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Accurate measurements of soil organic carbon (SOC) levels are essential to assess changes in C sequestration rates. To this end we conducted studies to evaluate laboratory variability in SOC concentration measured at USDA-ARS laboratories in Akron, CO, Cheyenne,WY, and Lincoln, NE. At the Akron laboratory we also evaluated field spatial variability within common cropping treatments in order to assess the potential to quantify significant changes in SOC content associated with rotations of varying cropping intensities. Our data showed very low coefficients of variation for SOC values from each of the three laboratories, and the same average SOC values for soils from each treatment. For mitigating spatial variability, the data showed that a 10-ha field required 10 cores, the 0.2-ha field, 2 cores, and the 0.02-ha field, one core, in order to achieve a difference of ≤10% from the mean 95% of the time. With respect to cropping intensity, all rotations with fallow contained statistically the same SOC levels, with the continuous cropping treatment [winter wheat-(Triticum aestivum L. )–corn-(Zea mays L.)–proso millet (Panicum miliaceum L.) showing higher levels of SOC than the conventional-till winter wheat summer fallow and the wheat–sunflower (Helianthus annuus L.)–fallow. Data indicate that net changes in SOC content over time as a result of management will be very difficult to assess, and will require a sufficient minimum elapsed time, as well as great attention to sampling protocol.