TITLE: USDA LTAR Common Experiment Measurement: Chamber-based fluxes of nitrous oxide (N2O) and methane (CH4) from soil
AUTHORS: G. Philip Robertson, Michel A. Cavigelli, Harold P. Collins, Curtis Dell, David R. Huggins, Virginia L. Jin, Kevin Kahmark, Mark A. Liebig

[DESCRIPTION]
Emissions of nitrous oxide (N2O) and methane (CH4) from soil are dynamic and spatially variable. Chamber based methods have been used since the 1980s to document fluxes and are widely used in both managed and natural ecosystems (Clough et al., 2020; Holland et al., 1999; Parkin and Venterea, 2010). They are well-tested, relatively inexpensive, and can be deployed extensively. They are also suitable for emissions of other gases, notably CO2 and NOx. Chambers can also be automated (e.g, Grace et al., 2020) to capture temporal dynamics. Here we provide hands-on, best-practice guidelines for the minimum and preferred chamber sampling plans sufficient to compare Common Experiment flux differences across all sites in the Long-Term Agroecosystem Research (LTAR) Network. Although each site is different, and exceptions will likely be necessary, following these guidelines will maximize the potential for making cross-site comparisons and provides those new to soil greenhouse gas sampling a straightforward path for successful measurement campaigns.

In general, chambers will need to be of a sufficient size and placed to capture representative fluxes, keeping in mind plant spacing, fertilizer placement, and other features of the site that affect spatial variability such as topography. Once placed, chambers will need to be sampled at intervals that capture episodic emissions that result from management events such as tillage and nitrogen fertilization and environmental events such as rainfall following dry periods. Fluxes are typically low during dry periods and in winter if freeze-thaw events are infrequent, and during these periods sampling frequencies can be relaxed. In general, we recommend a minimum of weekly sampling following events known to stimulate fluxes (tillage, N fertilization, rainfall following drought) and biweekly sampling otherwise except when soil is very dry or frozen, when sampling can be monthly. By convention, fluxes should be expressed as μg N2O-N m-2 h-1 or μg CH4-C m-2 h-1.

[GUIDELINES]
General Principles

Agriculture in general and agricultural soils in particular are responsible for much of the contemporary increase in atmospheric N2O, now increasing at the rate of 2% per decade (Tian et al., 2020). Understanding ecosystem-level controls on soil-borne fluxes and defining opportunities to mitigate emissions is fundamental to lowering atmospheric loading rates. Likewise, atmospheric CH4 concentrations are increasing at an alarming rate, and while agricultural soils – excepting paddy rice – do not contribute significantly to this increase (Robertson, 2004), soil CH4 oxidation (methanotroph consumption) represents a potential source of negative emissions, particularly in semiarid grazing lands and perennial herbaceous crops such as switchgrass (e.g., Bates et al., 2021; Liebig et al., 2012). Documenting the impact of different agricultural management strategies on emissions of both N2O and CH4 is important for evaluating the environmental performance of alternative production systems, for developing novel mitigation opportunities, and for validating simulation models that can be used to estimate global impacts of management change.

Chamber-based methods for capturing soil greenhouse gas emissions have been used for decades and are the preferred method for capturing flux differences among different experimental treatments and, when sampled frequently, can be used to estimate annual fluxes (see Clough et al., 2020; Holland et al., 1999; Parkin and Venterea, 2010).  Although eddy covariance methods that continuously sample large fetches are superior in many respects, they are expensive and, for N2O, sensors are currently limited to closed-path designs that require line power for precision pumps. Open-path N2O sensors, which require less power, are under development (Pan et al., 2022) but not yet commercially available. Eddy covariance methods are also not well-suited for plot-based research because of their need for large fetch areas. 

Sampling to detect differences in emissions among management systems such as those in the LTAR Common Experiment (CE) (Spiegal et al., 2018; Liebig et al., 2024) requires the deployment of appropriately sized chambers sampled at intervals sufficient to capture these differences. Because Common Experiment treatments typically differ in major ways with respect to crops and crop rotations; nitrogen fertilizer rate, timing, placement, and formulation; manure use; tillage and cultivation practices; cover crops; grazing and fire management; and other factors that can affect greenhouse gas emissions, it will be important to sample all treatments even when only one may be emitting detectable quantities. Moreover, to maximize statistical power for detecting treatment differences, it is more important to sample all replicate plots or fields rather than to sample multiple chambers in fewer plots or fields (Kravchenko and Robertson, 2015).

Chamber design

Chambers consist of two parts – a base that is inserted into the soil a few cm and left in place between field operations, and a lid that can be sealed to the base to provide a gas tight seal between the chamber headspace and the atmosphere. A pigtail vent in the lid or side of the chamber keeps atmospheric pressure changes from affecting flux estimates. The size of the chamber is important – usually 30 cm diameter or square is sufficient, and a height that does not preclude plants where present and does not overly dilute detectable headspace gas concentrations, e.g., 20 cm (Clough et al., 2020; Kahmark et al., 2020). The lid is left in place for up to an hour, and during this period the rate of accumulation of gas in the headspace represents the soil gas flux. In most agricultural soils the net flux is generally positive for N2O and negative for CH4.

Analysis methods

Two analysis methods for quantifying soil gas flux are available. For the first, called the syringe method, a several mL headspace sample is removed through a rubber septum (installed in the chamber lid) by a needle and syringe and transferred to a pre-labeled gas-tight vial that is then taken to the lab for gas chromatograph analysis; at least four samples need to be taken over the chamber closure period. For the second, called the flow-through method, a portable flow-through analyzer is connected by tubing to inlet and outlet ports on the lid to circulate headspace gas past a sensor that reports gas concentrations at 1 to 20 second intervals depending on instrument. From 2 to 5 minutes of sample time are typically needed per chamber once the sample lines have been flushed with headspace gas, which can take as little as 1 minute.

For syringe samples, vial contents must be analyzed in the lab using a gas chromatograph connected to an electron capture detector (ECD) for N2O analysis, a flame ionization detector (FID) for CH4 analysis, and either a thermal conductivity detector (TCD) or an infrared gas absorption (IRGA) analyzer for CO2 analysis. Lab-based flow-through analyzers can also be used to analyze vial contents. Autosamplers with valving that allows sequential gas analysis can efficiently analyze all three gases at once. CO2 analysis is especially useful for detecting sampling anomalies – the absence of a linear CO2 increase in the chamber headspace usually indicates chamber or vial leakage unless soils lack biological activity for some reason like freezing temperatures.

The choice of method will be dictated by available instrumentation and labor. In general, flow-through analyzers provide greater sensitivity but are expensive, require more expertise in the field, and for large sample campaigns can take more time in the field than syringe sampling. Sampling time in the field is dependent on the number of field personnel for the syringe method and, for the flow-through analyzer method, also dependent on the number of available analysis units. Several field personnel can sample more chambers at one time per sampling period than can one technician with a flow-through analyzer. This is an important consideration when attempting to sample multiple chambers quickly at a consistent time of day. 

Chamber placement

Place chambers within plots or fields to represent existing spatial variability. In topographically uniform experimental plots, this usually means ensuring that the chambers correctly represent row–interrow areas and proportionally sample areas where fertilizer, manure, or compost are banded or injected. Depending on the size and shape of the chamber, more than one chamber may be necessary to accommodate variability arising from row-interrow and fertilizer placement.  In fields, this principle also means calibrating for topographic positions and/or areas of high and low productivity. Placement is more complicated in grazed systems because sampling sites must account for animal movement (including congregation) and patches of urine and dung (including legacy effects). Chambers will periodically need to be removed to facilitate field operations. Reinstallation should be near the original sampling locations, while avoiding the exact location of prior chambers, and, where possible, waiting a day or longer to avoid sampling the disturbance associated with chamber installation.

Sample timing and frequency

Collect samples on a regular year-round basis supplemented with times of anticipated high fluxes. In cropland systems, this principle generally means weekly during the growing season with the potential for more intensive sampling at tillage, fertilization, cover crop incorporation, and substantial rainfall or irrigation following dry periods. In grazing land systems, this principle generally means more intensive sampling during active grazing, following rainfall after dry periods, or after prescribed fires. Sampling frequency can be relaxed to biweekly or even monthly during seasons with very low fluxes such as extended dry periods and deep winter.  If the object of sampling is to detect long-term treatment differences rather than to construct annual budgets, then it may be possible to relax sampling frequencies somewhat.

A general regular schedule is to sample weekly during the growing season (with greater frequency following N fertilization events—see table below), every other week in early and late seasons, and monthly in winter. Sample all treatments and chambers at each sampling event to avoid biasing cumulative fluxes, which may result from integrating fluxes over different time intervals or unequal sample sizes. For annual crops, the early season is spring thaw (or another end-of-winter event) to first field operations; the growing season is first field operations to peak crop biomass; the late season is peak crop biomass to winter onset; winter is characterized by near-continuous cold. For perennial crops and pastures, the early season is spring thaw (or another end-of-winter event) to plant green-up; the growing season is green-up to peak cumulative biomass; the late season is peak cumulative biomass to the onset of winter; winter = near-continuous cold. Peak cumulative biomass accounts for forage systems that are harvested multiple times per growing season.

Ancillary measurements

Useful ancillary measurements at the time of sampling include air and soil temperature and soil moisture; it can also be useful to sample for soil inorganic nitrogen content occasionally, especially following fertilization events. Chamber and ambient air temperatures can be different with some chamber designs, so this should be checked for a given design using temperature probes on a sunny day. Chamber air temperature is a required variable for gas flux calculations. Chamber height should also be measured in three to four locations along the inside perimeter of each chamber at sampling as the insertion depth of chambers can differ and settling can occur between sampling events; headspace volume is a crucial calculation term. The preferred soil sample depth is 0-10 cm for soil temperature and moisture; for soil nitrogen 0-10 or 0-25 cm is preferred but any depth to less than the A horizon thickness is acceptable. Soil moisture and temperature should be sampled outside of the chamber but within a 1 meter distance, distance in an area without foot traffic. Soil moisture probes can be placed permanently inside the chamber footprint, with care taken to minimize soil disturbance when connecting probes for recording data.

Table 1. Summary of recommendations or measurement of chamber-based emissions of nitrous oxide (N2O) and methane (CH4) from cropland and pasture.
 
 

[STEPS]
SECTION: Sample Collection
1. Chamber deployment (minimum of one day before sampling; one week preferred):

Place chamber bases in representative positions and pound into the soil ~5 cm using a flat board and mallet or other technique. Disturb the soil and surrounding plants as little as possible. As needed, clip plants to below top of chamber. A detailed working protocol is available in Kahmark et al. (2020).
SECTION: Sample Collection
1.1. Prepare lids for gas sampling – for syringe sampling, replace the rubber septum as needed: 

For portable analyzer sampling, calibrate/span occasionally and check for accuracy against typical ambient concentrations before using. 
Make sure tubing is not crimped or blocked and sample ports on lids are clear.
SECTION: Sample Collection
2. Gas sampling:

Gas sampling should occur at about the same time of day on each sample date, and the sampling sequence should be staggered by treatment (i.e. do not sample all replicates of one treatment together) in order to avoid any systemic time-of-day bias.  
Be careful to minimize trampling around chambers. 
A detailed working protocol with helpful visuals is available in Kahmark et al. (2020).
SECTION: Sample Collection
2.1. Measure and record:

Chamber height at 3-4 locations around its perimeter; these measurements are used to determine the chamber volume, important for areal flux estimates.
Soil and air temperature adjacent to chamber or, if chamber air temperature is different from ambient, air temperature within chamber at beginning and end of the closure period.
Soil moisture adjacent to chamber.
Time of day and time at which individual chambers are sampled if different than predetermined interval.
SECTION: Sample Collection
2.2. For syringe sampling:

Install the lid 
Add a vent needle to the first vial
Insert the sampling syringe needle into the chamber lid septum and mix the chamber headspace by gently pumping the syringe three times; then remove at least 10 mL of headspace gas.
Inject the headspace sample into the vented sample vial to flush the vial with sample. Repeat for a total of three flushes.  After flushing, remove the vent needle.
Re-insert the sampling syringe needle into the lid septum, pump the syringe three times to mix the chamber headspace, then withdraw at least 10 mL, inject into the sample vial, and record the stopwatch time. Adding 10 mL to a sample vial already at atmospheric pressure will over-pressurize the vial in order to prevent sample contamination, allow for the detection of vial leaks prior to analysis, and may be needed to flush the injection port of GCs with attached autosamplers. Samples should not be stored in plastic syringes because syringes can absorb and emit methane.
Repeat chamber sampling an additional three times at ~15 minute intervals (or other pre-determined interval time), recording the stopwatch time each time a headspace sample is removed from the chamber.  Between intervals several other chambers can be sampled.
SECTION: Sample Collection
2.3. For portable analyzer sampling:

Most analyzers require a warmup time.  Consult the manufacturer’s manual for recommended time frames and pre-test.
Connect the lid and tubing assembly to the analyzer input and output sampling ports .
Attach the lid to the chamber base and allow the system to recirculate with sufficient time to ensure chamber sample is entering the analyzer (~30 seconds depending on tubing length and flow rate). Sample for as long as needed to record a stable flux. Typically, the rate of change in concentration starts to stabilize in about a minute, after which ~2 minutes of additional sampling is required. If fluxes fail to stabilize after 3 minutes, suspect poor chamber closure or fluxes below detection limits. 
SECTION: Flux Calculation
3. For syringe samples:

Calculate a volume-based flux (αv) from the linear relationship between headspace gas concentration (ppmv or µL gas/L headspace) and time of sampling (min), to yield a flux of µL gas/L headspace/min. 
In these calculations gas is either N2O-N, CH4-C, or CO2-C. If fluxes are non-linear a non-linear flux calculation method may be necessary; see Venterea et al. (2020).
SECTION: Flux Calculation
4. For portable analyzer samples:

Determine time from chamber closure to even mixing of headspace (deadband) and remove from flux calculation.
SECTION: Flux Calculation
5. For both syringe samples and portable analyzers:

From αm, calculate the gas flux (fm) as microgram of element (N for N2O; C for CO2 and CH4) per square meter per hour), using the equation:

                                 fm = (αm  V  60 min/h) / A
	
where:
	fm is expressed in μg N or C/m2/h
        αm = as above, in μg/L/min
	V = volume of gas in chamber, in L
        A = soil surface area covered by chamber, in m2
SECTION: Flux Calculation
3.1. Convert αv with units based on volume to αm with units based on mass, in microgram per liter per minute, and correct for field temperature using the following application of the Ideal Gas Law:

                                                   αm = (αv  M  P) / (R  T)

where:
αm is expressed in μg N or C/L/min
M = molecular weight of GHG (28 μg N/μmol N2O or 12 μg C/μmol CO2 or 12 μg C/μmol CH4)
P = assumed atmospheric pressure = 1 atm
R = Universal gas constant = 0.0821 L-atm/mol-K = 0.0821 μL-atm/μmol-K
T = field temperature, in °K = °C + 273
SECTION: Flux Calculation
4.1. Determine the units reported by the analyzer and convert to αm: ug N2O-N/L/min for N2O, ug CH4-C/L/min for CH4, and ug CO2-C/L/min for CO2.
SECTION: Flux Calculation
5.1. Report:

for N2O: μg N2O-N m-2 h-1
for CH4: μg CH4-C m-2 h-1
for CO2: mg CO2-C m-2 h-1 ; note that CO2 is in mg not μg – typically fluxes of CO2 are much higher than fluxes of N2O and CH4; convert to mg by multiplying fm by 1000.

Upscale values as needed or appropriate, e.g., to report values as g ha-1 d-1, multiply μg m-2 h-1 by 0.24.