year. The study is based on analysis of soil underneath a human body that lay on the soil surface for 18 days before it was discovered. Soil samples were taken when the body was removed, and also 358 days later. Large amounts of the total FA concentration at the start of the measurement period were still present one year after the removal of the body. The FA profile suggested that extensive saturated FA reduction occurred during the first 18 days after deposition. 10-Hydroxystearic acid and FA salts, which are characteristic of adipocere, were abundant in all soil samples from beneath the body. Myristic acid and 10-hydroxystearic acid concentration declined over time. It was therefore concluded that FAs, still detectable one year after the temporal deposition of a body, can serve as an important indicator of human decomposition fluid in soil. Furthermore, the study suggests that in-situ processes such as the formation of adipocere promoted the preservation of human-derived FAs over the period of the study. (C) 2017 Elsevier Ltd. All rights reserved.

In a forensic investigation, the analysis of earth materials such as sediments and soils have been used as evidence at a court of law, relying on the study of properties such as color, particle size distribution and mineral identification, among others. In addition, the analysis of the organic composition of sediments and soils is of particular value, since these can be used as complementary independent evidence to the inorganic component. To investigate the usefulness of organic indicators in sediment characterization and discrimination, seventy-seven samples were collected during a period of one year in two river beaches located at the southern bank of the Douro River estuary in the North of Portugal. Isotopes of total carbon, pollen and plant wax-marker analyses were performed. In both beaches, an increase of the organic matter concentrations was noticeable, moving landward, related with the higher cover of associated plant material. The results obtained showed that the combination of all the techniques adopted showed a clear discrimination between samples from the two beaches, and also showed a differentiation of samples in relation to distance from the river in both beaches. The results also show that seasonality in these beaches was not a determining factor for discrimination, at the times considered. In addition, the effects of time was not marked.

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.

Disturbance from human recreational activities may affect the nutrition of free-ranging herbivores due to trade-offs between feeding in preferred habitats and perceived predation risk. To test this hypothesis, we estimated diet composition for red deer in the Scottish highlands in spring, when recreational activity tends to be high, and in winter when it is lower. We analysed faecal samples from three habitat types (grassland, heather moorland and woodland) collected at sites close to a busy track (disturbed) and at a distance from it (less disturbed). The diet consisted of 39% grasses, sedges, herbs and rushes (GSHR) and 58% Calluna vulgaris and Erica spp. (heather) in spring, compared with 14% grasses and 77% heather in winter, with small quantities of Vaccinium spp. (berry) and Pinus sylvestris (tree) in both seasons. In spring, faeces from disturbed grass and woodland sites indicated a diet with less GSHR and more heather and tree than faeces from less-disturbed sites, which could be due to an increased need for vigilance in exposed grassland and the need to seek cover. In contrast, faeces from all disturbed sites in winter indicated a diet with more GSHR and less heather than faeces from less-disturbed sites. This could be due to a seasonal decline in recreation and increase in hunting activity reversing the disturbance levels at the different sites, since hunting is not normally carried out in areas used by the public for recreation. We conclude that there may be nutritional benefits to deer of reducing disturbance near open grassland.

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. Copyright (C) 2011 John Wiley & Sons, Ltd.

Much of the forensic comparison of soils has focused oil mineralogy at the expense of soil organic components. This chapter discusses flow the nature of soil organic matter could be a useful tool in forensic comparison of, soil. The chemical diversity and resistant nature of most plant-derived wax markers in soil result in the potential to provide robust profiles that vary with the characteristics of the vegetation that contributed to its formation. The epicuticular wax of most plants contains mixtures of Ion-chain aliphatic lipids, Including hydrocarbons (mainly n-alkanes). long-chain fatty alcohols and fatty acids. The composition of these wax compounds differ between plant species, and these patterns persist in the soil. Plant wax markers may have value in characterising soils for forensic investigation where they could be used to indicate vegetation characteristics of an unknown sample, or for evaluative comparison of a 'questioned' evidence sample to that of a known scene of crime/reference site. We conducted a pilot study in North East Scotland to determine whether individual patches of garden planting bed soil could be discriminated from one another based on their plant wax marker profiles. Results indicated that both n-alkane and long-chain fatty alcohol profiles in garden soils were generally specific to the individual planting bed. The soils from the planting beds could be discriminated from one another. with the exception of two beds from within the same garden which differed little in broad-scale vegetation type. These results demonstrate the potential of plant wax markers in discriminating patches of soil at a scale relevant to forensic evaluative comparison. Further studies are required on soils and vegetation from I wider population of gardens and vegetation types. to extend the application of these plant wax markers to facilitate investigative approaches in potentially indicating plant community characteristics associated with an unknown source.

This paper explores the interaction of grazing pregnant dairy cattle (lactating and non-lactating) with contaminated pasture. Behavioural responses were divided into investigative and ingestive grazing events. Patches of sward were artificially contaminated with slurry (containing faeces and urine) and were used to test the behavioural interactions; faecal collection bags ensured no further contamination occurred throughout the two 1-week periods of the experiment. Investigative behaviour was quantified using an active transponder system with aerials buried around the contaminated patches. Ingestive behaviour was quantified using n-alkane markers sprayed on the contaminated patches. The cattle preferentially grazed the uncontaminated sward and this created a trade-off between the non-contaminated and contaminated areas. There was a large variation between cows in both investigative and ingestive behaviour, consequently the effect of physiological state was not significant (P>0.05). As the proportion of faecally contaminated to uncontaminated pasture increased, there was a significant (P<0.05) increase in investigative activity as quantified by both the total number and total duration of contacts in contaminated patches. The changes in proportion of contaminated to uncontaminated sward resulted in a greater proportion of contaminated sward selected in the diet on day 4 than day 1. The results from this study demonstrate investigative and ingestive behaviour can be reliably quantified by combining an active transponder system with sprayed n-alkanes, thus providing a useful tool for future studies.

Understanding of plant interactions is greatly limited by our ability to identify and quantify roots belonging to different species. We proposed and compared two methods for estimating the root biomass proportion of each species in artificial mixtures: near-infrared reflectance spectroscopy (NIRS) and plant wax markers. Two sets of artificial root mixtures composed of two or three herbaceous species were prepared. The proportion of root material of each species in mixtures was estimated from NIRS spectral data (i) and the concentration patterns of n-alkanes (ii), n-alcohols (iii), and n-alkanes +n-alcohols combined (iv). For each data set, calibration equations were developed using multivariate statistical models. The botanical composition of root mixtures was predicted well for all the species considered. The accuracy varied slightly among methods: alkanes < alcohols = alkanes + alcohols < NIRS. Correlation coefficients between predicted and actual root proportions ranged from 0.89 to 0.99 for alkanes + alcohols predictions and from 0.97 to 0.99 for NIRS predictions. These two methods provide promising potential for understanding allocation patterns and competitive interactions.

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.

This article introduces a hierarchical model for compositional analysis. Our approach models both source and mixture data simultaneously, and accounts for several different types of variation: these include measurement error on both the mixture and source data; variability in the sample from the source distributions; and variability in the mixing proportions themselves, generally of main interest. The method is an improvement on some existing methods in that estimates of mixing proportions (including their interval estimates) are sure to lie in the range [0, 1]; in addition, it is shown that our model can help in situations where identification of appropriate source data is difficult, especially when we extend our model to include a covariate. We first study the likelihood surface of a base model for a simple example, and then include prior distributions to create a Bayesian model that allows analysis of more complex situations via Markov chain Monte Carlo sampling from the likelihood. Application of the model is illustrated with two examples using real data: one concerning chemical markers in plants, and another on water chemistry.

Abstract Resistant compounds associated with vegetation have potential for understanding and uniquely describing soil. Although much of the forensic identification of soils has focused on the mineral component, this study illustrates how the origin of the organic component can be a useful tool in soil identification. The epicuticular wax of most plants containts mixtures of hydrocarbons (mainly n-alkanes) and plant species differences are persistent. Evidence from three separate studies is compiled to show the validity of this approach. In the first example, on upland grassland vegetation, the n-alkane pattern of the soil at one site reflected that of the overlying grass, whereas at another site, it reflected that of the previous vegetation, heather. In the second study, n-alkane analysis data indicated the presence of heather in a buried horizon, matching independent evidence from pollen identification. The third study was one covering the whole of Scotland, using an unbiased grid-sampling strategy. Results show that the patterns in the soil n-alkane profiles reflected the overlying vegetation. Where this was not the case, the profiles matched previously grown vegetation. Such biomarker information, derived from plant wax signatures, coupled with soil spatial information, has potential in the unique identification of soils.

Knowledge of the feeding ecology of mammalian herbivores is fundamental in predicting their responses to habitat change. Where native woodlands are newly established in open moorland, the extent to which trees form part of the diet of mountain hares (Lepus timidus) is unknown. This information is necessary for predicting the potential effects of mountain hare browsing on woodland establishment. The n-alkanes and a long-chain fatty alcohols found in the cuticular wax of diet plants and faeces (N = 240) were used as markers to estimate the composition of the diet of mountain hares in an area of moorland with newly established Pinus sylvestris and Betula pubescens woodland. During winter, the diet of mountain hares was dominated by Calluna vulgaris, but there was a seasonal shift to a diet dominated by grasses, sedges, and rushes in summer. Pinus sylvestris and B. pubescens were minor dietary components in all seasons. A higher proportion of grasses, sedges, and rushes was found in the diet of lactating females. Results suggest that when an alternative browse species such as C. vulgaris is widely available, mountain hares may not have a large impact on the establishment of native woodland. The dietary results from this study are in broad agreement with those from previous studies using other techniques.

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.

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.