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4. Davis_STP_Sterols

Description

Wastewater containing human sewage is often discharged with little or no treatment into the Antarctic marine environment. Faecal sterols (primarily coprostanol) in sediments have been used for assessment of human sewage contamination in this environment, but in situ production and indigenous faunal inputs can confound such determinations. Using GC-MS profiles of both C27 and C29 sterols, potential sources of faecal sterols were examined in near shore marine sediments, encompassing sites proximal and distal to the wastewater outfall at Davis Station, Antarctica. Faeces from indigenous seals and penguins were also examined. Several indigenous species faeces contained significant quantities of coprostanol but not 24-ethylcoprostanol, which is present in human faeces. In situ coprostanol and 24-ethylcoprostanol production was identified by co-production of their respective epi-isomers at sites remote from the wastewater source and in high total organic matter sediments. A C29 sterols-based polyphasic likelihood assessment matrix for human sewage contamination is presented which distinguishes human from local fauna faecal inputs and in situ production in the Antarctic environment. Sewage contamination was detected up to 1.5 km from Davis Station.

Abstract from - Rhys Leeming, Jonathan S. Stark, James J. Smith (2014), Novel use of faecal sterols to assess human faecal contamination in Antarctica: a likelihood assessment matrix for environmental monitoring.

Purpose

Sumary of methodology from - Rhys Leeming, Jonathan S. Stark, James J. Smith (2014), Novel use of faecal sterols to assess human faecal contamination in Antarctica: a likelihood assessment matrix for environmental monitoring.

The sewage effluent examined in this study came from Australia's Davis station situated on the coast of the ice-free Vestfold Hills (68.5746 S, 77.9671 E). Effluent at time of sampling consisted of macerated, un-treated sewage, which was being discharged into the intertidal zone near the station. Samples of approximately 100 ml of effluent were collected in acid-washed amber glass bottles from the end of the outfall line and stored at 1-5 degrees C in the dark. Effluent sub-samples were filtered through 45 mm Whatman GFF (nominal pore size 0.7 microns; heated at 450 degrees C overnight prior to use) and kept frozen until extraction.

Sediment cores were collected by divers from 25 sites around the Vestfold Hills using acrylic core sleeves and caps, at increasing distances from the sewage outfall (Fig. 1). Cores were subsequently stored frozen at -18 degrees C. Core sub-sampling was undertaken while cores were still frozen. For grain size analysis the outer 5 mm edge of the core was removed with a scalpel blade, placed in a clean, dried pre-weighed beaker, weighed, dried at 45 degrees C, reweighed, 2 mm sieved, and any residual sediment in the beaker weighed. The less than 2 and greater than 2 mm fractions were separately collected and weighed. A 5 g sample of the less than 2 mm fraction was used for grain size analysis, using a Malvern Mastersizer optical bench with Hydro 2000g accessory, and particle refractive index of 1.544 (quartz) and 1.333 (water). Total organic carbon (TOC) analysis was carried out using a 2 g homogenised wet sediment sub-sample weighed into a pre-combusted crucible, dried at 105 degrees C and reweighed before determination of total carbon by loss on ignition (LOI) at 550 degrees C for 4 h. For sterols analysis the top 1 cm was sliced from each core, and an approximately 3 g sediment sub-sample collected from the centre of the slice and placed in a pre-cleaned, pre-weighed glass vial. Weddell, Elephant, and Leopard seal, as well as Adelie Penguin faeces were aseptically collected into sterile 50 mL polycarbonate centrifuge tubes and kept refrigerated at 1-5 degrees C. An additional Leopard seal sample was also obtained from Taronga Zoo, Sydney for verification of that species sterol profile. All samples were kept frozen until extraction. Originally, sub-samples of animal faeces were composited and then analyzed. Subsequently, some individual Weddell seal faecal samples were analyzed to investigate their contribution(s) to differences between composites. Total organic carbon was estimated by loss on ignition (LOI) by heating dried sub-samples to 450 degrees C overnight and re-weighing samples.

All samples were extracted using a modified Bligh and Dyer solvent mixture (Bligh and Dyer 1959) utilizing dichloromethane (DCM) instead of chloroform. For each sample, 25 mL 1:2:0.8 v/v/v dichloromethane/methanol/Milli-Q H2O was added, shaken vigorously and left to extract for a minimum of 12 h. Total lipid extracts were recovered by breaking phase using 10 mL 1:1 DCM/NaCl-saturated Milli-Q H2O. Samples were shaken vigorously then centrifuged at 1000 rpm for 5 min. The solvent phase was pipetted from below the water layer into a round-bottomed flask. The samples were then re-extracted with another 5 ml DCM, and added to the corresponding round-bottom flask. Solvent from extracts was evaporated under reduced pressure using a rotary evaporator and water-bath at 35 degrees C. A small amount of methanol (2 mL) was added to help azitrope residual water. Total extracts were concentrated to near dryness (less than 0.5 mL), and transferred to a glass screw-cap 10 mL centrifuge tube using 3 X 1.5 mL DCM. Total extracts were reduced to near-dryness under N2 at 50 degrees C.

Saponification was carried out using ~ 2 mL 5% KOH in methanol/ Milli-Q H2O (4:1 by vol), added to the total extracts from sediments. Six mL 5% KOH in methanol/H2O (4:1 by vol) was added to total extracts from effluent and faecal samples. Tubes containing extracts were flushed with N2 gas and heated to 80 degrees C for 2 h. After cooling, 3 mL Milli-Q H2O and 2 mL 4:1 Hexane:DCM were added, extracts mixed using a vortex mixer and centrifuged at 2000 rpm for 2 min. The top solvent layer was transferred to borosilicate glass GC-vials. This extraction of the saponified neutral extracts (TSN) was repeated twice more for each sample. The combined TSN extracts were dried under N2 and derivatised using 100 micro-L bis(trimethylsilyl)trifluoroacteamide (BSTFA) containing 1% TMCS (trimethylchlorosilane; Alltech, Deerfield USA). Mixtures were heated to 60 degrees C for 60 min., BSTFA subsequently evaporated under N2, and TSN extracts prepared for GC analysis in DCM.

Gas chromatography with flame ionization detection (GC-FID) was initially performed using a Varian 3800 controlled by Galaxie chromatography software (Agilent Technologies) to establish the optimal concentrations to run samples for subsequent GC-MS analysis. Sterols were verified and quantified by GC-MS using a ThermoQuest Trace DSQ bench top MS fitted with direct capillary inlet. Data were acquired in selective ion monitoring (SIM) mode and processed using Xcalibur software. Both GC's were equipped with 50 m x 0.32 mm i.d. cross-linked 5% phenyl-methyl silicone (HP5) fused-silica capillary columns with He carrier gas and temperatre and operating conditions as described in Leeming (1996). Sterol fractions were quantified using 5?(H)-cholan-24-ol (Chiron AS, Norway) as the internal standard added prior to extraction. Peak identifications were based on retention times relative to standards for both GC-FID and GC-MS analysis (Table 2). Assessment of the method recoveries can be found in Borjesson et al. (1998)

Nonclementure: For brevity in the text, the term "5B/5a stanol ratio" refers equally to the ratios of coprostanol/5a-cholestanol and 24-ethylcoprostanol/24-ethylcholestanol unless a C27 or C29 prefix is given in which case it refers specifically to the former or latter ratio. Coprostanol is the C27 homologue of the C29 stanol 24-ethylcoprostanol just as cholesterol and 24-ethylcholesterol and epi-coprostanol and 24-ethyl-epi-coprostanol are also respective homologue pairs. Epi-isomers have the hydroxyl (OH) group in the a-configuration rather than B-configuration at carbon position three. Unless epi is stated, the stanol referred to is the non-epimerized 3B-OH stanol. When epi-isomers are compared to 5B-stanols, the relationship is always within the C27 or C29 group respectively. Cholesterol is the precursor to the suite of C27 stanols just as 24-ethylcholesterol is the precursor to the C29 stanols. Where error bars are shown, n=2.

Access

These data are publicly available for download from the provided URL.

Temporal Coverages

• Start date: 2009-11-01 - Stop date: 2010-04-01

Spatial Coverages

Science Keywords

• EARTH SCIENCE > BIOSPHERE > ECOSYSTEMS > MARINE ECOSYSTEMS > COASTAL
• EARTH SCIENCE > BIOLOGICAL CLASSIFICATION > ANIMALS/VERTEBRATES > BIRDS > PENGUINS
• EARTH SCIENCE > BIOSPHERE > ECOSYSTEMS > MARINE ECOSYSTEMS > BENTHIC
• EARTH SCIENCE > HUMAN DIMENSIONS > ENVIRONMENTAL IMPACTS
• EARTH SCIENCE > HUMAN DIMENSIONS > ENVIRONMENTAL IMPACTS > SEWAGE DISPOSAL
• EARTH SCIENCE > HUMAN DIMENSIONS > HUMAN SETTLEMENTS > COASTAL AREAS
• EARTH SCIENCE > OCEANS > MARINE SEDIMENTS
• EARTH SCIENCE > OCEANS > MARINE SEDIMENTS > SEDIMENT CHEMISTRY

Use Constraints

This data set conforms to the CCBY Attribution License
(http://creativecommons.org/licenses/by/4.0/).

Please follow instructions listed in the citation reference provided at http://data.aad.gov.au/aadc/metadata/citation.cfm?entry_id=Davis_STP_Sterols when using these data.