Final Report Research Report Evaluation of Disposal Options for Fusarium-Damaged Grain and Screenings

Project No. R8415 Date: February 17, 2017 Revised: February 28, 2017 Humboldt, Saskatchewan Final Report Research Report Evaluation of Disposal Options for Fusarium-Damaged Grain and Screenings For: Agriculture Development Fund

R8415 February 17, 2017 Revised: February 28, 2017 Humboldt, Saskatchewan ADF#20150030 Final Report Research Report Evaluation of Disposal Options for Fusarium-Damaged Grain and Screenings Joy Agnew, P.Eng., Ph.D. Project Manager Agricultural Research Services Joshua Kirsch Project Leader

Acknowledgements The Prairie Agricultural Machinery Institute (PAMI) acknowledges funding for this project through the Saskatchewan Ministry of Agriculture and the in-kind contribution of the Canadian Grain Commission for method development to assess the contamination levels in compost, digestate, and ash. PAMI also thanks Sheryl Tittlemier and her team at the Canadian Grain Commission for providing technical insights on the significance of the results. Finally, PAMI thanks the cooperation of local producers and business owners for providing access to fusarium-damaged grains and information on sorting equipment.

Table of Contents Page 1. Executive Summary... 1 2. Introduction... 3 3. Literature Review... 4 3.1 Sorting Technologies... 4 3.2 Disposal Options... 6 4. Materials and Methods... 9 4.1 DON Measurement Method Validation... 9 4.2 Composting Trial...10 4.3 Anaerobic Digestion Trial...11 4.4 Combustion Trial...12 5. Results and Discussion...14 5.1 Composting Trial...14 5.2 Digestion Trial...16 5.3 Combustion Trial...19 5.4 Economic Analysis of Disposal Options...21 6. Conclusions and Recommendations...25 7. References...26 Appendix A Mycotoxin Analysis Method Development Results A-1 Appendix B Mycotoxin Analysis Results...B-1 Appendix C Additional Data for Sorting Technologies C-1

1. Executive Summary The quantity of fusarium infested grain on the prairies has grown substantially over the past few years and is projected to continue to rise. In cases where the grain cannot be cleaned or the mycotoxin content is too high to be considered a blend for feed, the material needs to be disposed of. The purpose of this project was to better understand how various disposal options, such as cleaning, composting, combustion, and anaerobic digestion, can help Saskatchewan agricultural producers extract value from their fusarium infested grain and screenings. A review of grain cleaning technologies show that gravity sorters have a high capacity for sorting grain, but their effectiveness at removing fusarium damaged kernels may be limited depending on the level of contamination. Also, gravity separation tends to remove healthy kernels along with the contaminated kernels. Colour or optical sorters are more efficient, but more costly than gravity sorters. The newest technology involves near infrared transmission (NIT), but this technology is not yet readily available to farmers and has a relatively low capacity for handling large amounts of bulk grain. Since the review of literature on disposal options did not reveal many references on the effect of combining temperature and microbial treatments (such as composting and anaerobic digestion) on elimination of mycotoxins, a composting trial and an anaerobic digestion trial were conducted. A combustion trial was also carried out to determine if simple burning would deactivate the mycotoxins in heavily-infested grain. To assess the effectiveness of each of these processes, the Canadian Grain Commission had to develop and validate a methodology to assess mycotoxin concentrations in compost, digestate, and ash substrates since existing methodologies were designed for grain assessments. The results of these trials indicated that the composting process eliminated the mycotoxin of interest (deoxynvalenol, DON) since all composted samples assessed by the Canadian Grain Commission had undetectable levels of DON. Anaerobic digestion and combustion both reduced the concentration of DON in the substrate but did not eliminate it. It is hypothesized that a combination of the temperatures and microbial activity that occurred throughout the composting period deactivated the DON. Although DON is produced by Fusarium graminearum, the lack of DON in composted samples does not mean that the Fusarium graminaerum was also eliminated, but current measurement protocols do not allow an assessment of fusarium content in compost samples. A basic economic analysis of the pelleting and composting processes indicated that if the costs are in the low- to mid-range of expected pelleting and composting costs and Page 1 of 28

the value of the resulting product is in the mid- to high-range of the expected values, there may be a net positive return for highly infested grain and screenings. In addition to providing some return to the producer, disposal options other than dumping or landfilling may minimize the risk of further contaminating the soil. Page 2 of 28

2. Introduction Fusarium head blight (FHB) is a fungus caused by fusarium pathogens that affects cereal crops such as wheat, barley, and oats. The most common species is Fusarium graminearum, which is known to produce a toxin called deoxynivalenol (DON) or vomitoxin in the grain. DON reduces the quality of the grain as a feed and food source, so an infection of fusarium results in loss of grade as well as a reduction in yield. The amount of fusarium infested cereals on the prairies has grown substantially over the past five years, and the Canadian Grain Commission estimates that a third of all red spring wheat was downgraded due to fusarium damage in 2014. Due to the spread of this fungus, producers are experiencing grade losses, restricted marketing opportunities, added costs, and lost income. According to the Ministry of Agriculture and Forestry of Alberta, losses in Canada have ranged from $50 million to $300 million annually over the past two decades while future losses are projected to be between $30 and $132 per acre depending on the crop and area. Some of these losses can be recovered if the grain is cleaned or disposed of in a way that generates some revenue. However, most of the research conducted on FHB is focusing on the breeding and crop management to help prevent the spread of FHB. Others, such as researchers at the Feed Processing Institute and the Canadian Feed Research Centre, are assessing technologies for sorting and cleaning infested grain so it can be blended with uncontaminated grain and used for livestock feed. Research on methods of extracting value from heavily damaged grain or screenings is lacking. The objective of this project was to better understand how various disposal options (such as composting, combustion, and anaerobic digestion) can help Saskatchewan agricultural producers extract value from fuasarium damaged grain and screenings that are not suitable for other uses such as feed. Information on grain sorting technologies and disposal options will help producers understand the costs and benefits associated with each alternative. Page 3 of 28

3. Literature Review Existing information on sorting technologies and disposal of grain via composting and anaerobic digestion was reviewed and is summarized in the following sections. 3.1 Sorting Technologies The characteristics of fusarium damaged kernels (FDK) in wheat can make them difficult to remove using traditional seed cleaning methods. According to the Canadian Grain Commission, FDKs are typically thin, shrunken, and chalky in appearance. The infested kernels can also have a white or pinkish mold in the crease and sometimes the germ of the seed (Canadian Grain Commission, 2013). Although these are the typical characteristics of FDKs, they are not a true measurement of DON levels in the grain. In fact, kernels may show little to no visible symptoms even though DON is present. A basic review of the most common sorting technologies are included in the following sections. Additional information from grain cleaning contractors in Saskatchewan can be found in Appendix C. 3.1.1 Gravity Separation While FDKs are generally smaller than healthy wheat kernels, there is typically not enough of a difference to make sorting by size an effective method of removal. However, FDKs are relatively less dense than healthy kernels, which make gravity separation an option for removal. Gravitational beds use a combination of blower fans and mechanical shaking to sort the grain based on density. An article in the Food Science and Technology Journal found that gravity separation removed anywhere from 7% to 63% of the FDKs depending on the initial level of DON, which ranged from about 0.2 to 15 ppm. Gravity separation was most effective at higher DON levels and less so at lower concentrations (Cheli, 2013). Gravity tables are relatively simple, low cost, and can process grain at relatively high capacities. For example, the Oliver Max-Cap series gravity table can process up to 20 m 3 /h or 550 bu/h (Oliver Manufacturing, 2016). Gravity tables are also a common method of removing ergot, which like FDKs, are typically less dense then healthy kernels. One negative aspect of gravity sorters is that they remove a considerable amount of healthy grain with the FDKs. Due to the high DON levels in these screenings they are usually unmarketable and have to be disposed of. Another option is to store the screenings and blend them with next year s crop, if it is fusarium free, and sell it as feed. Page 4 of 28

After conducting a survey with grain cleaners throughout Saskatchewan, it was found that the use of gravity tables as part of a mobile seed cleaning unit was the most common method of removing FDKs from wheat. Cleaners are able to process anywhere from 5.3 to 20 m 3 /h (150 to 550 bu/h), depending on the percentage of FDK. Having a mobile cleaning unit come to a farm will typically cost approximately $18 to $36 per tonne ($0.50 to $1.00 per bushel) depending on the level of infection and demand for grain cleaning in a given year. 3.1.2 Optical Sorters Colour sorters use optics to analyze seeds and eject the ones that do not fall within a predetermined colour spectrum. Colour sorters were originally introduced to sort larger grains such as rice, coffee, and peas. However, the technology has now evolved to a level where it can be effective at processing smaller grains such as wheat (Rempel, 2014). According to Flaman Grain Cleaning, colour sorters can process up to 28 m 3 /h (800 bu/h) of grain depending on the model and the number of chutes. Monochromatic colour sorters are a cheaper option with higher capacity, and can still be very effective at removing ergot and pink-tinted fusarium damaged wheat. For example, the Satake Alpha Scan II High Capacity from Flaman can process up to 40 m 3 /h (1100 bu/h) when removing ergot from wheat (Flaman, 2016). It should be noted that fusarium infested kernels vary in their shade of colour from whitish to black or pink. Due to the different colours indicators, sorters must perform multiple tasks at once to determine which kernels should be discarded. This slows down the sorting process. The removal rate of infested kernels will also affect the capacity of the colour sorter. Therefore, the higher the rate of infection the slower the process will become (King, 2016). Based on consultations with grain cleaners throughout Saskatchewan, the relatively low capacity was found to be the main disadvantage of optical sorting machines. Most stated that the sorting process was generally too slow to be an effective method of removal for large batches of grain. However, due to their increased accuracy, colour sorters may be an option to retrieve healthy kernels that were rejected after sorting on a gravity table. A few grain cleaners were considering adding a colour sorter into their mobile grain cleaning unit, however, this option does not seem to be readily available in Saskatchewan at this time. 3.1.3 Near Infrared Transmission (NIT) Technology The newest and most accurate method of removing DON from wheat is NIT. This method involves sending infrared light through individual kernels and measuring the Page 5 of 28

response. The machine can then analyze individual kernels and sort them by chemical characteristics such as protein content, hardness, and virtuousness (Scott, 2015). One of the first commercially available sorters to use NIT technology is the Bomill TriQ. The TriQ uses infrared technology to remove DON infested grain based on crude protein level. Bomill claims that the TriQ has a capacity of 3 tonnes/hour (approximately 4 m 3 /h or 110 bu/h) and that up to ten machines can be combined to achieve a capacity of at least 30 tonnes per hour approximately 40 m 3 /h or 1100 bu/h (Bomill, 2016). In a study published in the Journal of Animal Nutrition, the Bomill TriQ was used to separate wheat samples into ten fractions based on crude protein content. The DON levels of the five unsorted samples varied from 0.9 to 8.4 ppm. In each case the grade was improved by removing the two fractions with the lowest crude protein content (the lowest 20%). In the four lower DON content samples, the grade was improved from feed to either No. 1 or No. 2, while in the highest DON content sample the grade was improved from salvage to No. 3 (Kautzman, Wickstrom, & Scott, 2015). NIT technology is still being evaluated in Saskatchewan, but is showing promise for removing DON infested kernels and improving the grade of fusarium infested wheat and other disease infested grains. Discussions with local grain cleaners revealed that this technology is currently not readily available to farmers. However, a few cleaners were aware of the technology and would be interested in adding the technology to their mobile seed cleaning unit once it becomes more economical and provides higher capacities. 3.2 Disposal Options In addition to the lack of information on cleaning technologies, there has been very little work done to identify suitable methods of disposing of the screenings or highly contaminated wheat, or determining the environmental impacts of different disposal methods. Jard et al. (2011) compiled a review of literature on chemical and physical transformations and deactivation of mycotoxins including DON. The reviewed methods included the use of absorbants, binders, and enzymes that are added to the infested feed and work in the animal's intestinal tract to prevent resorption of the mycotoxins. The authors also discussed physical and chemical treatments for transformation of mycotoxins, including thermal treatment, degradation by extrusion, radiation, oxidation (ozone treatment), reduction, and ammoniation. These physical and chemical treatments could theoretically be used to "clean" food or feed before use. However, Jard et al. (2011) stated that the use of chemical or physical processes to decontaminate food is limited by loss of nutritional quality, poor efficiency, and consumer concern about chemical treatment of food. Some of these chemical or physical processes may deactivate the mycotoxins and allow the resulting product (compost, ash, etc.) to be Page 6 of 28

used as a soil amendment, for example, and allow the producer to extract some value from the infested grain. The use of ozone, a powerful oxidizing reagent, as a method of reducing mycotoxin concentration in stored grain, has generated some interest recently. McKenzie et al. (1997) reported that numerous mycotoxins were deactivated after 15 seconds of treatment with ozone up to 20% concentration. However, the authors noted that the cost of producing the required quantity of ozone (using 1997 technology) was prohibitive (McKenzie et al., 1997). Wang et al. (2010) reported that a wet method of ozone fumigation better degraded mycotoxin-spiked grain samples than the dry method. These results were of interest because previous trials using a dry method of fumigation required high concentrations and long retention times to achieve satisfactory deactivation of mycotoxins (Wang et al., 2010). More recently, others have reported successfully detoxifying aflatoxins using ozone at a range of concentrations and exposure times (Chen et al., 2014; Geovana et al., 2014). Microbial degradation of mycotoxins outside of the animal's gut has received some attention recently. Preliminary work in Germany noted the toxins in mouldy grain were inactivated after 12 hours of digestion in a solid-state batch system, but it is unclear if the oxygen-free environment or microbial degradation was responsible for the deactivation (Frauz et al., 2006). Garda Buffon et al. (2011) evaluated degradation of DON due to fermentation with fungal species and found that a 48-hour fermentation interval presented the highest DON degradation velocity. In a very interesting study from France, Goux et al. (2010) digested DON-contaminated wheat flour in a liquid mesophilic system using wastewater digestate as an inoculant. The authors conducted digestion trials involving seven different levels of DON-contaminated flour ranging from 0 to 80,000 µg/kg (0 to 80 ppm) and found no effect of contamination level on biogas production. Average methane (CH 4) production was equivalent to approximately 350 L/kg VS (Goux et al., 201 0). For reference, liquid manure digestion generates 200 to 400 L CH 4/kg VS and PAMI's solid-state anaerobic digestion of manure generated 100 to 150 L CH 4/kg VS. More importantly, Goux et al. (2010) monitored the progress of DON concentration during the 32-day digestion period and noted a reduction in DON concentration of 25% after 15 days and non-detectable levels of DON after 30 days. The authors concluded that the anaerobic digestion process may present a good alternative to incineration to treat such substrates since it allows recovery of energy and nutrients, particularly nitrogen. A review of literature on the use of composting to deactivate mycotoxins did not reveal any replicated trials. However, numerous factsheets stated that the composting environment actually promotes the production of fungi that cause mycotoxins. Based on the results in Goux et al. (2010), the microbial processes that occur during anaerobic digestion effectively deactivate DON. Although the microbial processes that occur during Page 7 of 28

composting (aerobic) are entirely different from those that occur during digestion (anaerobic), it may be worth investigating if composting can deactivate DON and result in a nutrient-rich material that can be land applied. The main benefit of employing composting to generate a value-added product from fusarium damaged grain rather than anaerobic digestion is the low cost and relative simplicity of the composting process. Thermochemical conversion processes such as gasification or pyrolysis have not been evaluated in detail with respect to mycotoxin deactivation. However, the review by Jard et al. (2011) stated that temperature-based treatments (with no microbial activity) were ineffective at removing mycotoxins when the temperature was below 210 C. Pyrolysis and gasification processes occur at temperatures well above 200 C and produce energy rich products such as bio-oil and syngas. Page 8 of 28

4. Materials and Methods Composting, digestion, and combustion processes were investigated to see what effect each process would have on mycotoxins present in fusarium infested wheat. Samples of wheat with high and low DON levels were secured from local growers to provide two different substrates to compost, digest, and burn. Samples from each source were then sent to the Canadian Grain Commission to determine the actual DON content. The highly-infested sample had an average DON concentration of 38 ppm and lower sample had a DON concentration of 16 ppm. Both samples were obtained from screenings, which explains the relatively high DON concentrations compared to what is typically seen in the field. The Canadian International Grain Institute (CIGI) notes that grain used for feed should have less than 5 ppm DON in unsorted grain and grain used for food should have less than 1 ppm DON in unsorted grain. Grain with greater than 10 ppm DON is usually considered salvage. A summary of the initial DON analysis is found in Table 1. A full list of mycotoxins detected can be found in Appendix B. Table 1. Initial DON analysis of wheat samples used during composting, digestion, and combustion trials. DON Concentration (ppm) Sample Sample A (high DON) Sample B (low DON) 1 40.1 15.8 2 35.8 17.6 3 38.2 14.4 Average 38.0 15.9 Standard Deviation 1.70 1.60 4.1 DON Measurement Method Validation In order to analyze the DON content in compost, digestate, and ash, a blank sample of each type of substrate was sent to the Canadian Grain Commission for method development. These samples were then fortified with mycotoxins and monitored to see how well the method worked. The method for detecting DON yielded very good results. The recovery rate and limit of quantification (LOQ) for DON can be seen in Table 2. Page 9 of 28

Table 2. Quality indicators for method of measuring DON in wheat, ash, compost, and digestate substrates developed by Canadian Grain Commission. Substrate Recovery Rate (%) LOQ (ppb) Wheat 95 0.5 Ash 86 0.7 Compost 89 0.6 Digestate 68 0.9 A complete list of mycotoxins detected and their respective recovery rate and LOQs can be found in Appendix A. 4.2 Composting Trial For the composting trial, four compost piles were created consisting of a 50/50 mixture by volume of cow manure and wheat (Figure 1). Two piles contained the high DON wheat and two contained the low DON wheat. As well, two control piles were created, which contained cow manure only for a total of six piles. Each pile contained approximately 10 tonnes of material. Figure 1. Compost piles used to determine the effect of composting on DON concentration in the final product. After the compost piles were formed, samples were collected and sent for total carbon (C), total nitrogen (N), and moisture content analysis (Table 3). The optimum C:N ratio for composting is between 15:1 and 30:1 and the optimum moisture content is between 50% and 60%. The piles containing wheat had sufficient carbon to bring the C:N ratio within the ideal range, but the C:N ratio in the piles with only manure were lower than ideal. Since the moisture content of all piles was below the optimum level for composting, water was added to bring the moisture up to 55%. Page 10 of 28

The DON content for each pile at the start of the composting period was estimated by considering the dilution effect of mixing the grain with the manure. The manure was assumed to have no DON since the feedlot from which the manure originated does not feed DON infested grain. Table 3. Carbon, nitrogen, moisture, and estimated DON content of compost piles at the start of composting trial. Pile Treatment Total Carbon (kg/tonne) Total Nitrogen (kg/tonne) C:N Ratio Moisture Content (%) DON Content (ppm) 1 Low DON 289 16.9 17.1 37 5.33 2 Low DON 185 10.1 18.3 49 5.40 3 High DON 247 11.8 20.9 33 11.42 4 High DON 280 13.3 21.1 24 8.05 5 No DON 86 5.7 15.1 46 0.00 6 No DON 41 3.91 10.5 25 0.00 The difference in estimated DON content between piles 3 and 4 (both with high DON wheat) was due to the fact that the mixing ratio for pile 4 was slightly more than 50% manure. The composting trial ran from June 23, to October 12, 2016, (111 days), and the piles were turned with a front-end loader once a week for the first month. Moisture content was periodically assessed and water was added as required to bring each pile up to approximately 55% moisture. The temperature of each pile was monitored on a weekly basis to ensure that the piles were composting effectively. Samples were collected from each of the six piles at the end of the composting period and sent to the Canadian Grain Commission for mycotoxin assessment to determine if the composting process affected the mycotoxin concentration in the substrate. 4.3 Anaerobic Digestion Trial A pre-digestion trial with dairy manure was performed in order to obtain digestate to be used as an inoculant for the actual trial involving the DON infested wheat. This pre-trial consisted of 18 vessels divided into two separate incubators. One of the incubators is shown in Figure 2. The temperature in each incubator was maintained at 38 C ±2 C. Vessel temperature and total gas production (via wet tip gas meters attached to each vessel) were monitored for the duration of the trial. The pre-digestion trial ran from May 19, to June 29, 2016 (42 days). Page 11 of 28

Figure 2. One of two incubators used for the bench scale digestion trials. Each incubator holds nine vessels. For the actual trial, six vessels contained a mixture including high DON wheat, six vessels contained a mixture including low DON wheat, and six vessels contained manure only. The vessels containing grain consisted of 1.5 kg of wheat, 1.5 kg of manure, 1 kg of digestate, and 1 L of water. The manure only vessels consisted of 3 kg of manure, 1 kg of digestate, and 1 L of water. Three of the six vessels within each treatment were recirculated periodically throughout the digestion period. During recirculation, the liquid at the bottom of the vessel was redistributed to the top to promote good contact between the microbes and the substrate. The location of each vessel was randomized between the two incubators. Vessel temperature and biogas production were monitored throughout the trial. The trial ran from August 8, to October 3, 2016 (56 days). Post-digestion samples of the substrate (digestate) were collected from 11 vessels based on their estimated amount of microbial activity. These samples were then sent to the Canadian Grain Commission for analysis to determine if the digestion process affected the mycotoxin concentration in the substrate. 4.4 Combustion Trial To determine if combustion eliminated mycotoxins from the resulting ash, samples of both the high and low DON content wheat were taken to Prairie Fire in Bruno, Page 12 of 28

Saskatchewan. Each 10 kg sample was burned in a grain burning stove and the ashes were collected. The ash samples were then sent to the Canadian Grain Commission for mycotoxin analysis. The grain burning stove used can be seen in Figure 3 below. It was estimated that the temperature during combustion was between 150 C and 300 C. Figure 3. Grain burning stove used to generate ash from low and high DON content wheat. In addition to generating ash in the grain burning stove, samples of high and low DON content wheat were sent to the analytical lab of Alberta Innovates in Vegreville, Alberta, for an assessment of the higher heating value (HHV) of the grain using bomb calorimetry. The HHV provides an indication of the total energy content in the material. Page 13 of 28

19-Jun-2016 9-Jul-2016 29-Jul-2016 18-Aug-2016 7-Sep-2016 27-Sep-2016 17-Oct-2016 Temperature ( C) 5. Results and Discussion The results of the composting, digestion, and combustion trials are presented in the following sections. 5.1 Composting Trial A plot of the temperatures of the compost piles throughout the trial for each pile is shown in Figure 4. 80 70 60 50 40 30 1 (Low Fusarium) 2 (Low Fusarium) 3 (High Fusarium) 4 (High Fusarium) 20 10 Piles turned 5 (No Fusarium) 6 (No Fusarium) 0 Date Figure 4. Compost pile temperatures throughout the composting period. The temperature of one of the lower DON content piles peaked at 70 C after one week of composting, while the higher DON pile peaked at 55 C after the second week. The manure-only piles also reached temperatures around 55 C early in the trial. On October 12, samples were taken from each pile and sent to the Canadian Grain Commission for mycotoxin analysis. It should be noted that at the end of the trial the temperature of the piles containing wheat still ranged from 45 C to 51 C while the temperatures of the manure only piles had dropped considerably. The carbon, nitrogen, moisture, and DON content of samples collected from each pile at the end of the 111-day composting period are shown in Table 4. Page 14 of 28

Table 4. Carbon, nitrogen, moisture, and estimated DON content of compost piles at the end of the composting trial. Pile Treatment Total Carbon (kg/tonne) Total Nitrogen (kg/tonne) C:N Ratio Moisture Content (%) DON Content (ppm) 1 Low DON 360 25.4 14.2 14 0 2 Low DON 280 15.1 18.5 23 0 3 High DON 168 19.5 8.6 21 0 4 High DON 180 16.0 11.25 26 0 5 No DON 48 3.34 14.37 37 0 6 No DON 36 3.08 11.68 27 0 The piles containing wheat seemed to compost better than the manure only piles based on the compost temperatures and high reduction in C:N ratio in the piles containing grain. This is likely due to the higher initial C:N ratio in those piles resulting from the carbon content in the wheat. After 111 days of composting, DON was undetectable in all piles. Since the DON content pre-composting in the piles were estimated based on mixing ratios, and the DON content in the composted piles were actually measured (but were all zero), a statistical analysis of the data is not warranted. But the results certainly indicate that the composting treatment had an effect on the DON content in the substrate. Although it seems as if the composting process completely deactivated the DON, it is unclear if the Fusarium graminearum fungus, which produces DON, is still present in the composted samples. Unpublished research by the Canadian Grain Commission indicates that 50 C heat treatment did not reduce Fusarium load whereas at 65 C to 70 C, Fusarium graminearum was almost eliminated after 10 to 12 days (T. Graefenhan, personal communication). These investigations were done with dry heat, so the moist conditions within a compost pile should increase heat transmission and efficiency of the heat treatment. On the other hand, it was noted that fusarium is still present in malt after kilning at 70 C to 80 C for many hours (T. Graefenhan, personal communication), so heat treatment alone is not a consistent method of eliminating fusarium spores. It s possible that the combination of heat treatment and microbial activity resulted in the elimination of DON in the compost samples analyzed in this study. This study focused on DON as an indicator of fusarium contamination, but the analysis completed by the Canadian Grain Commission included several other mycotoxins that are by-products of fusarium. A full list of the DON and other DON related mycotoxins present in the samples can be found in Appendix B. One of those mycotoxins, Enn A, was present in the composted piles with high fusarium wheat although Enn A was not present in the original wheat or manure samples. This observation indicates that the fusarium species that generated Enn A had a chance to proliferate during the Page 15 of 28

Biogas Production (L) composting process. So composting may deactivate DON and, possibly, the fusarium species that produces DON, but it likely does not eliminate all fusarium species. Based on these findings, composted grain with fusarium may not be land applied riskfree, unless it can be demonstrated that composting deactivates the fusarium spores. But other methods of disposing of fusarium infested grain should also be used with caution. Fusarium spores are thick-walled structures that can survive ultraviolet radiation and cold temperatures (T. Graefenhan, personal communication), so dumping the screenings in the bush may result in environmental atmospheric dispersal and further contamination. 5.2 Digestion Trial The amount of biogas produced throughout the digestion trial varied from vessel to vessel (Figure 5 and Figure 6) and was used as an indicator of microbial activity within that vessel. Microbial activity typically fell into four categories: early activity, steady activity, late activity, or no activity. There are various reasons why some vessels produced little to no biogas. Even though all vessels were pressure checked prior to the trial starting, some vessels may have developed a leak which prevented the vessel from maintaining anaerobic conditions. The ph levels within some of the vessels may not have been at optimum levels to promote digestion. Also, the inoculant used may not have had the correct bacteria cultures required to jump-start the digestion process. Incubator 1 contained more vessels with early activity, while vessels in Incubator 2 seemed to have late or slow and steady activity. The discrepancy between the incubators was unexpected since the incubators were identical and the temperature difference between the incubators was less than 2 C. 60 50 40 30 20 10 0 0 20 40 60 Time (days) low DON, no recirc, 1 high DON, recirc, 1 low DON, no recirc, 2 low DON, recirc, 1 high DON, recirc, 2 no DON, recirc, 1 no DON, no recirc, 1 high DON, recrirc 3 Figure 5. Cumulative biogas production from vessels in Incubator 1. The legend information includes DON level (high or low), use of recirculation (yes or no), and repetition (1, 2, or 3). Page 16 of 28

Biogas Production (L) 120 100 80 60 40 20 0 0 20 40 60 Time (days) low DON, no recirc 3 high DON, no recirc, 1 no DON, no recirc, 2 no DON, recirc, 2 low DON, recirc, 3 high DON, no recirc, 2 no DON, recirc, 3 high DON, no recirc, 3 no DON, no recirc, 3 Figure 6. Cumulative biogas production from vessels Incubator 2. The legend information includes DON level (high or low), use of recirculation (yes or no), and repetition (1, 2, or 3). For comparison, previous bench-scale digestion trials with manure generated between 100 and 300 L of biogas per vessel. Since two of the manure only vessels in Incubator 2 appeared to recover and begin generating larger quantities of biogas around day 50, the issue may have been due to ineffective inoculum or a ph imbalance in the vessels. This highlights the need for monitoring and diligent management of the microbial population and indicates that anaerobic digestion may not be a simple solution that can be implemented on-farm. A summary of the degree of microbial activity for each treatment category and the predigestion and post-digestion DON concentration is presented in Table 5. The predigestion DON content was calculated by taking into account the DON concentration in the wheat, the dilution effect of the manure (which was assumed to contain no DON), water, and inoculant that was added to each vessel. Due to the general lack of microbial activity, samples from selected vessels were collected and sent for mycotoxin analysis. A full list of the mycotoxins detected can be found in Appendix B. Page 17 of 28

Table 5. Summary of microbial activity for all vessels and DON concentration pre-digestion and post-digestion for selected vessels. Treatment Level of microbial activity DON concentration of grain in substrate (ppm) Pre-digestion Post-digestion Reduction in DON concentration (%) Low DON, no recirculation Low DON, recirculation High DON, no recirculation High DON, recirculation No DON, no recirculation No DON, recirculation No activity 15.93 14.50 9.0 Early activity 15.93 8.93 43.9 Steady activity 15.93 9.82 38.4 Early activity 15.93 11.75 26.2 Early activity 15.93 n/a n/a No activity 15.93 7.55 52.6 No activity 38.03 30.93 18.7 Early activity 38.03 28.25 25.7 Early activity 38.03 n/a n/a Early activity 38.03 26.05 31.5 Early activity 38.03 n/a n/a Early activity 38.03 33.73 11.3 No activity 0.00 n/a n/a Late activity 0.00 0.00 n/a Steady activity 0.00 n/a n/a No activity 0.00 n/a n/a Late activity 0.00 0.00 n/a Late activity 0.00 n/a n/a The results show that there was a reduction in DON concentration in every case where a sample was collected and sent for analysis. The average post-digestion DON concentration is lower than the average pre-digestion DON concentration, but this difference is not statistically significant at a 95% confidence level (P=0.110). In other words, you would have to reduce the confidence level to 85% to state that the difference between pre- and post-digestion DON concentration was due to the digestion treatment rather than natural variability. There does not appear to be any correlation between the degree of microbial activity and reduction in DON concentration. As an example, one vessel with no activity had a low reduction in DON concentration (9%) while another vessel with no activity had a high reduction in DON concentration (52.6%). There also does not appear to be any correlation between level of DON contamination or presence of recirculation and microbial activity or reduction in DON concentration. These results did, however, validate the assumption that there was no DON in the manure mixed with the grain since the post-digestion samples from the manure only vessels contained non-detectable levels of DON. Page 18 of 28

These results also indicate that simply exposing DON infested wheat to anaerobic conditions is not sufficient to deactivate the DON. The reduction in DON concentration observed by Goux et al. (2010) after digestion of DON infested flour was likely due to the specific microbial activity achieved in their system rather than exposure to anaerobic conditions. 5.3 Combustion Trial Combustion of the low DON grain in the grain burning stove generated 507 g of ash from a 13.2 kg sample (3.84% ash) while combustion of the high DON grain in the grain burning stove generated 310 g of ash from a 12.4 kg sample (2.5% ash). It was noted that the low DON grain did not burn as easily due to a high quantity of wild oats in the sample which is likely the reason for the higher ash content from the low DON sample. The results from the DON analysis of the ash produced by burning the high and low DON content wheat samples are presented in Table 6. The ash content using both the standard protocol and the observations from the grain stove combustion test are noted. The ash samples for DON content analysis were produced in a grain burning stove because the standard ash content test did not generate a sufficient quantity of ash for mycotoxin assessment. Table 6. Heating value of wheat with high and low DON contents and DON concentration in ash samples. Treatmen t High DON Low DON Higher heating value of wheat (MJ/kg) Using standard protocol (750 C) 18.91 1.93 18.83 2.04 19.74 2.94 19.16 3.05 Ash content (%) From combustion in grain stove (approx. 300 C) DON content in ash (ppm) 2.50 6.9 3.84 4.1 While there was a significant reduction in the DON content for both the high DON and low DON samples due to combustion (34 ppm reduction for high DON and 11.8 ppm reduction for low DON), these results indicate that simple combustion may not completely deactivate DON in wheat. Due to lack of replicated samples, a statistical analysis is not possible for this data. The fact that DON remained in the ash of burned screenings may limit the disposal options of the ash to landfilling to prevent the risk of spreading DON infested material. However, if the heating value generated during the combustion of DON infested wheat can be utilized, there may still be some value in burning fusarium damaged grain that would otherwise be disposed of. Page 19 of 28

Using the average heating value of DON infested wheat of 19.2 MJ/kg and a reasonable value for fusarium infested grain of $0.037 to $0.11 per kg (equivalent to $1 to $3 per bu), the cost per GJ of heat using wheat equates to $1.91 to $5.73 per GJ. Assuming a conversion efficiency of 70% for appliances that can burn grain, the effective fuel value of fusarium infested grain is approximately $2.72 to $8.18 per GJ. It can be seen in Table 7 that when compared to traditional fuels, the value of fusarium damaged grain as a heating fuel is competitive, provided the infrastructure required to burn it is available. The appliance required to burn grain for heat is comparable to the equipment needed to burn wood chips, wood pellets, or straw, so fusarium damaged grain could be considered a lower cost alternative if no other disposal option is available for the grain. Table 7. Energy costs of various fuels (adapted from PAMI, 2014). Fuel Energy content Fuel cost ($/unit) Energy cost ($/GJ) Conversion Efficiency (%) Energy Cost ($/effective GJ) Electricity 3.6 MJ/kWh $0.07672/kWh 21.31 100 21.31 Natural gas 37.5 MJ/m 3 $0.28516/m 3 7.6 92 8.26 Fuel oil 38.7 MJ/L $0.807/L 20.85 86 24.25 Propane 25.3 MJ/L $0.49/L 19.36 90 21.52 Bituminous coal Lignite coal Wood pellets Wheat straw Wood chips Fusarium wheat 24,000 MJ/tonne 14,000 MJ/tonne 19,700 MJ/ tonne 13,700 MJ/tonne 18,000 MJ/tonne 19.2 MJ/kg $125/tonne 5.21 75 6.94 $98/tonne 7 75 9.33 $245/tonne 12.44 75 16.58 $40 to 100/tonne $50 to 100/tonne $0.037 to $0.11 per kg 2.92 to 7.30 65 4.50 to 11.23 2.78 to 5.55 70 3.97 to 7.92 1.91 to 5.73 70 2.72 to 8.18 If the value of fusarium damaged grain as a heating fuel is compared to all other heating fuels (regardless of the type of appliance), the cost of grain as a fuel is comparable to coal and natural gas, and lower cost than fuel oil and propane. If the cost of a carbon tax is also considered, then the cost savings of burning grain or other biomass rather than fossil fuels would increase even more (Table 8). Page 20 of 28

Table 8. Energy costs of various fuels, assuming two levels of carbon tax ($10/tonne and $50/tonne). Carbon tax at $10/tonne CO 2 Carbon tax at $50/tonne CO 2 Carbon tax ($/GJ) Total cost of fuel ($/GJ) Carbon tax ($/GJ) Total cost of fuel ($/GJ) Natural gas 0.50 8.76 2.52 10.78 Fuel oil 0.70 24.95 3.47 27.72 Propane 0.60 22.12 2.99 24.52 Lignite coal 0.93 10.26 4.64 13.97 Wood chips 0.00 3.97 to 7.92 0.00 3.97 to 7.92 Fusarium wheat 0.00 2.72 to 8.18 0.00 2.72 to 8.18 Although burning biomass such as wood chips and grain does emit carbon dioxide (CO 2), this carbon release is not considered a net positive addition to the carbon in the atmosphere, since the biomass sequestered that carbon during the growing cycle. Therefore, depending on how carbon tax legislation is defined, carbon tax should not apply to biomass fuel sources. 5.4 Economic Analysis of Disposal Options Currently, the cost of disposing of highly infested grain or screenings is relatively low and only involves the cost to dump the material in a slough, bush, or landfill. However, with these disposal methods, the producer is unable to recover any value from the material. In fact, there is a chance that improper disposal can result in additional contamination of adjacent land. Fusarium head blight is estimated to cost producers approximately $20/ha due to downgraded quality (Geddes, 2017), so care must be taken to minimize the risk of further contamination. Both composting and combustion may turn the grain or screenings into a saleable product that minimizes the risk of further contamination. The final compost may be used or sold as a soil amendment (provided the risk of spreading the fusarium can be proven to be eliminated) and screenings may be used or sold as a solid biofuel to co-fire with other solid biofuels such as grain (in grain burning appliances) or wood pellets (if the screenings are pelleted) provided the ash can be disposed of in a safe manner. 5.4.1 Economic analysis of combustion Highly infested grain or screenings may be utilized as a solid biofuel without pelleting in an appliance such as a grain stove, so the value of the screenings would be similar to the grain to be burned (refer to Section 5.3). If the grain or screenings are expected to replace other biofuels, such as wood pellets or coal, the material will need to be densified. Although there is a cost associated with densification, pelleted screenings would have a larger market potential. Page 21 of 28

The cost of densifying or pelleting screenings was estimated based on previous research conducted by PAMI. The capital and operating cost of pelleting switchgrass was reported to be $40/tonne at a mid-scale (7 tonne/h) by The Research Park, 2009 (as cited in PAMI, 2012). In the same report, it was noted that Aung et al. (2011) stated that pellets created from cereal straw cost $115/tonne including the cost to collect, densify and transport the material. The cost of pelleting municipal solid waste was determined to be approximately $10/tonne at a large scale (20 tonne/h) based on capital and operating costs of the equipment (PAMI, 2017). Therefore, a range of pelleting costs from $10 to $115 per tonne (or $0.27 to $3 per non-densified bushel) were used in this analysis. The value of pellets made from screenings was estimated based on the cost of other solid biofuels (Table 7 and Table 8). If the fusarium damaged grain pellets are considered equivalent to wood pellets, they would have a value of approximately $245/tonne. If the fusarium damaged grain pellets are meant to replace lignite coal as a fuel, their value could be as high as $14/GJ (assuming a $50/tonne CO 2 carbon tax). This converts to a value of $269/tonne based on the HHV of the fusarium damaged grain (Table 6). Realistically, pelleted screenings will not be considered as high value as wood pellets or lignite coal. So a range of values from $100/tonne to $200/tonne were used in the analysis (Table 9). Table 9. Estimated value of screenings ($/tonne) based on cost of pelleting and value of product. Cost of pelleting ($/tonne of screenings) Value of pellets ($/tonne of pelleted screenings) Low (100) High (200) Low (10) 90 190 Mid (60) 30 140 High (115) -15 85 The costs of transportation and ash disposal are not considered in this analysis, but similar costs would be incurred if wood pellets or coal were utilized. It was assumed that the combustion appliance is already in place so the capital and operating cost of the combustion equipment was not considered. Based on this rough analysis, fusarium damaged grain or screenings could have a net positive value if the cost of pelleting is in the low- to mid-range of expected pelleting costs and the value of the resulting biofuel is approximately $100 per tonne (which is reasonable based on current costs of other solid biofuels) or higher. The lowest net value in this analysis ($30/tonne) translates into a value of approximately $0.81/bu of non-densified screenings while the highest net value in this analysis ($190/tonne) translates into a value of approximately $5.17/bu. This also assumes that a local pelleting plant is available for processing and a market for pelleted biofuels exists. Page 22 of 28

5.4.2 Economic analysis of composting Fusarium damaged grain or screenings can also be turned into a usable or saleable product through composting. The results of this study indicate that the composting process deactivated the mycotoxin of interest, DON, but further work is required to determine if composting eliminates fusarium graminearum. This economic analysis assumes that the resulting compost can be land-applied without risk of further fusarium contamination. To estimate the cost of composting fusarium damaged grain and screenings, it was assumed that the producer would have access to sufficient quantities of manure, land base, and equipment (tractor with front end loader) required for pile construction and management. It was also assumed that each pile would be comprised of approximately 4 tonnes of grain or screenings and 8 tonnes of solid manure. Based on this study, this mixing ratio resulted in a good C:N ratio for composting and the size of piles were easily managed with a tractor and front end loader. The labour required to prepare the composting pad, handle the feedstocks, build six piles and periodically turn six piles was estimated based on PAMI s experience during this study. This labour requirement was approximately 20 hours (for six piles). Additional labour and equipment use would be required to utilize the end product. This labour requirement is estimated to be 10 hours (for six piles of product). The Saskatchewan Custom and Rental Rate Guide states that the hourly rate for a 150 hp tractor with front end loader and operator at $20/h is approximately $133/h (Saskatchewan Ministry of Agriculture, 2016). Therefore, the total cost to manage six piles is estimated to be $3,990. Since six piles contain a total of 24 tonnes of screenings, the cost of composting is estimated to be $166 per tonne of screenings (approximately $4.51/bu). Depending on the scale of the composting operation, the actual cost per tonne may be higher or lower than this value so a range of costs (from $100 to $200 per tonne of screenings) was used in this analysis. The value of compost was estimated based on the retail value of bulk compost of $137.50/tonne (Mother Earth News, no date). The actual value of manure compost as a soil amendment was calculated for PAMI Research Report E8310 (PAMI, 2013). The value of the nutrients alone (based on retail value of nitrogen, phosphorous and potassium) was estimated to be $3/tonne. The value of compost in terms of organic matter content and other soil health benefits was undefined but is estimated to be $50/tonne. Therefore, the range of values of $50/tonne to $140/tonne of compost was used for this analysis. Since composting results in a volume and mass reduction of substrate, this must be accounted for in the analysis of value of final product. Assuming the ratio of 2:1 by mass of manure:screenings is suitable and assuming a mass reduction of 33% due to Page 23 of 28

composting, every tonne of screenings will generate approximately two tonnes of final product. So the range of compost values in the analysis shown in Table 10 is $100 to $280 per tonne of composted screenings. Table 10. Estimated value of screenings ($/tonne) based on cost of composting and value of product. Cost of composting ($/tonne of screenings) Value of compost ($/tonne of screenings) Low (100) High (280) Low (100) 0 180 Mid (150) -50 130 High (200) -100 80 Based on this rough analysis, fusarium damaged grain or screenings could have a net positive value if the value of the resulting compost is relatively high (similar to retail compost) and the cost of composting involves only labour and equipment use. This analysis also assumes that the value of the manure used to generate the compost was negligible when, in fact, it would have a value approximately equivalent to the value of the composted screenings. Grain or screenings could be considered a carbon amendment which helps improve the C:N ratio and the overall composting process, which may generate a higher value product. But, based on this limited data, it is not clear if the incremental increase in value of compost justifies the cost of adding grain or screenings to manure. Considering that the alternative use of screenings has zero value and additional contamination could cost up to $20 per hectare of contaminated land, composting may be a viable disposal alternative. Page 24 of 28