*2.1. Experimental Design*

A laboratory study using a randomized complete block design with three replicates was set up using one soil and six residue treatments, plus an untreated soil control (*n* = 21). The soil was a Forman soil (fine-loamy, mixed, superactive, frigid Calcic Argiudolls) [38], similar to many soils of glaciated parts of the region. The six residue treatments were corn (CR), soybean (S), forage radish (R), pea (P), spring wheat (SW), and winter wheat (WW) (Table 1). Fresh individual crop residues were collected immediately in the fall following harvest and analyzed for carbon (C) and nitrogen using an Elementar Vario Max® CN analyzer (Ronkonkoma, New York, NY, USA) during a previous study conducted by Aher et al. [26]. Upon collection, the residue was oven-dried at 60 ◦C and ground in a Wiley mill to pass a <2 mm screen. Bulk Forman soil was collected at the Conservation Cropping System Project site [26] near Forman, North Dakota (97◦38 38" N 46◦05 05" W). The bulk soil sample was air-dried, crushed, and sieved through a 2 mm screen. Residue treatments (*n* = 18) were prepared containing 15 g of soil mixed with 15 g of quartz sand (20 mesh) with 0.50 g of residue placed on the soil surface following the procedure of Stanford and Smith [39]. The quantity of residue represents the equivalent of 6.25 Mg/ha in a field environment [7]. Soil only (*n* = 3) contained 15 g of soil mixed with 15 g of sand. Samples were then transferred to labeled glass leaching tubes where the soil was placed into the tube, and crop residue was then placed on top of the soil surface to simulate no-till residue accumulation. Leaching tubes were kept in a constant temperature room at 22 ◦C simulating average soil temperatures in the region during the growing season and incubated as described by Stanford and Smith [39]. Before the soil samples were transferred to the leaching tubes, a small amount of glass wool was placed at the bottom of the leaching

tubes to prevent soil sediment loss during leaching and was placed at the top to prevent soil and residue disturbance during leaching solution application.

**Table 1.** Six crop residue treatments used for the five incubation cycles, their scientific name, abbreviation, and C/N ratios.


**<sup>1</sup>** C/N ratios for corn, soybean, forage radish, winter pea, spring wheat, and winter wheat were determined using near-infrared reflectance (NIR).

Five incubation cycles and four freeze–thaw periods were conducted for the experiment. Each cycle represented an annual growing season. Between each cycle, the soil treatments were frozen for three weeks to represent an annual freeze–thaw cycle. Following each freeze (*n* = 4), an additional 0.5 g of individual crop residue was placed on the soil surface (total = 2.5 g per leaching tube over the course of the study). Biweekly leachings were conducted at 2, 4, 6, 8, and 10 weeks for the first cycle. For the second cycle, additional leaching was conducted at 12 weeks. For cycles three, four, and five, additional leachings were conducted at 12 and 14 weeks to determine the potential of cumulative effects of the simulated growing seasons. For the first leaching of the samples and due to the air-dry nature of the soil/sand/residue column, 50 mL of 0.01 M CaCl2 was added to the glass tubes in 10 mL increments, followed by 10 mL of nutrient solution as described by Stanford and Smith [39] to remove ambient levels of NH4+-N and NO3-N. Subsequent leachings used 30 mL of 0.01 M CaCl2 and 10 mL nutrient solution. Parafilm® (Menasha, WI, USA) was used to cover the incubation tubes between leachings to prevent contamination and preserve soil water content during this study. Air vents were created in the film to allow for soil respiration. At the end of an incubation period, the leaching tubes were then transferred into the freezer for three weeks at 0 ◦C. Leachate was collected, covered, and refrigerated (8–24 h) if necessary until mineral N (NO3-N and NH4-N) analysis was conducted using a Timberline TL 2900 NH4/NO3 analyzer (Timberline Instruments Inc., Boulder, CO, USA). Results are adjusted for soil mass and reported as mg N kg−<sup>1</sup> soil in order to relate our results to quantities that might be experienced in field environments.
