DOI: 10.5061/dryad.fj6q573rf
title: Data from: Quantifying and correcting for pre-assay CO2 loss in short-term carbon mineralization assays

description: Abstract. The active fraction of soil organic carbon is an important
 component of soil health and often is quickly assessed as the pulse of CO2
 released by re-wetting dried soils in short-term (24–72 h) assays.
 However, soils can lose carbon (C) as they dry and, if soil samples vary
 in moisture content at sampling, differential C loss during the pre-assay
 dry-down period may complicate the assay's interpretations. We
 examined the impact of pre-assay CO2 loss in a long-cultivated
 agricultural soil at initial moisture contents of 30 %, 50 %, and 70 %
 water-filled pore space (WFPS). We found that 50 % and 70 % WFPS
 treatments lost more C during drying than did those in the 30 % WFPS
 treatment and that dry-down losses led to a 26 %–32 % underestimate of
 their CO2 pulses. We developed a soil-specific correction factor to
 account for these initial soil moisture effects. Future C mineralization
 studies may benefit from similar corrections.
description: Site
 description We
 collected soil using a shovel from the Ap horizon (0−20 cm) of an arable
 grass field at the W.K. Kellogg Biological Station (KBS) in Hickory
 Corners, MI (42°41’02” N, −85°37’34” W). KBS soils are mixed, mesic Typic
 Hapludalfs of co-mingled Kalamazoo and Oshtemo series (Crum and Collins,
 1995) developed on glacial outwash with intermixed loess (Luehmann et al.,
 2016). Soil collected in September 2019 for this experiment was from the
 Kalamazoo series, which are well-drained fine-loams (43% sand, 38% silt,
 19% clay) with ~2% total C (Grandy and Robertson, 2006) and a pH of 7.2
 (Robertson et al., 1993). Average annual precipitation at KBS is 1005 mm,
 with an average of 1.3 m of snowfall per year, and mean annual temperature
 is 10.1°C (Robertson and Hamilton, 2015). The site was in various
 corn-soybean-wheat rotations for the prior 40 years and before that,
 corn-soybean-small grain rotations for at least 60 years. Experimental
 design To
 examine the influence of initial soil moisture on the pre-assay loss of
 CO<sub>2</sub> during dry-down, we pre-wet recently collected
 soil to three different initial water-filled pore space (WFPS) levels: 30,
 50, and 70%. Then we measured gravimetric soil moisture (GSM) and
 CO<sub>2</sub> loss while soil was air-drying, after which we
 re-wet them and measured the 24-h CO<sub>2</sub> pulse by
 standard methods (Robertson et al., 1999; Franzluebbers et al.,
 2000). Laboratory
 analyses After
 collection, soil was sieved through a 4-mm mesh and mixed. We measured GSM
 and calculated the target volumetric water content (VWC, g
 H<sub>2</sub>O cm<sup>-3</sup> soil) for each
 treatment following Eq. 1 (Elliott et al., 1999): VWC<i> = </i>WFPS/100
 * (1− SBD/2.65)                                          
                                                            
                               (1) where soil bulk density (SBD) is
 1.5 g soil cm<sup>-3</sup>, previously assessed(Robertson,
 2016). Then we divided VWC by SBD to obtain a target GSM and thereby
 determined the amount of water to add to the field-moist soil (11% WFPS;
 GSM = 0.032 g H<sub>2</sub>O g<sup>-1</sup> dry
 soil). We then weighed 40 g of soil into each of 75 polyethylene cups.
 Each cup was randomly assigned to an initial WFPS treatment (30, 50, or
 70%), for a total of 25 replicates per treatment. We added sufficient
 deionized water to each cup to achieve the target initial WFPS and stirred
 to evenly distribute water. After soil was wet and stirred in the cups,
 the contents of each cup were transferred to a labeled paper bag. The soil
 was spread evenly across the bottom of the bag, and the top portion of the
 bag was removed to increase air flow. Afterwards, the soil was immediately
 weighed and set on a laboratory bench to air-dry. Immediately after wetting, as
 well as 1, 3, and 8 days later, we assessed GSM and
 CO<sub>2</sub> loss rates for five replicates per initial WFPS
 treatment. GSM, which was determined after drying the soil at 105°C for 24
 h, stabilized at 1.5% in the air-dried soil (Fig. 1a), but did not reach
 zero even when soil was completely air-dry. Because soil in all initial
 WFPS treatments were air-dry by day 3, with CO<sub>2</sub>
 loss rates close to zero, we terminated GSM and CO<sub>2</sub>
 measurements after day 8. CO<sub>2</sub> loss
 rates at each sampling interval were measured by placing 10 g of soil into
 a 235 mL mason jar fitted with a gas-sampling septum. Then we sampled 5 mL
 of headspace from each jar at 4 intervals (0, 0.5, 1, and 2 h), injected
 it into an evacuated 3 mL exetainer (Labco Limited, Lampeter, Wales,
 United Kingdom), and replaced the jar headspace with laboratory air.
 CO<sub>2</sub> samples were analyzed within 24 h using a
 LI-820 CO<sub>2</sub> Gas Analyzer (LI-COR Biosciences,
 Lincoln, NE, USA)<i>.</i> On day 15 we re-wet the remaining
 five replicates of air-dried soil from each initial WFPS treatment to 50%
 WFPS (Franzluebbers et al., 2000). We then assessed subsequent 24-h
 CO<sub>2</sub> pulses by sampling headspaces at 0, 2, 4, 8,
 and 24 h.  2.4 Statistical
 analyses and correction factor CO<sub>2</sub> pulses
 were calculated as the positive slope of the linear regression of
 CO<sub>2</sub> concentrations through time after accounting
 for headspace dilution, and then converted to a standardized rate using
 the ideal gas law. In 17 of 75 cases, we omitted one of the four data
 points within a jar, which were clear visual outliers. In two cases, we
 rejected jars with leaks. CO<sub>2</sub> loss rates during the
 dry-down period were analyzed with a two-way analysis of covariance
 (ANCOVA), where initial WFPS treatment and days elapsed since wetting
 (Day) were factors and GSM at the time of sampling was a covariate.
 Additionally, a one-way analysis of variance (ANOVA) was used to determine
 whether initial WFPS treatment significantly affected the 24-h
 CO<sub>2</sub> pulses upon re-wetting the air-dried
 soil. We also
 calculated a correction factor to account for pre-assay
 CO<sub>2</sub> loss prior to the 24-h
 CO<sub>2</sub> pulse assay. To calculate the total amount of
 CO<sub>2</sub> loss during dry-down for each initial WFPS
 treatment, we calculated a best-fit exponential decay curve:
 Y =
 α<sup>βX</sup> + θ                                        
                                                                                                                                        (2) where Y = daily CO<sub>2</sub>-C loss and X = length of dry-down period, until soil was air-dry (i.e., immediately after wetting through day 3). Total C loss was equivalent to calculating the area under the curve, using bootstrapping to allow for error estimates. Because we used sacrificial sampling, we could not calculate standard deviation or standard error in the usual way. Instead, we used a bootstrapping approach in which we computed predicted values for CO<sub>2</sub> losses (<i>Ŷ<sub>i</sub></i>) and residuals (<i>e<sub>i</sub></i> = <i>Y<sub>i</sub></i> – <i>Ŷ<sub>i</sub></i>). All zeroes for CO<sub>2</sub> losses were set to 1 for fitting the regression because an exponential decay curve can approach but never attain 0 and because 1 was lower than any value we observed. Then we created a bootstrap sampling of residuals specific to each dry-down interval (0, 1, or 3 days), sampled randomly from each interval with replacement, and added randomly sampled residuals to predicted values (<i>Y<sub>i</sub><sup>*</sup></i> = <i>Ŷ<sub>i</sub> + e<sub>i</sub><sup>*</sup></i>) for each dry-down interval (after Hesterberg, 2015). Residuals were bootstrapped 10,000 times to derive multiple estimates of coefficients for the exponential decay curve (α, β, and θ). We also integrated under the curve 10,000 times to get an error estimate (i.e., coefficient of variation) associated with the total amount of pre-assay CO<sub>2</sub> loss during dry-down. Then we divided the total CO<sub>2</sub> loss by three days to obtain the daily rate used to calculate a correction factor following Eq. 3: CF = (daily CO<sub>2</sub> loss during dry-down / 24-h CO<sub>2</sub> pulse after re-wetting) + 1                                                                   (3) The correction factor for each treatment was then multiplied by each replicate’s 24-h CO<sub>2</sub> pulse following re-wetting. Finally, we verified that the correction factors worked by conducting a one-way ANOVA to determine whether initial WFPS treatment still had an effect on the corrected pulses. For all analyses, we confirmed that assumptions of normality and homogeneity of variance were not violated.