Impacts of a polyethylene silage pile underlay plastic with or without enhanced oxygen barrier (EOB) characteristics on preservation of whole crop maize silage, as well as a short investigation of peripheral deterioration on exposed silage faces

Accepted Manuscript Title: Impacts of a polyethylene silage pile underlay plastic with or without enhanced oxygen barrier (EOB) characteristics on preservation of whole crop maize silage, as well as a short investigation of peripheral deterioration on exposed silage faces Author: P.H. Robinson N. Swanepoel PII: DOI: Reference:

S0377-8401(16)30051-7 http://dx.doi.org/doi:10.1016/j.anifeedsci.2016.02.001 ANIFEE 13462

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Received date: Revised date: Accepted date:

11-9-2015 22-1-2016 2-2-2016

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Please cite this article as: Robinson, P.H., Swanepoel, N., Impacts of a polyethylene silage pile underlay plastic with or without enhanced oxygen barrier (EOB) characteristics on preservation of whole crop maize silage, as well as a short investigation of peripheral deterioration on exposed silage faces.Animal Feed Science and Technology http://dx.doi.org/10.1016/j.anifeedsci.2016.02.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Impacts of a polyethylene silage pile underlay plastic with or without enhanced oxygen barrier (EOB) characteristics on preservation of whole crop maize silage, as well as a short investigation of peripheral deterioration on exposed silage faces P.H. Robinson* [email protected], N. Swanepoel Department of Animal Science, University of California, Davis, CA, 95616, USA *

Corresponding author.

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Highlights - Use of thin inner (i.e. between the silage and the main plastic cover) plastic films with enhanced oxygen barrier (EOB) properties are recognized by some governmental agencies as a mitigation of silage deterioration. - In four large maize silage piles, underlay film with or without EOB properties had no impact on silage fermentation parameters in the outer 25.4 cm of the silage pile, or in 25.4 to 50.8 cm depth below the surface of closed silage piles through 6 mo post pile building. - In a 5th pile, there was clear evidence of deterioration in the surface silage to a 25.4 cm depth immediately behind the exposed silage face, which was not impacted by type of underlay film. - In a 6th pile, surface spoilage only occurred behind the exposed silage face, and it moved into the pile at a similar rate as silage removal from the face. - Results do not support use of a thin plastic underlay film with EOB properties, versus one without, since air ingress to the silage mass through the silage pile cover was minimally causative of silage deterioration, which was associated with the exposed face.

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Abstract Large silage piles, up to 15,000 tonnes, common in some dairy areas, present challenges since their large surface area creates an enhanced potential for oxygen to penetrate the mass. Use of thin inner (i.e. between the silage and main plastic cover) plastic films with enhanced oxygen barrier (EOB) properties are recognized by some governmental agencies as a mitigation of silage deterioration even though underlay films are generally accepted to only potentially impact the outer 30 to 50 cm of the silage in a pile. In four large maize silage piles, underlay film with or without EOB properties had no impact on silage fermentation parameters indicative of spoilage in the outer 25.4 cm of the silage pile, or in the 25.4 to 50.8 cm depth below the surface of closed silage piles at ~3 and at ~6 months post pile building. In contrast, in a 5th pile, there was evidence of deterioration in the surface silage to a 25.4 cm depth immediately behind the exposed silage face, which was not impacted by type of underlay film. A final experiment in a 6th pile showed that surface spoilage occurred well behind the exposed silage face, and that it moved into the pile at a similar rate as silage was removed from the face. Results do not support use of a thin plastic underlay film with EOB properties, versus one without, since air ingress to the silage mass through the silage pile cover appeared minimally causative of silage deterioration, which was associated with the exposed face. Maize silage deterioration of exposed face silage would likely be minimized by increasing speed of exposed face movement, and/or use of weight lines directly behind the exposed face, as has been recommended by others. Keywords: silage; maize; plastic underlay film; oxygen barrier; polyethylene

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1. Introduction Maize silage is the most important ensiled crop in most developed dairy areas. However spoilage of silage while it is ensiled are an economic loss to dairy farmers. One of the critical points to control spoilage in silage is to limit, as much as possible, oxygen ingress to the silage mass since it supports growth of aerobic microorganisms and the resulting heat production can lead to silage with degraded nutritional quality (Woolford, 1984).

Indeed Bolsen et al. (1993)

demonstrated the beneficial impacts of ‘sealing’ the surface of an ensiling mass with a plastic cover weighted with tires on several measures of silage spoilage. Since that time, many management practices have been suggested to reduce maize silage spoilage by reducing oxygen availability in the ensiled mass (e.g., Bolsen, 2006; Wilkinson and Davies, 2012), and many are now very commonly used commercially. These include creating a high pack density at silage pile building, rapid covering of the ensiled mass with a plastic cover, sealing the periphery of the pile with soil or weights and using ‘weight lines’ along the area of plastic overlap on the pile surface. However a relatively simple practice, recommended by Bernardes et al. (2012) and Wilkinson and Davies (2012), as well as others in the commercial literature, which has gained wide use on commercial maize silage piles, is use of a thin inner plastic film (i.e., between the silage mass and the main plastic cover), generally containing enhanced oxygen barrier (EOB) properties. The meta-analysis of Wilkinson and Fenlon (2013) seems very convincing that use of inner plastic cover films with EOB characteristics on silage, versus non-EOB plastics, reduced dry matter (DM) losses and increased aerobic stability, amongst many other beneficial impacts. However 9 of 21 studies in Wilkinson and Fenlon (2013) did not specify the covering of the plastic silage covers on the piles (i.e. on top of the outer plastic cover), and 6 of 21 studies confounded the plastic silage covers with its protective covering (i.e., lines of ½ car tires or protective nets – which is important since nets transmit less solar radiation to the silage surface - and protect the plastic from bird and rodent damage - than ½ tire chains which are associated with dark ‘ring shadows’). Thus it appears that at least some of the benefits attributed by Wilkinson and Fenlon (2013) to EOB characteristics in plastic films and covers may have been due to the protective cover, or in reality the ‘silage covering system’, which was the case in 6 of the 21 Wilkinson and Fenlon (2013) studies. While the Wilkinson and Fenlon (2013) dataset included many studies with maize silage (13 of 21), most were laboratory scale or small bunkers, and there were only two studies (i.e., Kuber et al. 2008; Basso et al. 2009) with large surface area maize silage piles which create an enhanced potential for oxygen to penetrate the ensiled mass. While these 2 studies reported strong treatment effects, what the studies actually compared was a single layer of 125 micron polyethylene versus a layer of the same polyethylene over an inner 45 micron EOB film, which confounds use of an inner film with the EOB characteristics of the film. Thus there are no studies comparing a thin underlay

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film with EOB properties to a polyethylene film with a similar thickness without EOB properties in large maize silage piles (or any silage structures). The objectives of our study were to measure fermentation characteristics of maize silage indicative of silage spoilage, as impacted by use of thin plastic underlay films with or without EOB characteristics. In Experiment 1, two separate silage pile geographic orientation experiments, each with 2 maize piles, were completed to examine impacts of these films on silage fermentation characteristics in the outer 50.8 cm of the surface (as the 0 to 25.4 and 25.4 to 50.8 cm depths) at 2 times after pile building, but before silage feedout. In Experiment 2, underlay film impacts were measured on silage at a 60 cm depth below the surface and in the outer 25.4 cm of the surface, as well as on silage from deep in the pile, all during silage pile feedout. Due to unanticipated results of these two experiments relative to the underlay folms, Experiment 3 was completed in which impacts on silage deterioration of the distance of the silage surface core point from the exposed silage face at different times relative to silage pile feedout was assessed, in order to identify the reasons for peripheral deterioration on exposed silage faces.

2. Materials and Methods 2.1. Experiment 1 Four large ‘wedge type’ maize silage piles were constructed between September 17 and October 13 (2013) in the Northern San Joaquin Valley of California (USA) to examine surface spoilage of silage under the cover plastic at ~3 and at ~6 months post pile building (i.e., indicative of relatively short and long times of silage fermentation prior to feedout) as impacted by plastic underlay film. Based upon long-term pre-experiment visual experiences which suggested there were different impacts of the side face orientation of East/West versus North/South silage piles, two of the piles had an East/West orientation and two had a North/South orientation. 2.1.1. Experiment 1a The two piles with an East/West orientation (average 5,600 tonnes as built) were Experiment 1a. After pile construction was complete, all piles were covered within 48 h near one end with alternate coverage (Figure 1) of a clear, pliable polyethylene film (POLY) of 40.6 microns (ARI Co., Belmont, CA, USA; trade name ‘HiTec Underlay’) or an enhanced oxygen barrier plastic film (EOB) of 45.7 microns (Industria Plastica, Mongralese, Italy; trade name ‘Silostop’). Experimental sections on the pile surfaces were created with 15 m wide plastic sheets with ~0.8 m overlaps at each side and the top of the piles. All piles were covered with 127 micron white/black plastic, white side out, and covered with side-by-side rings of ½ tires – with treatment overlaps covered with 2 rings of ½ tires.

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At 2.4 months after covering, and again at 6.0 months, the pile surfaces were core sampled. On each core sampling occasion in each of the four sections of each pile (Figure 1), sampling was by coring through the silage cover plastic four times at two levels, being 1/3 of the way up each side (Low level; 2 cores/section) and 2/3 of the way up the side (High level; 2 cores/section), where side heights were ~13 m. Cores were separated by a minimum of 1 m. The coring device was a 4.76 cm (inside diameter) by 61 cm length stainless steel tube driven by an 18 volt cordless Ridgid drill (Model R86008; Ridgid Tool Co., Elyria, OH, USA) and with 2 marked segments of 25.4 and 50.8 cm from the tip. Each coring event consisted of first coring to a depth of 25.4 cm (outer core), followed by re-coring of the same hole to 50.8 cm (inner core). The stainless steel core was cleared with a plunger after each sample and any remaining silage in its teeth was removed prior to the next coring. Immediately upon removal of each silage core, the temperature of the exposed end of the core was measured with a handheld infrared thermometer (Fluke-561, Everett, WA, USA), and a small portion, ~8 g, of silage was placed in a 150 ml plastic cup, 80 ml of distilled water added and, after 2 min, the pH was read on an ‘Oyster 15’ pH meter (Extech Instruments, Nashua, NH, USA). Each core was then put into a 3.8 L plastic bag from which, after mixing in the bag, 15 g was removed to a 0.5 L plastic bag and placed in an ice chest. The balance of the sample (2 samples from each level within each section by core depth) were combined in a single 3.8 L plastic bag, mixed, and put in an ice chest. Thus each of the 4 experimental treatment sections on each pile resulted in 4 individual inner core and 4 individual outer core samples for mold and yeast count analysis (the 15 g samples), as well as 2 pooled (by level) inner core and 2 pooled (by level) outer core samples for analysis of DM, volatile fatty acids (VFA) and ethanol, by methods described below, at each coring time post pile construction. The silage piles were treated as replicates within sub-experiment. As sampling ‘level’ on the piles sides (Figure 1) was not statistically important, pile side level values were arithmetically combined prior to final statistical analysis.

Data were statistically analyzed using the GLM

Procedure of SAS (2014) and included time of sampling (i.e., months post covering), side face exposure (i.e., North and South), core depth (i.e., inner and outer cores) as well as treatment (i.e., POLY and EOB underlay films) as fixed effects. Interactions of underlay film with side face and sample depth were also examined. 2.1.2. Experiment 1b The two piles with a North/South orientation (average 6,990 tonnes as built), were Experiment 1b. After pile construction was complete, all piles were covered as described for Experiment 1a. At 3.2 months after covering, and again at 5.7 months, pile surfaces were core sampled as described for Experiment 1a.

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The silage piles were treated as replicates within sub-experiment. As sampling ‘level’ on the piles sides (Figure 1) was again not statistically important, pile side level values were arithmetically combined prior to final statistical analysis.

Data were statistically analyzed using the GLM

Procedure of SAS (2014) and included time of sampling (i.e., months post covering), side face exposure (i.e., East/West), core depth (i.e., inner and outer cores) as well as treatment (i.e., POLY and EOB underlay films) as fixed effects. Interactions of underlay film with side face and sample depth were also examined. 2.2. Experiment 2 A pile of maize silage (8,620 tonnes as built) was constructed in a single filling episode in September (2014) to examine surface, face edge and deep mass spoilage of silage during pile feedout as impacted by underlay film. The pile had an East/West orientation and, near one end of the pile, was alternately covered (Figure 2) with a clear pliable polyethylene film (POLY) of 40.6 microns or an enhanced oxygen barrier plastic film (EOB) of 45.7 microns, as described in Experiment 1, within 48 h in order to create one section of each treatment on each side of the pile. The balance of the covering process was as described for Experiment 1. Thirty days after filling (i.e., in October), the East end of the pile was opened and silage was removed daily with a front end loader while maintaining a straight and flat exposed silage face. At 6 months post filling (i.e., in March when the exposed silage face reached experimental Section 1 (Figure 2). Approximately 0.4 and 0.6 of the distance into each of Sections 1 and 2, the exposed face and surface behind the face (i.e., 0.5 m into the plastic cover, or ~1.5 m from the exposed face, since ~1 m of silage had been exposed by surface plastic removal) were core sampled, according to a grid (Figure 3), to create silage samples which represented the deep mass (i.e., cores 8, 9), the outer edge of the face (i.e., cores 2, 4, 6; 60 cm below the surface), and the surface behind the exposed face (i.e., cores 1, 3, 5, 7) for each side of each section. Thus samples 2, 4, 6, 8 and 9 were cored horizontally into the exposed silage face while samples 1, 3, 5 and 7 were cored into the pile surface perpendicular to the outer surface. Each of the 4 coring events (i.e., 2 per experimental section; Figure 2) used the same coring device described in Experiment 1, except that it was marked into 4 segments of 12.7 cm from the tip. Each coring event consisted of coring all 9 pre-marked locations per treatment side of each section (Figure 3). For deep mass samples 8 and 9, each core consisted of 4 samples of 12.7 cm segments with an initial depth of 12.7 cm (A core), followed by re-coring of the same hole to 25.4 cm (B core), re-coring the same hole to 38.1 cm (C core) and a final re-coring to 50.8 cm (D core). Upon removal of each 12.7 cm core sample, the temperature of its exposed end was immediately measured with the infrared thermometer, and a small portion of silage from the exposed end was measured for pH as described in Experiment 1. Samples were pooled by core depth (i.e., the 2 ‘A’

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cores etc.) to create 4 samples (depths) per coring event per treatment side, which were then subsampled into two 3.8 L plastic bags, to create replicate samples (i.e. one remain fresh and frozen while other is dried). For edge (of the face) cores 2, 4 and 6, each sample consisted of a single 50.8 cm core which was 60 cm under the surface. Immediately upon removal of each core sample, the temperature of the exposed end was measured with the infrared thermometer, and a small portion of silage from the exposed end was measured for pH as described above. All edge samples were pooled to create one edge sample per coring event per treatment side, which was then subsampled into two plastic bags. For surface cores 1, 3, 5 and 7, each sample consisted of a single 25.4 cm core. Immediately upon removal of each core sample, the temperature and pH were measured as described above. All surface samples were pooled to create 1 surface sample per coring event per treatment side, which was then subsampled into two plastic bags. Thus a total of 48 duplicated samples were created (i.e., 6 samples per treatment side (2) for each coring event (2) in each of Sections 1 and 2). Each of the 48 final wet (i.e. as sampled and frozen) samples described above were analyzed for DM, VFA’s and ethanol after the final coring event was complete. Their replicate samples, dried at 55oC, were analyzed for N, neutral detergent fibre (NDF) and acid detergent fibre (ADF). Data were statistically analyzed using the GLM Procedure of SAS and included location (i.e. main mass, edge, surface) as well as treatment (i.e. POLY and EOB underlay films) as fixed effects, as well as their interaction. Prior to statistical analysis, main mass cores ‘A’, ‘B’, ‘C’ and ‘D” data were arithmetically pooled to create a single main mass sample per treatment side within each section by coring event. Thus all statistical analyses had 8 observations. 2.3. Experiment 3 A pile of maize silage (2,270 tonnes as built) was constructed in a single filling episode in September (2014) to examine the progression of spoilage into the silage pile from the exposed face during pile feedout, as determined by sampling the silage under the undisturbed plastic cover up to the exposed face in two events which were separated by face silage removal. The pile had a North/South orientation and was completely covered within 48 h of pile building with an enhanced oxygen barrier plastic film (EOB) of 45.7 microns, as described in Experiment 1. The balance of the covering process was as described for Experiment 1. At the point when the exposed face was ~23.5 m from the beginning of the back ramp slope, both sides and the back ramp of the pile were cored to 50.8 cm in two segments of 25.4 cm each according to the grid in Figure 4, using the coring procedure described in Experiment 1. This created a total of 10 sampling sites, each being ~0.5 of the way up both sides, and back ramp, with 2 sites on the back ramp and 4 sites on each side. The cores on the back ramp were ~0.33 and 0.67 of the distance across the back ramp, while the cores on each side were 0.5, 7, 13.5 and 20 m back

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from the cut line of the silage cover of EOB plastic and 127 micron black/white coverage plastic on each side of the silage pile. There was ~2 m of exposed silage between the cut line of the plastic and the actual exposed face, and ~0.3 m (depth) of silage had been removed from the ~1 m of exposed silage directly behind the exposed face by the dairy feed crew due to their perception of an unacceptable extent of deterioration. The pile was re-cored 69 d later using the same procedures, except that core points 1, 2, 9 and 10 (see Figure 4) no longer existed due to planned silage removal and core points 3 and 8 now had the same pile face spatial relationships (i.e., being closest to the exposed face) that the now nonexistent core points 1 and 10 had had 69 d earlier. Thus core sites on each side of the pile were 0.5 and 7 m (previously 13.5 and 20 m for the same core sites) behind the cut line of the silage cover EOB plastic and 127 micron black/white coverage plastic on each side of the silage pile. Face management at the second coring was similar to that at the first. Data were statistically analyzed using the GLM Procedure of SAS and included location (i.e., distance from the exposed face) as well as core depth (i.e., outer and inner) as fixed effects, and their interaction. Pairs of core points with the same spatial orientation to the face from the two sides of the pile were treated as replicates. Due to the planned removal of 4 of the original core points due to feedout between sampling events, each coring event was statistically analyzed separately. 2.4. General Measurements and Calculations 2.4.1. Temperature records A portable weather data logger (HOBO U23; Onset, Bourne, MA, USA) recorded ambient temperatures every 30 min throughout the period that silage piles were active at each pile site. Each station was placed in the area as near as possible to the pile that was protected from direct sunlight. Weather stations were ~2 m above ground level on poles. 2.4.2 Mold and Yeast counts/scores As mold and yeast counts can vary from zero to >5,000,000 cfu/g wet weight (WW) in silage, presentation of their counts as average cfu/g WW can result an unrepresentative average of the bulk of the samples due to one very high value. Thus while mold and yeast counts are listed in tables as cfu/g WW, values were also converted to scores reflective of contemporary assessments of the meaning of silage mold and yeast counts relative to impacts on animals fed the silage. While numerous mold and yeast scoring systems are available in the commercial literature, and used by commercial laboratories, we melded several with our own experiences to create the score conversion: 1 = <0.5 million cfu/g WW (low) 2 = <1.0 million cfu/g WW (safe to feed) 3 = <2.0 million cfu/g WW (caution in feeding)

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4 = <3.0 million cfu/g WW (observe cattle closely) 5 = <5.0 million cfu/g WW (dilute with other silage) 6 = >5.0 million cfu/g WW (do not feed to cattle) For yeast counts, the score conversion was: 1 = <0.1 million cfu/g WW (low) 2 = <1.0 million cfu/g WW (reduced aerobic stability) 3 = <5.0 million cfu/g WW (no aerobic stability) 4 = >5.0 million cfu/g WW (excessive aerobic instability) 2.4.3. Density calculation The density of each core was calculated as weight of the cored silage to the volume of the core. 2.5. Analytical procedures Samples of silage were dried at 55oC for 48 h and air equilibrated for 24 h before chemical analysis. All samples were ground to pass a 1 mm screen on a model 4 Wiley Mill (Thomas Scientific, Swedesboro, NJ, USA). Oven DM was determined as the gravimetric loss when the air equilibrated sample was dried at 105oC for 3 h in a forced air oven. Total N was determined by the Leco method (#990.03, AOAC, 2006). Neutral detergent fibre (NDF) was determined using neutral detergent and heat. Heat-stable amylase (aNDF) was used to remove starch (#2002.04, AOAC, 2006). Acid detergent fiber (ADF) was determined as the residue after AD extraction. Both NDF and ADF are reported ash-inclusive. All acid and ethanol assays were assayed using high performance liquid chromatography (HPLC; Muck and Dickerson, 1988). The HPLC system used a refractive index detector (MA model 2414; Waters Corporation, Milford, CN, USA) and a Bio-Rad Aminex HPX-87H column (Bio-Rad Laboratories, Hercules, CA, USA) at 35oC. Mold and yeast counts were determined according to Adesogan et al. (2004).

3. Results 3.1. Experiment 1 3.1.1. Impacts in North/South sited piles (Table 1) The average ambient temperature from the time that the North/South sited piles were constructed until the end of the second coring event was 11.6oC. Wet density was higher for inner versus outer cores (P <0.01), but the extent of this difference was less (P <0.01) at 5.7 versus 3.2 months. That our values are generally lower than those reported by others (e.g., Kohler et al. 2013 of 691 kg/m3) is because they were on the surface of the silage mass. The pH was not impacted at 3.2 months, but higher (P =0.05) for West versus East sides at 5.7 months. Temperatures were higher (P <0.01) at 5.7 versus 3.2 months, and higher (P

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<0.05) at 3.2 months for inner versus outer cores only. The DM was higher (P <0.01) for inner versus outer cores, and were lower (P <0.01) at 5.7 months. Yeast counts were higher (P =0.02) at 5.7 months, but much more so for outer versus inner cores (P <0.05). Yeast scores also were higher (P <0.01) at 5.7 mo, and tended (P =0.06) to be higher for West versus East sides and outer versus inner cores. Lactic acid concentrations were higher (P =0.02) for inner versus outer cores, but not impacted by coring time, whereas acetic acid concentrations increased with time, although the time*depth interaction (P =0.06) suggested lower concentrations in outer versus inner cores at 3.2 months, but the reverse at 5.7 mo. Succinic and butyric acid concentrations were higher at 5.7 months (P <0.01), and the latter tended (P =0.05) to be higher on West versus East sides. Ethanol was much higher at 5.7 months, but much more (P =0.05) in outer versus inner cores at 5.7 versus 3.2 months. 3.1.2. Impacts in East/West sited piles (Table 2) The average ambient temperature from the time that the East/West sited piles were constructed until the end of the second coring event was 14.8oC. Wet density was lower (P =0.02) at 6 months, although its extent was small since it was solely (P <0.05) in the inner versus outer cores. The pH was lower (P <0.01) in North versus South sides, and in inner versus outer cores. Temperatures increased (P <0.01) with coring time post pile building, but only differed (P <0.01) for South versus North sides, and for outer versus inner depths. In addition, they were higher (P <0.05) on South versus North sides at 6 months, and higher for inner versus outer cores at 2.4 months. The DM decreased with time, although it was solely (P <0.05) in outer versus inner cores. Mold counts and scores increased with time (P <0.01 and 0.02), although values were very low in general. Yeast counts were not impacted, but yeast scores were higher (P <0.01) on North versus South sides and in outer versus inner cores. Lactic acid concentrations were higher (P =0.01) for inner versus outer cores, and higher (P =0.02) at 6 mo. Acetic acid concentrations were also higher (P <0.01) at 6 mo, and higher (P =0.01) for inner versus outer cores. Propionic acid levels were lower (P =0.03) at 6 months, but the time*depth interaction (P =0.05) suggested this was primarily in inner versus outer cores. Succinic acid concentrations were higher at 6 months (P =0.01), but this was only (P <0.05) on the South side which differed from the North side at 6 mo. Formic acid, detectable at 2.4 months, was lower (P =0.02), and no longer detectable, at 6 months. 3.2. Experiment 2 (Table 3) It took 68 days for the pile face to move through both experimental sections, or 2.9 m/wk. The average ambient temperature during this period was 19.2oC. As there were no location by treatment interactions (i.e., P >0.20), results are summarized by main effect.

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The underlay film had no impacts on any response parameter, except that the aNDF level of the silage under the POLY film was higher (P <0.05) than that of the silage under the EOB film. However the numerical extent of this difference was modest. Silage wet density was much higher (P <0.05) in the deep mass versus at the surface or edge, although that of edge silage was higher (P <0.05) than that of surface. Surface silage pH was lower (P <0.05) than that in edge and deep mass, whereas the temperature was highest (P <0.05) in surface silage. The DM content of the silage, as well as concentrations of lactic acid and all VFA, were not impacted by location of silage in the pile. In contrast, ethanol concentrations were highest (P <0.05) in silage from the deep mass, with edge and surface silage having similar concentrations. Crude protein concentrations were highest (P <0.05) in surface silage, with edge and deep mass silage having similar concentrations. While deep mass aNDF concentrations were lower (P <0.05) than in edge and surface silage, the extent of the differences were numerically modest and, in light of similar values for ADF, unconvincing of a real difference.

3.3. Experiment 3 (Table 4) It took 69 days for the pile face to move from the first to the second coring date, or 1.7 m/wk. The average ambient temperature during this period was 16.2oC. 3.3.1. Impacts at first coring (t=0 d). Wet density was higher (P <0.01) in inner versus outer cores. The pH tended (P =0.06) to be higher for outer versus inner cores, but this was solely due to higher values at locations 1 and 10 and 2 and 9 (i.e., those core points nearest the exposed silage face). In spite of no overall statistical impact of the location*depth interaction (i.e., P =0.21), outer versus inner cores had higher (P =0.05) pH at these locations. Temperatures were highest (P <0.05) at locations 1 and 10 nearest the exposed face. In contrast, mold and yeast counts and scores were higher (P <0.05) at locations 2 and 9 than at 3 and 8, 4 and 7 (i.e., on the back ramp of the pile), with values at locations 1 and 10 intermediate. 3.3.2. Impacts at second coring (t=69 d) Wet density was higher (P <0.01) in inner versus outer cores. The pH was higher (P <0.01) for outer versus inner cores, but this was solely due to higher values at locations 3 and 8 and 4 and 7 (i.e., those nearest the exposed face).

In spite of a weak overall statistical impact of the

location*depth interaction (i.e., P =0.11), outer versus inner cores had higher (P =0.05) pH at locations 3 and 8, and 4 and 7. Temperatures were lowest (P <0.05) at locations 5 and 6 (i.e., on the back ramp). Mold counts and scores tended to be lower (P =0.09) at locations 5 and 6, and outer versus inner cores had higher (P =0.05) mold scores and counts at locations 3 and 8, and 4 and 7. In contrast, yeast counts and scores were statistically similar at all locations and at both

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depths. In general, impacts of location and depth relative to the exposed face at scoring #1 had simply ‘migrated’ into the pile with its use due to silage removal from the exposed face.

4. Discussion Use of a thin underlay plastic film with EOB properties had no impact on the extent of deterioration of maize silage under the plastic cover prior to pile feedout in either the outer 25.4 cm of the surface or the 25.4 to 50.8 cm core depths at either ~3 or ~6 6months after pile building. Indeed the lack of underlay*depth and underlay*time interactions in Experiment 1 make it unequivocal that the EOB properties of the underlay film were either too small to matter, not required, or both, for up to 6 months post pile covering.

Based upon the relatively normal

fermentation profiles (i.e., within the ranges reported by Weissbach and Strubelt, 2008), and lack of substantive differences between the inner and outer surface cores, it is also clear that the silage at, and immediately under, the silage surface was well fermented with little evidence of deterioration, even 6 months after pile building. That these results appear to contrast findings of the metaanalysis of Wilkinson and Fenlon (2013), which suggested less silage deterioration under EOB versus POLY plastics, may be because none of those meta-studies actually compared inner films with or without EOB properties. In addition, some of the meta-studies did not specify the outer surface protective covering of the silage plastic covers, such as lines of ½ car tires or protective nets, and some of the studies used different protective coverings for the films with and without EOB characteristics. Thus it seems that some of the benefits attributed to EOB plastics were due to a ‘silage covering system’, which included EOB plastics, rather than the EOB film per se. Results of Experiment 2, where silage was sampled during pile feedout, are consistent with those of Experiment 1 in that there was no discernable effect of underlay film on fermentation characteristics of the outer 25.4 cm of silage in the pile. However in contrast to Experiment 1, there is evidence that silage in this outer 25.4 cm surface core had deteriorated (i.e., higher temperatures and CP, lower pH, and numerically lower lactic and acetic acid concentrations), although there was no differences between silage 60 cm under the surface or in the deep silage mass. This contrasts to Borreani and Tabacco (2012) which suggests that the 2.9 m/wk rate of exposed pile face movement in this pile should have been fast enough to prevent face spoilage. Nevertheless the differences between Experiments 1 and 2 in the extent of silage deterioration in the outer 25.4 cm of the pile surface, where the only substantive differences in piles between experiments was that Experiment 1 cored the surface of closed piles whereas Experiment 2 cored the surface of a pile in direct proximity to the exposed face, suggests that it was the exposed face per se which facilitated silage deterioration. Indeed the exposed face, and the likelihood of air ingress between the cut plastic and the surface of the silage seemed likely to be causative of silage deterioration.

Thus the

14

recommended mitigation of surface silage spoilage to use ‘weight lines’ where the cover plastic sheets overlap (Bolsen, 2006) could be extended to use of moveable ‘weight lines’ directly behind the exposed silage face, even with relatively rapid face movement relative to Borreani and Tabacco (2012) suggestions. The impact of our exposed face, not sealed with a weight line, on deterioration of silage at and directly behind the face was confirmed in Experiment 3. The slower face movement rate on this pile, 1.8 m/wk, should have been fast enough to prevent face spoilage (Borreani and Tabacco, 2012). Nevertheless, silage as far as 7 m from the exposed face had deteriorated, at least in the outer 25.4 cm core, regardless of the location in the pile of the coring event per se. It seems that deterioration began on the silage surface under the plastic cover before penetrating the silage mass as the exposed face approached the core site. Thus it appears that when silage removal rate from a pile, and therefore face movement rate, is too slow relative to the fermentation characteristics of the silage that the rate of penetration of molds and yeasts into the silage mass caused by air ingress between the cut plastic and the silage surface will exceed the rate of removal of silage from the exposed face. This process will lead to continuous removal and discarding of spoiled silage from the surface of the silage pile close to the exposed face even though silage 13.5 m (in our study) behind the face had not deteriorated at all. The POLY and EOB silage underlay films had similar impacts on indices of silage deterioration in the outer 25.4, and the 25.4 to 50.8 cm depth, of maize silage pile surfaces prior to opening, or during pile feedout ~60 cm under the pile surface at the exposed face, or in the deep silage mass of the pile. However the surface 25.4 cm in direct proximity to the exposed face had signs of deterioration (which was not impacted by underlay film) based upon increased temperatures and ethanol concentrations, and declining lactic and acetic acid concentrations. Further investigation of surface spoilage during unloading showed that it was ‘moving’ with the exposed face such that silage 7 m back of the cut edge of the cover plastic had deteriorated in the outer 25.4 cm core, with no deterioration 13.5 m back of the cut edge of the cover plastic, regardless of the amount of silage which had been removed from the pile. It seems clear that deterioration of maize silage (often visible on the periphery of the exposed face at pile unloading) is primarily a function of exposure at the face rather than on the surface of the pile while it is under the plastic silage pile cover behind the exposed face. That this EOB film had no advantage over a similar polyethylene film without EOB properties is likely because oxygen ingress to the pile through the surface plastic was not causative to silage deterioration.

15

5. Conclusions Taken together, results do not support use of a thin plastic underlay film with EOB properties, versus one without EOB properties in maize silage piles, at least if silage feedout is prior to ~6 mo after pile building, since it seems that ingress of air to the silage mass through the plastic cover of the silage pile during this period is minimally, or not at all, causative to silage deterioration, which was only associated with the exposed silage face. Silage deterioration at the exposed face appears likely to be minimized by increasing the speed of exposed face movement over the ground, and/or use of moveable weight lines directly behind the exposed face. However further research is required to investigate the interaction of these factors.

Conflict of Interest The authors declare that they have no conflict of interest regarding this manuscript.

Acknowledgements The authors thank Mr. Jim Rallies of ARI (Belmont, CA, USA) for assistance in organization of the creation of most of the silage piles utilized in this study. The authors are also grateful to the cooperating dairy farmers who allowed us to use their maize silage piles for this project.

16

References Adesogan, A.T., Krueger, N, Salawu, M.B., Dead, D.B., Staples, C.R., 2004. The influence of treatment with dual purpose bacterial inoculants or soluble carbohydrates on the fermentation and aerobic stability of bermudagrass. J Dairy Sci. 87, 3407-3416. AOAC, 2006. Official methods of analysis of AOAC International, 18th ed., AOAC International, Arlington, VA, USA. Basso, F.C., Bernades, T.F., Casagrande, D.R., Lodo, B.N., Roth, A.P.T.P., Reis, R.A., 2009. Aerobic deterioration in corn silage sealed with oxygen barrier film under farm conditions. In: Proc. XV International Silage Conference, pp 213-214. Madison, WI, USA. Borreani, G., Tabacco, E., 2012. Effect of silo management factors on aerobic stability and extent of spoilage in farm maize silages. In: Proc XVI International Silage Conference. Ed: K. Kuoppala, M. Rinne, A. Vanhatalo. pp 71-72. Bernardes, T.F., Nussio, L.G., do Amaral, R.C., 2012. Top spoilage losses in maize silage sealed with plastic films with different permeabilities to oxygen. Grass Forage Sci. 67, 34-42. Bolsen, K.K., 2006. Silage management: Common problems and their solution. In: Proc. Tri-State Dairy Conference, pp 83-94. Bolsen, K.K., Dickerson, J.T., Brent, B.E., Sonon, R.N., Dalke, B.S., Lin, C., Boyer, J.E., 1993. Rate and extent of top spoilage losses in horizontal silos. J Dairy Sci. 76, 2940-2962. Köhler, B., Diepolder, M., Ostertag, J., Thurner, S., Spiekers, H., 2013. Dry matter losses of grass, lucerne and maize silages in bunker silos. Agric. Food Sci. 22, 145-150. Kuber, R., Bolsen, K.K., Wigley, S., Wilkinson, J.M., Bolsen, R.E., 2008. Preservation efficiency and nutritional quality of whole-plant corn sealed in large pile silos with and oxygen barrier film (Silostop) or standard polyethylene film. In: Proc. XIII International Conference on Forage Conservation, pp 178-179. Nitra, Slovak Republic. Muck, R.E., Dickerson, J.T., 1988. Storage temperature effects on proteolysis in alfalfa silage. Trans. of the ASAE, 31, 1005-1009. SAS Institute. 2014. SAS/STAT Users Guide, Release 9.1. SAS Inst. Inc., Cary, NC, USA. Weissbach, F., Strubelt, C., 2008. Correcting the dry matter content of maize silage as a substrate for biogas production. Landtechnik, 63, 82-83. Wilkinson, J.M., Fenlon, J.S., 2013. A meta-analysis comparing standard polyethylene and oxygen barrier film in terms of losses during storage and aerobic stability of silage. Grass Forage Sci. 69, 385-392. Wilkinson, J.M., Davies, D.R., 2012. The aerobic stability of silage: key findings and recent developments. Grass and Forage Sci. 68, 1-19. Woolford, M.K., 1984. The Silage Fermentation. Marcell Dekker, Inc., New York, NY, USA.

17

Figure Captions Figure 1. Aerial view of the alternate sections of underlay film coverage in each of the four maize silage piles used in Experiment 1. The four dots in each section indicate the approximate locations of the coring points which were about 0.4 and 0.6 of the way across a section (crosshatch shading is the area of underlay film overlap).

18

Figure 2. Aerial view of the alternate sections of underlay film coverage in the maize silage pile used in Experiment 2. [Coring events 1 and 3 were ~0.4 of the distance across each section and coring events 2 & 4 were about ~0.6 of the distance (crosshatch shading is the area of underlay film overlap).]

19

Figure 3. End view of the face of the maize silage pile used in Experiment 2 showing the sampling pattern used on each of four occasions (i.e., twice in each section as shown in Figure 2). [Sample points 2, 4, 6, 8 and 9 were cored horizontally into the face to 50.8 cm, whereas core points 1, 3, 5 and 7 were cored laterally into the pile through the surface plastic to 25.4 cm. Core points 8 and 9 were 1.4 and 2.8 m above grade respectively, core points 2, 4 and 6 were 0.6 m below the surface of the pile and core points 1, 3, 4 and 7 were ~1.5 back of the exposed face and ~0.5 m back of the cut plastic cover. The pile was 5 m in height and 29 m wide at grade (note that the drawing is not to scale in that there was much more distance under the peak than is visually depicted, as indicated by the distance in boxes at the base of the pile).]

20

Figure 4. Aerial view of the maize silage pile used in Experiment 3. Coring events 1 (top) and 2 (bottom) were separated by 69 days.

21 Tables

Table 1 Impacts of pile side, core depth and underlay on aspects of silage fermentation quality in North/South sited piles (Expt. 1a). Pile side

Time

Core depth b

Pc

Underlay

(moa)

East

West

Outer

Inner

POLY

EOB

SEM

Time

Side

Depth U'lay Time* Time* Side Depth

Wet density (kg/m3)

3.2 5.7

433 368

409 356

280 269

559 457

433 364

407 362

9.0

<0.01

0.16

<0.01

0.31

0.66

<0.01

pH

3.2 5.7

3.82 3.68a

3.78 3.82b

3.88 3.84

3.73 3.66

3.77 3.74

3.84 3.76

0.025

0.14

0.18

<0.01

0.21

0.01

0.64

Temperature (oC)

3.2 5.7

22.0 27.5

23.6 26.5

20.3a,c 27.4d

25.2b 26.6

23.1 26.7

22.4 27.4

0.50

<0.01

0.71

<0.01

0.98

0.07

<0.01

Dry matter (g/kg)

3.2 5.7

322 299

333 310

318 292

338 317

323 303

333 306

8.9

<0.01

0.10

<0.01

0.30

0.96

0.70

Lactic

3.2 5.7

11.3 13.0

10.5 11.4

10.7 11.3

11.1 13.0

10.9 12.5

10.9 11.9

1.09

0.56

0.28

0.02

0.94

0.66

0.33

Acetic

3.2 5.7

6.0 10.2

5.7 7.8

5.6 10.1

6.1 8.0

6.1 9.1

5.6 8.9

0.80

<0.01

0.07

0.68

0.63

0.11

0.06

Propionic

3.2 5.7

0.6 0.4

0.8 0.2

0.8 0.5

0.7 0.1

0.7 0.3

0.7 0.3

0.38

0.08

0.91

0.57

0.83

0.36

0.53

Butyric

3.2 5.7

<0.1 0.4

0.3 0.5

0.2 0.4

0.1 0.4

0.1 0.4

0.2 0.4

0.12

<0.01

0.05

0.71

0.94

0.39

0.50

Succinic

3.2 5.7

0.2 0.8

0.2 0.5

0.2 0.7

0.2 0.6

0.3 0.7

0.1 0.6

0.13

<0.01

0.10

0.50

0.34

0.22

0.74

Formic

3.2 5.7

0.1 0.1

0.1 0.2

0.1 0.2

0.1 0.0

0.1 0.1

<0.1 0.2

0.12

0.46

0.78

0.32

0.74

0.69

0.38

3.2 5.7

0.9 3.5

0.9 3.0

0.8c 3.8d

1.0 2.7

0.9 2.9

0.9 3.6

0.40

<0.01

0.63

0.38

0.26

0.42

0.05

3.2

0.01

0.01

0.01

0.01

<0.01

0.01

0.08

0.16

0.20

0.26

0.26

0.22

0.26

Acids (g/kg wet weight)

Ethanol (g/kg wet weight)

Mold million cfu/g wet

22

weight

score (1-6) Yeast million cfu/g wet weight

score (1-4)

5.7

0.03

0.30

0.29

0.04

0.29

0.04

3.2 5.7

1.0 1.0

1.0 1.2

1.0 1.2

1.0 1.0

1.0 1.2

1.0 1.1

0.06

0.17

0.17

0.17

0.33

0.17

0.17

3.2 5.7

0.02 1.23

0.08 1.96

0.08c 2.77a,d

0.03 0.42b

0.07 2.39

0.04 0.81

0.46

0.02

0.54

0.07

0.22

0.61

0.08

3.2 5.7

1.0 1.6

1.2 1.9

1.1 2.0

1.1 1.6

1.1 1.8

1.1 1.7

0.08

<0.01

0.06

0.06

0.70

0.38

0.17

a

- Months post building of the pile.

b

- 'Outer' is the surface 25.4 cm and 'inner' is the core from 25.4 to 50.8 cm.

c

- The interactions of Time*U'lay and Depth*U'lay were all P>0.17 and are not listed.

a, b

- Means with different superscripts on rows within factors differ (P <0.05).

c, d

- Means with different superscripts in columns within factors differ (P<0.05).

23

Table 2 Impacts of pile side, core depth and underlay on aspects of silage fermentation quality in East/West sited piles (Expt. 1b). Pile side

Time

Core depth b

Pc

Underlay

(moa)

South

North

Outer

Inner

POLY

EOB

SEM

Time

Side

Depth U'lay Time* Time* Side Depth

Wet density (kg/m3)

2.4 6.0

338 303

324 324

210 240

453c 386d

332 317

330 309

5.3

0.02

0.63

<0.01

0.57

0.02

<0.01

pH

2.4 6.0

3.85 3.83

3.77 3.72

3.87 3.80

3.75 3.76

3.79 3.80

3.84 3.76

0.022

0.27

<0.01

<0.01

0.83

0.65

0.18

Temperature (oC)

2.4 6.0

25.0c 37.0a,d

23.9 28.1b

21.5a,c 32.6d

27.4b 32.5

24.7 32.0

24.2 33.1

0.50

<0.01 <0.01

<0.01

0.67

<0.01

<0.01

Dry matter (g/kg)

2.4 6.0

288 268

290 256

282c 244a,d

297 280b

288 258

290 266

7.3

<0.01

0.36

<0.01

0.33

0.17

0.04

Lactic

2.4 6.0

9.8 13.4

10.5 13.1

9.5 12.7

10.7 13.8

10.2 12.2

10.0 14.3

1.21

0.02

0.97

0.01

0.20

0.35

0.61

Acetic

2.4 6.0

6.0 13.1

6.0 12.1

5.1 12.2

6.8 12.9

5.7 12.9

6.3 12.3

1.42

<0.01

0.47

0.03

0.79

0.43

0.90

Propionic

2.4 6.0

1.4 0.4

1.2 0.5

0.8 0.7

1.8 0.2

1.5 0.5

1.1 0.4

0.58

0.02

0.93

0.41

0.55

0.79

0.05

Butyric

2.4 6.0

0.2 0.4

0.2 0.3

0.2 0.5

0.2 0.3

0.2 0.5

0.2 0.3

0.16

0.19

0.40

0.29

0.26

0.54

0.63

Succinic

2.4 6.0

0.5c 1.1a,d

0.6 0.7b

0.3 0.6

0.7 1.1

0.6 1.0

0.5 0.8

0.16

0.01

0.08

<0.01

0.53

0.02

0.46

Formic

2.4 6.0

0.2 <0.1

0.1 <0.1

0.2 <0.1

0.1 <0.1

0.1 <0.1

0.2 <0.1

0.10

0.02

0.79

0.27

0.77

0.79

0.27

2.4 6.0

3.2 3.3

2.1 3.4

2.4 3.7

2.9 3.0

3.4 3.2

1.9 3.4

1.00

0.60

0.38

0.77

0.40

0.46

0.53

2.4

0.01

0.02

0.02

0.01

0.02

0.01

0.024

<0.01

0.23

0.25

0.88

0.29

0.33

Acids (g/kg wet weight)

Ethanol (g/kg wet weight)

Mold million cfu/g wet weight

24

score (1-6) Yeast million cfu/g wet weight

score (1-4)

6.0

0.08

0.15

0.15

0.08

0.12

0.12

2.4 6.0

1.0 1.1

1.0 1.2

1.0 1.1

1.0 1.1

1.0 1.1

1.0 1.1

0.03

0.02

0.31

0.73

0.73

0.31

0.73

2.4 6.0

0.11 0.16

0.43 1.49

0.50 1.46

0.03 0.18

0.50 1.40

0.03 0.30

0.354

0.27

0.10

0.08

0.11

0.32

0.42

2.4 6.0

1.3 1.3

1.5 1.6

1.6 1.6

1.2 1.3

1.5 1.5

1.3 1.4

0.08

0.69

0.01

<0.01

0.23

0.50

0.50

a

- Months post building of the pile.

b

- 'Outer' is the surface 25.4 cm and 'inner' is the core from 25.4 to 50.8 cm.

c

- The interactions of Time*U'lay and Depth*U'lay were all P>0.07 and are not listed. The sole exception was that EOB higher (P<0.05) than POLY

a, b

- Means with different superscripts on rows within factors differ (P<0.05).

c, d

- Means with different superscripts in columns within factors differ (P<0.05).

at 2.4 mo (2.48 vs. 3.62), but not different at 6.0 months.

25

Table 3 Impacts of silage location and underlay on aspects of silage fermentation quality (Expt. 2). Location Underlay

Surface a

Deep Mass

Edge

P

EOB

POLY

EOB

POLY

EOB

POLY

SEM

U'lay

Loc

Loc*U'lay

Wet Density (kg/m3) c

296

309

412

421

892

884

8.5

0.62

<0.01

0.64

pHb Temperature (oC)b

3.50 38.2

3.48 39.7

3.62 30.9

3.67 30.6

3.66 31.9

3.69 32.0

0.048 1.22

0.69 0.73

0.02 <0.01

0.81 0.85

Dry matter (g/kg)

288

268

298

314

303

310

6.9

0.97

0.15

0.44

Acids (g/kg wet weight)d Lactic Acetic Propionic Butyric Succinic

9.5 7.9 1.5 <0.1 0.2

10.3 7.1 0.7 <0.1 0.2

14.1 11.6 1.7 0.1 0.2

17.1 9.6 1.3 0.5 0.2

14.3 9.1 0.5 0.4 0.5

13.4 9.00 0.9 0.4 0.7

1.16 0.63 0.31 0.11 0.13

0.62 0.38 0.59 0.46 0.81

0.13 0.14 0.45 0.27 0.26

0.68 0.74 0.62 0.67 0.94

Ethanol (g/kg) e

3.0

2.4

2.0

1.9

4.3

4.8

0.39

0.92

0.05

0.74

Nutrients (g/kg dry weight) Crude proteinb 85 aNDFe, f 422 ADF 262

93 433 276

79 408 281

70 423 280

75 387 266

74 406 264

2.1 3.6 6.7

0.82 0.05 0.37

0.03 0.02 0.93

0.20 0.86 0.69

a

- EOB = enhanced oxygen barrier characteristics, POLY = polyethylene.

b

- Surface differs from edge and deep mass (P<0.05). Deep mass and edge do not differ.

c

- All locations differ from each other (P<0.05).

d

- Formic acid was not detected in any sample.

e

- Deep mass differs from surface and edge (P<0.05). Surface and edge do not differ.

f

- POLY differs from EOB (P<0.05).

26

Table 4 Impacts of location on the side of the pile and time of coring on some aspects of silage preservation quality (Expt. 3).

Coring

Wet density (kg/m3)

pH

Temperatur e (oC)

Mold counts (million cfu/g wet weight)

score (1-6)

Yeast counts (million cfu/g wet weight)

score (1-4)

Side of Pilea

Back of Pile Locations 5&6

Locations 4&7

Core depthc

Core depthc Inner Outer

Locations 3&8 Core depthc

Locations 2&9b Core depthc

Locations 1&10b Core depthc

P

Inner

SEM

Loc

Depth

Location* Core depth

184 -

256 -

9.3 22.4

0.09 0.85

<0.01 <0.01

0.27 0.90

4.21i -

6.57h -

4.26i -

0.291 0.258

0.16 0.08

0.06 <0.01

0.21 0.11

31 -

29 -

47 -

44 -

0.8 2.6

<0.01d 0.03g

0.21 0.29

0.85 0.84

<0.01 2.3i

1.3 -

1.3 -

1.4 -

0.2 -

0.21 1.39

0.07e 0.09

0.29 0.07

0.66 0.30

1.0 6.0h

1.0 3.0i

2.5 -

3.0 -

2.5 -

1.0 -

0.24 0.83

0.02e 0.09

0.54 0.14

0.39 0.51

<0.1 <0.1

<0.1 0.6

<0.1 0.2

5.4 -

3.3 -

1.5 -

0.1 -

0.97 0.15

0.23f 0.25

0.60 0.45

0.97 0.58

1.0 1.0

1.0 2.0

1.0 1.5

2.5 -

2.5 -

2.0 -

1.0 -

0.35 0.29

0.26f 0.24

0.68 0.67

0.94 0.82

Event

Outer

Inner Outer

Inner

Outer

1 2

142 139

272 288

171 141

316 303

1 2

4.09 4.23

3.98 4.18

4.24 6.17h

1 2

29 28

29 27

1 2

0.2 0.1

1 2

187 168

348 298

173 -

280 -

4.31 4.31i

4.16 6.08h

4.21 4.24i

6.11h -

30 40

29 34

28 45

28 40

<0.1 0.1

0.2 4.8h

<0.1 0.3i

0.1 9.8h

1.0 1.0

1.0 1.0

1.0 3.5h

1.0 1.0i

1 2

<0.1 <0.1

<0.1 <0.1

<0.1 <0.1

1 2

1.0 1.0

1.0 1.0

1.0 1.0

a

- See Figure 4 for the meaning of the location numbers, but location 1/10 is closest to the exposed face and location 4/7 is closest to the back of the pile.

b

- Locations 2, 9 and 1, 10 no longer existed on coring event 2 due to the pile face moving back to location 3 and 8.

c

- 'Outer' is the surface 25.4 cm and 'inner' is the core from 25.4 to 50.8 cm.

d

- Locations 1&10 were higher (P<0.05) than all others.

Inner Outer

Core

27 e

- Locations 1&10 and 2&9 were higher (P<0.05) than all others.

f

- Locations 2&9 were higher (P<0.05) than locations 5&6, 4&7 and 3&8.

g

- Locations 5&6 were lower (P<0.05) than all others.

h, i

- Values with different superscripts within location differ (P<0.05).