Workshop Report: SF6 tracer technique guidelines

9-10 March 2011 Palmerston North, New Zealand

Draft Report June 2011 Keith Lassey NIWA Client report WLG2011-29

GLOBAL RESEARCH ALLIANCE International Workshop 9-10 March 2011 Palmerston North, New Zealand Compiled by Keith Lassey1 1 NIWA, P.O. Box 14-901, Kilbirnie, Wellington, New Zealand. Phone +64-4-386 0300
Fax +64-4-386 0574

Funding sources New Zealand Government to support the goals and objectives of the Global Research Alliance on Agricultural Greenhouse Gases Disclaimer This report has been commissioned by the New Zealand Government to support the goals and objectives of the Global Research Alliance on Agricultural Greenhouse Gases. While every effort has been made to ensure the information in this publication is accurate, the Global Research Alliance does not accept any responsibility or liability for error of fact, omission, interpretation or opinion that may be present, nor for the consequences of any decisions based on this information. Any view or opinion expressed does not necessarily represent the view of the Global Research Alliance.

Contents
Background Workshop participants Workshop Agenda Outcomes Table of contents for the Guidelines Proposed content/outline for each chapter in the guidelines 1. Introduction 2. Purpose of Manual 3. Overview of the SF6 tracer technique and its evolution 4. Pre-experimental planning: how many animals are needed? 5. Permeation tubes: the source of SF6 6. Breath sampling systems 7. Special considerations for fistulated animals 8. Backgroundair sampling 9. Analyses of breath samples 10. 11. 12. 13. 14. 15. 16. 17. 18. Next steps Acknowledgements Glossary of abbreviations and terms References Appendix 1. Workshop presentations Workshop discussion SF6 permeation tubes Breath sampling apparatus Background air sampling Animal management and feed intake Data quality assurance and quality control What SF6 detail should be reported? Future issues and potential improvements Unresolved puzzles [aka Enigmas] Information available on web Acknowledgements References Annex 6 6 7 10 10 11 11 11 12 12 13 13 14 14 15 16 18 18 18 18 18 19 19 19 19 19 20 20 22 22 22 23 24

Sample handling and gas analysis Gas analysis Animal management, diet and feed intake Estimation of CH4 emission rates, yields, and emission factors Other considerations

24 25 25 26 26

Statistical considerations for pre-experimental planning and data analysis 26 Acceptance and inclusion in guidelines What SF6 tracer technique detail should be reported? Future issues and potential improvements Enigmas Information available on the internet 27 27 27 27 28

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Background
The SF6 tracer technique pioneered by Johnson et al. (1994) has been used widely for determining methane (CH4) emission rates by individual ruminant animals. It is a technique available for robustly and accurately determining CH4 emissions by individual grazing animals. The New Zealand Government in support of the goals of the Global Research Alliance for Agricultural Greenhouse Gases funded a workshop held in Palmerston North, 810 March 2011 to bring together key researchers and organisations to discuss the diverse and innovative deployments of the SF6 tracer technique, various technical aspects of the technique and the techniques strengths and weaknesses. The goals of the workshop were to: 1. Discuss and detail the content of the proposed guidelines for the measurement of CH4 emissions by individual animals using the SF6 tracer technique. 2. Allocate responsibilities to the authors of each section of the guidelines 3. To commence drafting key sections of the guidelines, focussing on those sections that benefit from face-to-face consultation or where participants have individual critical expertise 4. Develop an agreed timeframe that would assure completion of draft guidelines by the end of June 2011. This report details the draft outline of the chapters and their content as it was developed at the workshop and identifies the authors for each chapter. Next steps are to build on the chapter outline proposed in the workshop report after consideration of the objectives of the guidelines, their target audience and how the writing process will ensure that the final guidelines will be relevant and accessible to the target audience.

Workshop participants
Personal invites to attend the workshop were sent to all scientists currently known to work in this area of science; of particular importance when selecting the participants was their experience applying innovative approaches to the SF6 tracer technique. In total 17 participants attended the workshop from 7 countries (Argentina, Australia, Brazil, Canada, France, Ireland, and NZ), each brought considerable individual experience with the technique and interpretation of results. Table 1 lists workshop participants, alphabetically by country. One participant (Alan Iwaasa) was unable to participate in person, but supplied presentations and expressed a keenness to be involved in the overall project. Table 1:
Participant Jos Gere

Workshop participants, with contact details updated where necessary.

Host institution Grupo de Fisicoqumica IFAS-UNCPBA, Tandil, ARGENTINA Ambiental, Email address jgere@exa.unicen.edu.ar

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Chris Grainger Roger Hegarty

Peter Moate Richard Williams Alexandre Berndt Alan (absent) Iwaasa

Ccile Martin Tommy Boland

Matthew Deighton

Keith Lassey Ross Martin German Molano Cesar Pinares-Patio Natasha Swainson Ben Vlaming Garry Waghorn

(formerly) Dept Primary Industry, Ellinbank, Vic., AUSTRALIA Dept Animal Nutrition, Environmental and Rural Sciences, University of New England, Armidale, NSW, AUSTRALIA Dept Primary Industry, Ellinbank, Vic., AUSTRALIA Dept Primary Industry, Ellinbank, Vic., AUSTRALIA Brazilian Agricultural Research Corporation, Embrapa, So Carlos, So Paulo, BRAZIL Semiarid Prairie Agricultural Research Centre, Agriculture and Agri-Food, Swift Current, SAS, CANADA INRA, Unit de Recherches sur les Herbivores, Saint-Gens-Champelle, FRANCE School of Agriculture, Food Science and Veterinary Medicine, University College Dublin, IRELAND Dept Animal and Bioscience Research, Animal & Grassland Research and Innovation Centre, Teagasc Moorepark, Fermoy, Co. Cork, IRELAND NIWA Ltd, Wellington, NZ NIWA Ltd, Wellington, NZ AgResearch Ltd, Palmerston North, NZ AgResearch Ltd, Palmerston North, NZ AgResearch Ltd, Palmerston North, NZ (formerly) AgResearch Ltd, Lincoln, NZ Dairy NZ, Hamilton, NZ

chrisgraingercg@gmail.com roger.hegarty@une.edu.au

peter.moate@dpi.vic.gov.au richard.williams@dpi.vic.gov.au alberndt@cppse.embrapa.br alan.iwaasa@agr.gc.ca

cecile.martin@clermont.inra.fr Tommy.Boland@ucd.ie

Matthew.Deighton@teagasc.ie

k.lassey@niwa.co.nz r.martin@niwa.co.nz german.molano@agresearch.co.nz cesar.pinares@agresearch.co.nz natasha.swainson@agresearch.co.nz ben@agrivet.co.nz garry.waghorn@dairynz.co.nz

Workshop Agenda
Tuesday 8 March 9:15 10:00. Welcome and Introduction 10:00 10:20. Morning tea 10:20 10:40. Overview of the SF6 technique [Keith] Principles of a tracer, and choice of SF6 10:40 12:00. SF6 PERMEATION TUBES [Keith] Permeation tubes in NZ: their fabrication, charging with SF6, and performance monitoring [Peter] Permeation tubes at DPI, Vic, and SF6 release kinetics [Tommy,Matthew]Permeation tubes in Ireland [Ccile] Permeation tubes in France [Alexandre] Permeation tubes in Brazil, esp. effect of washer size [Roger] Permeation tubes with very high release rates [others?] Permeation tubes as developed at home research institution 12:00 12:50. LUNCH

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12:50 15:00. [Keith] [Richard] [Jos?] [others?] [Ross/Keith] [Ccile] [Peter] [others?]

BREATH SAMPLING APPARATUS Halter with capillary-based flow limiter, inlet design to maximise breath interception efficiency Variations on capillary systems, such as crimped capillary; cleaning and maintenance of sampling equipment The Argentine ball-and-socket system for limiting flow rate Any further variations on passive breath collection apparatus? Pumped system to deliver sample into a bag, canister (evacuated), or even direct to an analyzer Sampling from fistulated animals Rumen headspace gas composition, from fistulated and non-fistulated cows Any special considerations for sampling from a fistulated animal, whether problems with gas loss at fistula or opportunities to sample directly from rumen headspace

15:00 15:20. 15:20 16:00. [Keith]

AFTERNOON TEA BACKGROUND AIR SAMPLING Introduce the issues: selecting background site(s), and how to cope with spatial gradients in background [Richard] Experiences with spatially variable indoor backgrounds [others?] Other experiences with background siting caused by spatially variable backgrounds 16:00 17:00. GAS ANALYSIS: SAMPLE HANDLING [Keith] The Johnson et al. approach: over-pressure by dilution [Ross] Extracting an under-pressured sample: a double-ended piston approach developed by NIWA [Ross] Pumping a sample from a bag [Richard, others?] Using vials for intermediate storage Wednesday 9 March 9:00 9:10. 9:10 10:00. [Ross] [Ross/Keith] 10:00 10:20. 10:20 11:10. [Keith] [others?] 11:10 12:00. [Roger?] 12:00 12:50. 12:50 15:00. [Garry et al.] [Csar et al.] [Ccile] 15:00 15:20. 15:20 17:00. [Csar et al.] SYNOPSIS OF DAY 1 GAS ANALYSIS: GAS CHROMATOGRAPHY Plumbing configurations of GC, examples of chromatograms Detector (FID, ECD) responses to CH4, SF6, choice of gas standards in NZ MORNING TEA GAS ANALYSIS: GAS CHROMATOGRAPHY (CONTINUED) Inferring CH4, SF6 mixing ratios from chromatograms: the NIWA procedure other experiences with GC analyses GAS ANALYSIS: OTHER TECHNOLOGIES Gas analysis other than by GC eg, IR or FTIR spectroscopy LUNCH ANIMAL MANAGEMENT AND FEED INTAKE Determination of feed intake under grazing and indoor situations, and of feed properties (eg, NIR techniques) Management of animals and feed under grazing and indoor situations, including insertion of permeation tubes Management of animals and analyses AFTERNOON TEA TOUR OF AGRESEARCH RUMINANT METHANE FACILITY Will lead a tour of AgResearch facility; transport will be provided to AgResearch and return

Thursday 10 March

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9:00 9:10. 9:10 10:00. [Keith] [Keith] [anyone?] 10:00 10:20. 10:20 12:00. [Keith] [all] 12:00 12:50. 12:50 15:00. 15:00 15:20. 15:20 16:30. 16:30 17:00.

SYNOPSIS OF WORKSHOP TO DATE ESTIMATION OF CH4 EMISSION RATES, CH4 YIELDS, EFS Equations linking CH4 and SF6 mixing ratios, in both sample and background, in combination with SF6 permeation rate with estimated CH4 emission rate. Definition of CH4 yield both in units such as g(CH4)/kg(DM) and in energy terms, % of GEI (IPCC definition). Any other perspectives MORNING TEA DRAFTING OF MANUAL How to proceed from here Participants to please share key papers with others (with scanning facilities available if necessary), and make available photo-files intended for manual. LUNCH DRAFTING OF MANUAL, CONTINUED AFTERNOON TEA DRAFTING OF MANUAL, CONTINUED WRAP-UP

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Outcomes
Table of contents for the Guidelines
A draft outline of the proposed guidelines was prepared and supplied in advance of the workshop to the participants. The workshop sessions were set-up to allow participants to give scientific presentations that focused on the topics proposed to be included in the guideline. Each participant summarised the current understanding of the topic and in-depth discussions followed each session where participants considered i) agreed minimum requirements, ii) site specific requirements or iii) evolving requirements, for each topic. The proposed outline of the guidelines and key issues to be covered was revised as a result of the workshop and authors/co authors were nominated and agreed. Following these presentations and discussions, the participants agreed on a revised Table of Contents and the authors for the Guidelines (see below). The participants also agreed that the Guidelines should be as succinct and clear as possible, so that they would be of use to a wide audience. Agreed table of contents:
Chapter Title
Introduction Purpose of the manual Overview of the SF6 tracer technique and its evolution Pre-experimental planning: how many animals are needed? Permeation tubes: the source of SF6

Lead author
Keith Lassey Keith Lassey Keith Lassey Chris Grainger

Author (s)

Roger Hegarty Ben Vlaming, Natasha Swainson, Peter Moate, Tommy Boland, Cesar Pinares Peter Moate, Richard Williams, Matthew Deighton, Ccile Martin, Roger Hegarty, Alex Berndt, Alan Iwassa Peter, Richard, Jos, Ccile, Cesar, Ross, Alan Ccile Martin, Alan Iwassa, Garry Waghorn, Alex Berndt Mathew Deighton Cesar Pinares, German Molano, Ccile Martin, Tommy Boland, Alex Berndt, Roger Hegarty Keith Lassey, Mathew Deighton, Ben Vlaming Richard Hegarty, Ccile Martin, Garry Waghorn, Natasha 10 of 28

Keith Lassey

Breath sampling systems

Mathew Deighton

Special considerations fistulated animals

for

Tommy Boland Peter Moate, Richard Williams Keith Lassey and Ross Martin Garry Waghorn

Background air sampling Analysis of breath samples Animal management and feed intake Estimation of methane emission rates and methane yield Acceptance and inclusion guidelines International Workshop Report

Peter Moate

Cesar Pinares

What SF6 detail should be reported? Issues and potential improvements Information available on the web

Richard Williams All is this a separate section or a part of all chapters Peter Moate

Swainson, Alan Iwassa, Alex Berndt, German Molano Peter Moate

Proposed content/outline for each chapter in the guidelines

The participants agreed that because of the need for reliability of information about the SF6 tracer technology, the development of the guidelines requires more than a literature review and summary of currently adopted approaches. Instead, a critical analysis and thorough discussion of all the issues involved with the methodology is required in each relevant chapter to ensure the guidelines are credible, pass the scrutiny of the wider science community, and when adopted, provide the best possible emissions estimates. Consideration will be given to grey literature, unpublished material and peer reviewed publications during the analysis.

1.

Introduction

[Lead: Keith Lassey: NIWA] [est. 2pp] Short introduction to the topic: why measure ruminant methane; what is the SF 6 tracer technique; and under what circumstances is it implemented in preference to (say) enclosure techniques? Mention that this manual was the product of an international workshop (place and date) with authorship comprising active participants. The manual supplies a composite of the varied proven implementations of the technique that the participants of the workshop are prepared to share for inclusion in this manual.

2.

Purpose of Manual

[Lead: Keith Lassey: NIWA] [est. 2pp] What does this manual seek to achieve? What readership does it try to reach? Basically everything to do with the SF6 technique that will help get new converts to this technique up and running, including photographs as well as text.

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3.

Overview of the SF6 tracer technique and its evolution

[Lead: Keith Lassey: NIWA, supported by Roger] [est. 3pp] Summarise here the main facets of the technique summarised under headings that comprise subsequent chapters. This introduction should give users confidence that irrespective of known limitations and problems with SF6, there are currently no viable alternatives for high-precision measurements from individual animals, so we need to make the best of the method. In particular, cover the following: the principles for operation, constant SF6 emissions over time a short history of the technique citing articles that have been pivotal to the development of the technique; cross-reference Appendix 1 which comprises a list of papers identified in a search of papers relating to the SF 6 technique. report exit points of CH4 from ruminants, and that nose/mouth dominates SF6 physical properties, molecular weight and other characteristics; industrial uses and properties (insulator), increasing atmospheric concentrations. Why is SF6 used for ruminal methane measurements; non toxic, low atmospheric background concentrations, behaviour that makes it a useful marker gas? What is the fate of rumensourced SF6? Why is SF6 better than ethane, for example? Finally, the importance of a system for measuring methane in grazing animals. What deployments of SF6 have been done relative to global SF6 emissions inventory (about 12kg released over 17 yrs from ruminant tracer measurements vs 6800t released annually from all sources worldwide: Williams et al., submitted) and especially its place in the future; especially farmlet trials, and application in regions where use of chambers, tents or laser technology is not possible. This could include hilly or arid regions, and might involve collections over 510 days. The technique is relatively inexpensive and can be applied to more animals than some other measurements. Consideration of other techniques available, cost of technique vs other methods, ease of use versatility, staff capabilities in using technique.

4.

Pre-experimental planning: how many animals are needed?

[Lead: Chris Grainger, supported by Ben, Natasha, Peter, Tommy, Cesar] [est. 5pp] Required animal numbers are affected by the required precision of measurement across a herd being sought and the expected variation associated with different treatments and measurement techniques. Ben Vlaming has tabulated data that summarise animal number requirements and there are comparisons that show variance associated with both the SF6 and chamber measurement techniques. This aspect of the report will be useful for researchers trying to plan experiments, because they will be forced to think about the magnitude of the difference they are trying to detect, and make an appropriate choice of animal numbers, and whether to use SF6 or chambers. May need some statistician input here. Will it cover animal welfare, ethics approval, types of animals, age of animals, etc?
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5.

Permeation tubes: the source of SF6

[Lead: Keith Lassey: NIWA, supported by Peter, Richard, Matthew, Ccile, Roger, Alex, Alan] [est. 9pp] This chapter covers all aspects of permeation tubes, including: Tube design and properties Describe the various design details (diagrams where possible) and how to charge with SF6. How to determine payload. Include description of designs by NIWA (Lassey et al., 2001), Teagasc (role of washer dimension in controlling permeation rate, large capacity tubes); INRA design, NSW high flowrate tubes. Special care should be given to design details, especially washers, Teflon thickness, and the importance of these variables. Refining and standardising permeation tube construction/washer squashing, Teflon thickness Tube calibration Considerations of calibration (oven temperature, use of gloves, scale calibration). Estimation of release rate and usebydate. There seems to be a range in preferred release rates. Make it clear that when approaching useby date, the issues surrounding the accuracy of release rate extrapolation might outweigh the value of the data. Tube performance How do tubes perform over time (eg, Lassey et al., 2001)? A quantitative understanding of how permeation rates change over time could help reduce uncertainty in CH4 emission estimates for serial experiments (eg, using MM kinetics of Moate et al.). Consider supplying details about places of manufacture, and the issues of shipping. Suppliers of the components could be a worthwhile inclusion. Insertion of Tubes How tubes are inserted, consideration of tried and tested methods?

6.

Breath sampling systems

[Lead: Matthew Deighton: Teagasc, supported by Peter, Richard, Jos, Ccile, Cesar, Ross, Alan] [est. 6pp] This covers all systems used to sample breath (respired + eructed) from grazing or housed animals, and to collect those samples for offline or online analysis. These can be divided into breath samples sucked passively into preevacuated canisters, and active pumping to bags or directly to the analyzer, the latter being a rare approach. Subheadings might be as follows:

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Halter, canister and plumbing design This involves design of systems to deliver breath samples into preevacuated canisters, including halter design, from the inlet and its position relative to the nostrils, to the tubing, protection from damage, crimping capillary tubing or alternative flow limiters (Argentine system), presence or absence of inline filter and of valves on yokes. Good designs are available, and points such as minimising the number of joints, use of stainless steel vs. brass, material used for halter construction are all important. Of equal importance are the collection canisters. Some recommendation to use either PVC, stainless steel or aluminium is relevant, and also positioning on the neck or back of the animal is important. Harness construction (Matt Deighton) is very important to ensure happy animals, happy ethics and reasonably content researchers. Information should apply to sheep and cattle, with comments about other species (goats, deer, camelids). Also, consider the importance of design for young (small) and mature individuals, and the impact on data. Active (pumped) sample delivery This delivery can be used for penned animals or otherwise when a uniform delivery rate needs to be assured, or for an automated system that analyzes bags sequentially (eg, NIWA Lung system: Lassey et al. (GGAA2010 special issue in AFST)).

7.

Special considerations for fistulated animals

[Lead Tommy Boland: Teagasc, supported by Ccile, Alan, Garry, Alex] [est. 3pp] Fistulated animals provide both a problem and an opportunity, which may also be influenced by cannula design. The problem relates to the potential for gas leakage at nonuniform rate around the cannula. This should be addressed by any participants who have compared fistulated and nonfistulated animals (Canadian group?). A useful opportunity relates to the potential for gas sampling directly from the rumen headspace. (eg,, INRA). Talk about the merits and demerits of this. Another opportunity relates to being able to remove permeation tubes and replace them with newlycalibrated tubes (could be the same tubes, recalibrated). If rumen fistulation does not affect methanogenesis, why is there an over estimate of methane. One suggestion was that both SF6 and methane could escape from the fistula, so less SF6 came out in the breath, and the methane from hind gut fermentation that is excreted via the lungs, in conjunction with low SF 6 concentrations, resulted in the overestimate. This could be modelled.

8.

Backgroundair sampling

[Lead: Peter Moate or Richard Williams, supported by Matthew] [est. 2pp] This section discusses both the apparatus used to collect background samples (usually identical or similar to that used for breath sampling), and the strategy for selecting
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sites for backgrounds. The latter is especially important when animals are housed indoors or in incompletely ventilated areas where there can be appreciable concentration gradients of CH4 and/or SF6. Need guidance on where to sample background air; height, proximity to an animal, number of samples in a paddock, in an enclosed environment. Also, some information on how to make these decisions. Also, indicate the importance of background gas concentrations on the accuracy of methane estimations, crossreferencing Chapter 11. Should there be canisters dedicated to backgrounds? Recommend if you need dedicated canisters for background collections.

9.

Analyses of breath samples

[Lead: Keith Lassey: NIWA and Ross, supported by Richard, Alex, Alan, Roger] [est. 10pp] This is really getting gas out of a canister into a GC, and GC operation (or alternative analyzer). Information in this section will apply equally for SF6 and methane. Sample extraction How to extract the sample from the canister at subambient pressure or the partially inflated bag. This will traverse the two main options: dilute the canister with gas that is free of CH4 and SF6 (usually N2) to provide superambient pressure; this allows the diluted sample to be either bled directly to the analyzer (the usual approach as advocated by Johnson et al.) or injected into a vial suitable for interim storage and/or transportation to the analytical laboratory pump the sample directly from the canister or bag to the analyzer (NIWAs piston system (being prepared for publication) or Lung system (latter reported by Lassey et al. in AFST/GGAA2010 special issue)). Analysis by gas chromatography This is the predominant means of analysis, using flame ionisation detection (FID) and electron capture detection (ECD) for CH4 and SF6 respectively. Discuss the merits of GC and potential configurations, including: specifications concerning columns, detectors, software columns and detectors in series or parallel GC plumbing such as sample loops and multiport valves what can go wrong? eg, dirty samples; excessive SF6 concentrations, power failure, etc. examples of chromatograms linearity and curvilinearity of FID and ECD respectively availability and choice of gas standards (CH4 and SF6) and their inclusion frequency during analytical runs how to infer CH4 and SF6 mixing ratios from chromatograms
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calibration - gas sources Analysis by other technologies Have nonGC technologies to measure CH4 and/or SF6 mixing ratios proven feasible? Note lessons learned by Rogers group. Intermediate sample storage Discuss special considerations for intermediate storage devices such as vials (eg, Alan, Alex) or bags (Richard). These could be motivated by need for longdistance transport of (sub)samples from field site to lab, by limited availability of canisters, and/or by capability for automated analysis of vialstored samples. Do gas ratios change if samples are stored for a week or a month? Settle on recommended methods for transporting gases.

10. Animal management and feed intake

[Lead: Garry Waghorn, supported by Cesar, German, Ccile, Tommy, Alex, Roger] [est. 10pp] Discuss aspects of animal and feed management that are germane to the SF 6 technique. This includes considerations when the animals concerned will be grazing (preparation of both animals and pasture), and for animals that will be housed or penned (preparation of animals and feeding regime). Discuss also the insertion of permeation tubes (per os, per fistula). Just what should be measured relates to objectives of the research, which need to be clearly defined in advance. This chapter has three components: managing animals, assessing the rate of feed consumption (DMI), and analysing samples of feed. The latter produces feed composition, including in particular the DM content and gross energy (GE) content which are usually closely related. Botanical or chemical properties of the feed are of peripheral interest to the estimation of CH4 yield, but will often be tested for their possible influence on CH4 yield, depending on the research objective. Animal preparation Discuss the handling of animals, including the need to acclimatize the animals to wearing breath collection gear, and considerations relating to dosing with permeation tubes. Determining intake Measurement of feed intake is inessential to the determination of CH4 emission rates using the SF6 tracer technique. However, CH4 emission rates by themselves are not amenable to extrapolation across a herd or flock to assess enteric emissions on a farm to country level. For this purpose, the CH4 yield is much more robust, but it does require knowledge of the intake of feed from which the CH4 is derived. Thus, in many situations intake will be an important component of research, because it will aid
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interpretation. This section will cover (in brief) the opportunities provided by measuring intake i.e. better interpretation of findings, opportunities for improved comparisons between diets, or feeding systems etc. A brief overview should be given of benefits and negative aspects of indoor feeding, the risks associated with estimating intakes from feeding tables, and the challenges (and uncertainties) associated with measuring intakes at grazing. Some mention needs to be made of rates of eating, meals per day and the rates of methanogenesis during (and immediately after) eating, compared to other times, because this may affect overall methane production. This section mustnt become a book, sticking to issues pertinent to use of the SF6 tracer technique for measuring methane. Provide references to literature that discusses feed intake measurement and monitoring in a broader sense without repeating here. The combination of DM (or GE) content plus the assessed rate of feed consumption allows estimation of DMI or GEI. In cases of grazing where feed intakes are notoriously difficult to measure, a third approach would be to apply an energy requirements model to asses GEI directly. All of these options will be addressed: available options to measure intake in penned/housed animals, and to estimate intake by grazing animals measurement of DM and GE content of diet assessment of GEI using energy requirement modelling Diet If intake is measured, it is equally important to measure the composition of what is eaten (say why); Routine analysis will be appropriate in some circumstances, but in other situations more detailed analyses may be appropriate; pectin, lipids nitrate etc, but also measures of digestibility, degradation rate etc. Duration of measurement Impact on accuracy.

11. Estimation of methane emission rates and methane yield

[Lead: Peter Moate, supported by Keith, Matthew, Ben] [est. 5pp] Present the formula for CH4 emission rate as a function of CH4 and SF6 mixing ratios in breath samples and in backgrounds. Discuss the underlying assumptions, the implications of variable or uncertain backgrounds, and the choice of period over which the breath samples are integrated (usually 24 hours, also multiday) and the number of replicate periods. Cite relevance of CH4 per unit liveweight or LW gain. Discuss CH4 yield and its major sources and appropriate means of quantification of uncertainty, and how it becomes a useful basis for estimating EFs, noting that IPCC Tier 1 EFs for cattle are computed this way.

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12. Data quality assurance and quality control

[Lead: Cesar Pinares, supported by Richard, Ccile, Garry, Natasha, Alan, Alex, German] [est. 4pp] QA/QC of SF6 data: how to recognise crook data (criteria?). Distinguish methane emissions and estimated methane emissions. Use IPCC standards here.

13. What SF6 detail should be reported?

[Lead: Richard Williams, supported by Peter] [est. 2pp] Make recommendations about what should be reported in publications and reports. These include following properties of permeation tube used in study: age since charged with SF6, SF6 payload, SF6 release rate, how tubes were calibrated and for how long, location of background collections and background magnitudes (CH4 and SF6).

14. Future issues and potential improvements

[Lead: all leads of other chapters] [est. 2pp] Ensure these are addressed in headings above where appropriate: Could recalculate old data based on Peters MichaelisMenton kinetics, enabling retrospective finetuning of permeation rates. This may lessen the variance within data sets and remedy overestimates of emissions. Figure out if changes to flow rates over time are proportionately greatest for tubes with high permeation rates Intraruminal temperature: does it matter?

15. Unresolved puzzles [aka Enigmas]

[Lead: all] [est. 1pp] Relationships between SF6 release rate and CH4 emission estimates (Ben, Cesar in peerreviewed papers) that is not seen by Matthew (but was Matthews experiment designed to detect it or capable of detecting it?) Much lower methane yields from sheep fed chicory or white clover measured by SF6 (1216 and 16.7 g/kg DMI) compared to chambers (2125 g/kg DMI). What is the reference to this (Garry)? How to explain much greater variation between animals when yield (with measured intakes) is measured by SF6 with 4 days of measurements, than chambers. Peter mentioned couching distribution. Matts use of better, stainless steel, connections to prevent leakage around capillary tubes reduced estimates of methane production and he doesnt know why.

16. Information available on web

[Lead: Peter Moate] [est. 1pp] Include crossreferences to various SOPs, Codes of Practice.
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17. Acknowledgements 18. References

These are references cited in this document. Use the AFST reference style. They will likely all be merged by the editor into a single alphabetised list rather than by chapter. It would be helpful if all authors could use Endnote in their individual contributions, or export their final reference list into an Endnote-compatible format.

19. Annex
This annex will be a list of citations of all known papers in the literature that report on the SF6 tracer technique. The list has been made available to workshop participants as a separate file, inviting participants to add to the list. As of 30 June 2011, the list comprises 94 references.

Next steps
The workshop participants agreed that the development of standardised guidelines is urgently needed to provide consistency, transparency and, probably most importantly, reliability of the measured emissions. Comprehensive guidelines will provide potential users of the SF6 tracer technique with a set of best practice guidelines drawn from the experiences of research groups around the world who have deployed the technique in diverse and innovative ways. The guidelines will help facilitate greater international uptake of this low cost method for obtaining CH4 emissions data from grazing ruminants, which in turn will provide greater confidence in CH4 emissions factors, national inventory estimates and the efficacy of mitigation approaches. The next step is for those experts who have indicated their interest in this project, whether it is as an author, lead author or editor, to seek appropriate resources from their national institution to enable them to participate. New Zealand agreed in principle to take the role of scientific coordinator and editor of the guidelines subject to necessary resources being made available. The guidelines should build on the chapter outline proposed in the workshop report after consideration of the objectives of the guidelines, their target audience and how the writing process will ensure that the final guidelines will be relevant and accessible to the target audience.

Acknowledgements
NZAGRC personnel did a great job in organising the workshop logistics, including travel arrangements for all participants, and with their hospitality. Harry Clark (Director NZAGRC) and Victoria Bradley (NZAGRC Operations Manager) personally attended some workshop sessions or checked that all was running smoothly which was much appreciated. Host institutions of all participants kindly permitted their respective participations.
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Glossary of abbreviations and terms

CH4 GC GL GRA LA MAF NZAGRC PT SA SF6 methane gas chromatography, or gas chromatograph guidelines and protocols for the measurement of methane emissions by individual animals using the SF6 tracer technique Global Research Alliance (http://www.globalresearchalliance.org/) lead author of a GL chapter NZ Ministry of Agriculture and Forestry (http://www.maf.govt.nz) NZ Agricultural Greenhouse (http://www.nzagrc.org.nz/) Gas Research Centre

permeation tube, with SF6 as the permeant supporting author (also known as contributing author) of a GL chapter sulphur hexafluoride

References
Gere, J.I., Gratton, R., 2010. Simple, low-cost flow controllers for time averaged atmospheric sampling and other applications. Lat. Am. Appl. Res. 40, 367376. Grainger, C., Clarke, T., McGinn, S.M., Auldist, M.J., Beauchemin, K.A., Hannah, M.C., Waghorn, G.C., Clark, H., Eckard, R.J., 2007. Methane emissions from dairy cows measured using the sulfur hexafluoride (SF6) tracer and chamber techniques. J. Dairy Sci. 90, 27552766. Johnson, K., Huyler, M., Westberg, H., Lamb, B., Zimmerman, P., 1994. Measurement of methane emissions from ruminant livestock using a SF 6 tracer technique. Environ. Sci. Tech. 28, 359362. Lassey, K.R., Walker, C.F., McMillan, A.M.S., Ulyatt, M.J., 2001. On the performance of SF6 permeation tubes used in determining methane emission rates from grazing livestock. Chemosphere: Global Change Sci. 3, 377381. Lassey, K.R., Pinares-Patio, C.S., Martin, R.J., Molano, G., McMillan, A.M.S., 2011. Enteric methane emission rates determined by the SF6 tracer technique: temporal patterns and averaging periods. Anim. Feed Sci. Technol. 166167, 183191. McGinn, S.M., Beauchemin, K.A., Iwaasa, A.D., McAllister, T.A., 2006. Assessment of the sulfur hexafluoride (SF6) tracer technique for measuring enteric methane emissions from cattle. J. Environ. Qual. 35, 16861691.

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Martin, R.J., Bromley, A.M., Harvey, M.J., Moss, R.C., Pattey, E., Dow, D., 2011. The Lung: a software-controlled air accumulator for quasi-continuous multi-point measurement of agricultural greenhouse gases. Atmos. Meas. Tech. Discuss. 4, 1935 2011. Pinares-Patio, C.S., Machmller, A., Molano, G., Smith, A., Vlaming, J.B., Clark, H., 2008. The SF6 tracer technique for measurements of methane emission from cattle effect of tracer permeation rate. Can. J. Anim. Sci. 88, 309320. Pinares-Patio, C.S., Lassey, K.R., Martin, R.J., Molano, G., Fernandez, M., MacLean, S., Sandoval, E., Luo, D., Clark, H., 2011. Assessment of the sulfur hexafluoride (SF6) tracer technique using respiratory chambers for estimation of methane emissions from sheep. Anim. Feed Sci. Technol. 166167, 201209. Vlaming, J.B., Brookes, I.M., Hoskin, S.O., Pinares-Patio, C.S., Clark, H., 2007. The possible influence of intra-ruminal sulphur hexafluoride release rates on calculated methane emissions from cattle. Can. J. Anim. Sci. 87, 269275. Woodward, S.L., Waghorn, G.C., Ulyatt, M.J., Lassey, K.R., 2001. Early indications that feeding Lotus will reduce methane emissions from ruminants. Proc. N.Z. Soc. Animal Prod. 61, 2326.

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Appendix 1.
Workshop discussion

Workshop presentations

The section presents workshop deliberations (presentations plus some discussion) under the headings of the workshop programme (Appendix 1) together with extra headings that were decided upon at the workshop to incorporate new and unanticipated material into the GL. Individual presentations are available as annexes to this report. More detail on the discussion points that are to be traversed in the GL chapters are relegated to Appendix 2. The remainder of this section presents workshop material and its relevance to the GL. Wherever a topic is mentioned, or portrayed as raised by a workshop participant, it should be taken as a recommendation that that topic be traversed in the GL. Thus in traversing the main content of presentations and the discussion stimulated by those presentations, the subject material of the GL is being defined and traversed. Accordingly, the major workshop presenters under each heading are also on the authorship of the corresponding chapter of the GL. More detail on the intended coverage of each chapter is provided in Appendix 2.

SF6 permeation tubes

Presentations were given by Keith, Peter, Matthew, Ccile, Alexandre and Richard, on various aspects of permeation tubes (PTs). Topics covered included tube fabrication, charging, calibrating and deployment. In addition, an investigation of a possible effect of tube orientation was reported by Richard (no effect detected). Presentations by Ccile, Alexandre, Ben and Alan each covered various aspects of the SF 6 tracer technique that included a component on PTs. Roger overviewed his experiences with high-emission PTs (and of infrared analysers, relevant to Gas analysis below), and also with tracers alternative to SF 6. The highemission PTs were motivated by the need for higher SF6 concentrations for detection by IR analysers. It is not a direction that Roger would recommend. A particularly novel approach to analysing PT performance was described by Peter: the application of Michaelis-Menten kinetics to describe SF6 interaction with the permeable membrane, as a possible refinement to determining SF 6 permeation rates by linear regression. In effect it furthers the revelation by Lassey et al. (2001) that SF 6 permeation rates gradually decline over time by offering a means to quantify that decline with sufficient ease and surety to retrospectively correct for tube performance. If this promise is fulfilled, it will greatly enhance the utility of the SF 6 tracer technique by permitting serial experiments on animals with unrecoverable permeation tubes in their rumens. This work is under preparation for publication. Peter raised the spectre that when retaining PTs for calibration in an oven or incubator with set-point at 39C, there should be an independent check of oven fidelity such as by using a simple mercury thermometer (such as designed for human body
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temperature) or temperature logger. Peter had found that the set temperature on their incubator was 2C out! No other workshop participant reported precautionary checks on set temperatures, and there was some alarm at the possibility of a 2C error because of the sensitivity of permeation rate to temperature of between 3 and 10% per C. All the above considerations will receive mention in the GL as part of best practice procedures, under sub-headings such as those in Appendix 2 (Section 5 therein). In the case of applying Michaelis-Menten kinetics, permission from Peter will be required if that development has not been published.

Breath sampling apparatus

Presentations were given by Keith, Ross, Richard, Peter, Jos and Cesar, on systems for sampling breath from grazing or housed animals. Topics covered include: the plumbing configuration between inlet and canister and its mounting on the facial halter, different means to restrict sample flow rates, bags as gas collection vessels, multi-day sampling, and any special considerations for fistulated animals. Presentations by Ben, Alan, and Ccile on their respective research covered some aspects of sampling, with Ccile specifically covering sampling from fistulated cattle, including use of a cannula to enable sampling directly from the rumen headspace. Richard described the approach at DPI Victoria for limiting sample flow rates by crimping capillary tubing as an alternative to requiring a long length of (expensive) capillary tubing that the original protocol called for (Johnson et al. 1994). Jos described the novel approach (described also by Cesar) of using a ball and socket to restrict flows to very low levels appropriate for multi-day sampling (Gere & Gratton 2010). Richard also described fabricating and using bags, each with a septum, as inexpensive yet reliable (but usually single-use) collection vessels. Without a sucking capacity, preevacuated bags require pumps for active sample collection, and a hand syringe was used for sample extraction. Keith described a variation of the tracer technique that automated the collection of successive breath samples (20-min accumulations every 20 mins for 6 days from each of 3 housed animals) by pumping into individual Tedlar bags from which samples are drawn, again by pump, directly into the analyser (gas chromatograph): Lassey et al. (2011). This is a particular application of a more general Lung system used to collect up to three samples in parallel and analyse them for trace gas content (Martin et al. 2011). The Lung system operates unattended, with automated valves diverting airflows to and from different bags under software control within the laboratory, avoiding disturbance of the plumbing on the animal halter. All of the different strategies to control sample flow rates and sample containment discussed above will be traversed in the GL under headings and with content such as in Section 6 of Appendix 2.

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Background air sampling

A presentation by Richard, on considerations for background sampling for both grazing and housed animals, included the siting of background samplers and the association between individual housed animals and individual background sites. In a presentation below (Estimation of CH4 emission rates, yields, and emission factors), Keith discussed some criteria for sample versus background mixing ratios. Richard showed a slide of the atmospheric growth in CH4 and in SF6 at Australias clean air station at Cape Grim, NW Tasmania, showing current clean-air atmospheric mixing ratios at 1.8 ppm and 6.7 ppt, respectively. (Those of us in atmospheric sciences are well acquainted with such time series, but graphs of such time series are still worth reiterating in the GL as many GL readers would know little about global background levels). In agricultural regions, local CH4 mixing ratios can be higher than clean-air backgrounds. Within a feeding environment for cattle hosting intra-ruminal SF6 permeation tubes, mixing ratios of both CH4 and SF6 can exceed double the clean-air values, and inside a feeding bin can be higher by more than an order of magnitude. Few, if any, of the workshop participants had investigated local gradients in background CH4 and SF6 as thoroughly as Richard and DPI-Vic colleagues. Keith reported some investigations of such gradients done in an experiment with housed cows at Dexcel (now Dairy NZ) in Hamilton with Sharon Woodward in 2000. The latter work was published in short form without mention of such investigations (Woodward et al. 2001). Recommendations about siting and elevation of background samplers, and about the ventilation of indoor areas will be made in the GL (see Section 8 of Appendix 2).

Sample handling and gas analysis

Ross demonstrated a piston system, co-designed by Ross and NIWA colleagues, which enables gas samples to be withdrawn from a canister without the need to dilute with nitrogen to super-ambient pressure as was proposed by the originators of the technique (Johnson et al. 1994). Super-ambient pressure simplified sample transfer to the GC by bleeding off part of the over-pressure. Using a piston system to extract an undiluted sample has two major advantages: (1) it avoids diluting the signal and so effectively decreases the low detection limit for measurements; and (2) it allows a smaller sample to be drawn into a pre-evacuated canister, avoiding concern that the collection rate falls off as sample pressure rises, and makes the entire sample available to the GC (instead of just the over-pressured portion). There was a lot of interest expressed in this system, which has been used for about 10 years in NZ experiments. It also lends itself to automation. A paper describing the piston system is presently under preparation for publication. Alans presentation covered sample storage in vials (exetainers) with transfer via syringe, necessitating placement of a septum near the valve of the canister. Such transfer is one of several manual transfers that become necessary (eg, syringe to GC), implying multiple handing, increasing the cost (and risk of handling error) of gas analysis.
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Discussion arose around sample containers for intermediate storage, such as to facilitate transportation to a distant laboratory for analysis, or to free up yokes when gas analysis cannot proceed imminently. An important property of such storage containers would be their retention of gas integrity. The candidates here were vials (eg, exetainers), or bags (see Breath sampling apparatus above). Both of the above methods of sample extraction together with the traditional overpressuring with nitrogen will be traversed in the GL (see Sample extraction, Section 9 of Appendix 2), as will recommendations about storage containers (see Intermediate sample storage, Section 9 of Appendix 2).

Gas analysis
A presentation by Keith covered aspects of gas chromatography (GC), including inference of SF6 mixing ratio from non-linear electron-capture detection (ECD) and the choice and application of gas standards. Rogers presentation covered infrared detection techniques (for which much higher concentrations of SF6 are needed than for GC detection), and detection techniques suited to alternative tracers. Roger has moved away from this approach, and appears to regard it as an investigation that didnt bear fruit. A view held in some of the community of SF6 tracer technique researchers is that little error is made in taking GC/ECD response to be linear (and in some cases linear through the origin). I am aware of some groups not represented at the workshop that use a single SF6 gas standard which leaves little choice but to presume linearity. So this section in the GL will cover aspects of GC pertinent to applying the SF 6 tracer technique, which will include typical detector response and the need for, and choice of, gas standards. For more detail, see Analysis by gas chromatography, Section 9 of Appendix 2.

Animal management, diet and feed intake

A presentation by Garry covered aspects of animal and feed management and estimation of feed intakes that are particularly pertinent when applying the SF 6 tracer technique to either grazing or housed animals. Input came from others including those whose broader presentations had also covered these topics (eg, Ccile, Alexandre, Ben, Cesar, Natasha, Chris). Garrys talk had some usefully-provocative points that generated discussion around: preparation of both animals and feed determining feed intake by grazing animals the merits of a small number of meals to housed animals versus more protracted feed delivery (ie, role of rate of feed consumption) determining feed composition and digestibility: what to measure and how to measure it (eg use of near-infrared reflectance spectroscopy)
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Garry stressed the importance of clearly defining in advance the objectives of the research, because this will determine experimental protocols and measurements. The above points are given more detail in Section 10 of Appendix 2 which summarises coverage of the relevant chapter of the GL.

Estimation of CH4 emission rates, yields, and emission factors

A presentation by Keith covered all topics in this heading, including the equations concerned, and criteria for mixing ratios in breath samples relative to those in background samples that would yield useful results. Keiths presentation raised a novel query about data analysis: estimating CH4 emission rate based on N consecutive days of sampling conventionally proceeds as an average (over N days) of quotients (of CH4 mixing ratio divided by SF6 mixing ratio, with each mixing ratio itself a daily average, and background corrected). An equally valid estimator is the quotient of 2 averages (the average CH4 mixing ratios over all N days divided by the counterpart average SF6). The latter is in principle the same as estimating CH4 emissions based on an N-day sample collection. Keith raised the query which estimator is the better? It was apparent that most workshop participants had not considered such alternative estimators. Note that this query has recently been traversed by Lassey et al. (2011). It will also be traversed in the GL. A useful oral contribution came from Peter, emphasising the need to properly scope backgrounds. He noted the dearth in published experiments of information on background collections their number and location(s), and representative CH4 and SF6 mixing ratios or in some cases a failure to report a background correction in the equation that estimates CH4 emission rate, including in the seminal paper by Johnson et al. (1994). Peter also reminded the workshop that underlying the estimation of CH 4 emission rate was the ideal gas law, PV = nRT, and that the data analyst should be mindful of applying consistent units. Caveats were noted for estimates of CH4 yield for grazing animals for which feed intakes are inferred and poorly quantified.

Other considerations
In general discussion, various other sub-topics not explicitly covered under the above headings were deemed worthy of explicit consideration. Accordingly, the GL will include short chapters or sections that explicitly address the following topics.

Statistical considerations for pre-experimental planning and data analysis

The intent is to establish the number of animals required if an experiment is to have sufficient power to detect the anticipated differences in CH4 yield between treatments. Such considerations are not novel to the SF6 tracer technique, but their consideration
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should take account of the inter-animal and inter-day variability that seems to be associated with the technique itself. Discussion of these considerations pointed to Bens PhD thesis which addressed this topic in some detail. This will be a fairly short but focused chapter, led by Chris, a keen proponent of the chapter (see Section 4 of Appendix 2).

Acceptance and inclusion in guidelines

It was felt that the GL should recommend criteria for identification of crook data and for data acceptance ie, should offer some QA/QC criteria or guidelines. Cesar offered to lead this chapter, through wide consultation with workshop participants (see Section 12 of Appendix 2).

What SF6 tracer technique detail should be reported?

Peter and Richard had compiled the results of a literature search of papers applying the SF6 tracer technique, which they shared with participants. They noted a large range in the type and level of detail reported in those papers on how the technique was applied. For example, some failed to report: how the PTs were calibrated or over what duration; the time between calibration and the experiment; the range of SF6 permeation rates; the delivery rate of breath sample; the siting of background samplers and the background mixing ratios encountered. Some supplied little or no detail on the gas chromatography (configuration, gas standards, etc), or crossreferenced other papers that supplied little detail. The GL will include a chapter led by Richard and Peter that recommends a minimum level of detail that should be reported, which would include addressing the particular examples above. The afore-mentioned list of references from a literature search is a useful resource that will be included as an annex to the GL, with all participants invited to add further publications. Additions to date have significantly lengthened the list, which as of 30 June 2011, contains 94 references.

Future issues and potential improvements

This would anticipate issues that could potentially arise, such as standardization of PT structure and fabrication, possible retrospective improvement to data interpretation (eg, in the light of applying Michaelis-Menten kinetics to recalculate SF6 permeation rates); whether temporal variation in intra-ruminal temperature matters. These and other considerations that will be traversed are proposed in Section 14 of Appendix 2.

Enigmas
Acknowledge that some puzzles about the SF6 tracer technique persist as unexplained. These include the following:

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1. Indications that CH4 emission estimates may depend upon SF6 release rate have been reported by the AgResearch group (Vlaming et al. 2007, Pinares-Patio et al. 2008), but apparently not confirmed in Ireland (Matthew, communicated at workshop). No mechanistic explanation has been forthcoming for such dependence. 2. Much lower estimates of methane yields from sheep fed chicory or white clover when measured using the SF6 tracer technique than when measured using chambers (Garry, communicated at workshop). 3. Why does variation in CH4 yield between animals appear greater when using the SF6 tracer technique over several days with measured intakes, than when measured using chambers (eg, McGinn et al. 2006, Grainger et al. 2007, Pinares-Patio et al. 2011)? Is this at least partially explained by Lassey et al. (2011) who demonstrate that SF6 is not emitted by a sheep at a uniform rate? 4. If rumen fistulation does not affect methanogenesis, why can it lead to an overestimated CH4 emission? For example, is it to do with escape of SF6 and CH4 at the fistula plus some hind-gut CH4 excreted via the lungs, with the result that proportionately more of the internally-sourced SF6 escapes at the fistula than of CH4? 5. Is the use of stainless steel componentry in the breath-collection plumbing better than brass? Matthew had found that the former (at least at the capillary junctions) had led to lower estimates of methane production, but no-one else could corroborate this. 6. Matthew reported that intramuscular injections of antibiotic reduced CH4 yields two weeks after injection. Why? Could other drugs have similar effects? For how long is this prolonged? Some or all of these puzzles, also addressed in Section 15 of Appendix 2, may be addressed in the GL, according to the editors discretion following finalization of the Chapters 114. Criteria to be applied for inclusion include whether it is important to the efficacy of the SF6 tracer technique, whether it no longer appears to be the enigma that it appeared to be at the workshop, and whether or not it is better addressedor is already addressedelsewhere in the GL.

Information available on the internet

It was felt appropriate to include under separate heading cross-references to any other manuals on the SF6 tracer technique, such as standard operating procedures or codes of practice. Peter would take the lead on seeking out and reporting such websites. This section is important.

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