Final Report for GNC14-186
Project Information
It is good practice for farmers to clean their grain bins before storing newly harvested grain. Unsanitary bins can harbor unwanted insects that infest grains, including the red flour beetle (Tribolium castaneum (Herbst)), the saw tooth grain beetle (Oryzaephilus surinamensis (Linnaeus)), and the lesser grain borer (Rhyzopertha dominica (Fabricius)). Due to the fact that it is difficult and labor intensive for farmers to clean under the perforated floor of steel grain bins, where many insects thrive due to the presence of grain dust and broken kernels, it is necessary to employ other methods to control stored-product insects prior to refilling the bin. The use of elevated temperatures has been previously documented as an effective method to kill stored-product insects within 2-4 hours when temperatures of 50-60°C are reached and maintained in empty bins. Previous research has shown that the efficacy of residual insecticides or inert dusts against stored-product pests can be increased after application followed by heat treating the bin with temperatures below 50°C. Laboratory trials will be conducted to determine the efficacy of a low toxicity insecticide (Diacon) and the inert dust, diatomaceous earth (DE), on concrete dishes to simulate the floor of a grain bin against 20 adults of T. castaneum, and then held at 28, 32, 36, 42, 44 and 46°C for 4-24 h. Based on the results, Diacon or DE will be chosen for further validation in field trials using empty 3000 bu bins, with and without heat treatments. Bioassays containing adult insects will be placed inside the bins to determine the efficacy of treatment. The end result of this research will be to provide guidelines to farmers about the optimal time to apply residual insecticides or inert dusts to bins prior to storage based on temperatures inside the bins. In addition, the relationship of Diacon or DE in combination with heat treatments on the mortality of stored-product insects will be determined.
Introduction:
Several species of stored-product insects have been reported from empty bins (Chao, 1954; Wright, 1991; Reed et al., 2003; Arthur et al., 2006; Hagstrum et al., 2008). Removal of grain and grain debris from empty bins prior to storing newly-harvested grain can help in reducing insect numbers in the newly-stored grain (Reed et al., 2003; Arthur et al., 2006). Some stored-product insects are long-lived and removal of residual grain and grain debris, which serves as their food, may not be sufficient to control them. The use of an approved insecticide (Arthur and Subramanyam, 2012) after sanitation of empty bins is shown to provide effective control of insects. Bridgeman (1994) conducted tests with an amorphous silica (Dryacide®, A & R McLaughlin Private Limited, Wembley Downs, Western Australia) applied to storage surfaces at 6-8 g/m2 in four 100 m long x 15 m wide x 5 m high rectangular storage structures in Australia. Treatment efficacy was verified by using 30 flour-baited cardboard traps (Wright, 1991) in each storage facility. Trapping three weeks before sanitation and three weeks after sanitation showed no significant difference in the percentage of traps with beetles and psocids. However, after application of Dryacide®, trapping over the next 11 weeks showed a decrease in the percentage of traps with beetles and psocids from 18 to 3% and from 90 to 40%, respectively.
Clean, empty bins can also be treated with several alternatives to chemical insecticides. Two approved alternatives to chemical insecticides include the use of diatomaceous earth or DE and the use of high temperatures (Subramanyam and Roesli, 2000; Tilley et al., 2007; Subramanyam et al., 2011), or a combination of DE and heat (Dowdy and Fields, 2002).
There are numerous studies documenting the effectiveness of DE dusts against stored-product insects, mostly on grains (McGaughey, 1972; Korunić et al., 1996; Subramanyam and Roesli, 2000; Kavallieratos et al., 2005; Vardeman et al., 2007; Kavallieratos et al., 2010). There are limited published studies that examined the efficacy of heat treatment of empty bins against adults of stored-product insects. Tilley et al. (2007) reported 100% mortality of adults of the red flour beetle, Tribolium castaneum (Herbst), lesser grain borer, Rhyzopertha dominica (F.), and rice weevil, Sitophilus oryzae (L.), by raising temperatures of the bin’s floor to a minimum of 50°C for up to 2-4 h. Moog and Maier (2007) reported 77-91% mortality of adults of the maize weevil, Sitophilus zeamais (Motschulsky), when exposed for 3 h at 55°C in the plenum area of empty bins. The mortality of T. castaneum adults in the plenum area at this temperature and exposure time was 72-87%. However, similar exposure in areas 1.83 m above the plenum resulted in 100% mortality of both species. The authors inferred that the lack of uniform distribution of hot air at the plenum may have resulted in less than 100% mortality of beetles.
Previous research has shown that heat treatments in combination with DE increased mortality of stored-product insects. Dowdy (1999) reported mortality of unfed and fed adults of T. castaneum adults exposed to untreated glass Petri dishes and dishes treated with 5 g/m2 of four DE dusts at 34 and 50°C and 65% r.h. The DE formulations used were Concern® (Necessary Organics, Inc., New Castle, Virginia, USA), Natural Guard® (VPG Co-op Gardening Group, Inc., Bonham, Texas, USA), Insecto® (Natural Insecto Products, Inc., New Castle, Virginia, USA), and Protect-It® (Headley Technologies, Vancouver, British Columbia, Canada). Exposure of unfed insects for 15-30 minutes to 34°C alone resulted in 0-1.3 and 42.5-55.0% mortality, when mortality assessments were made 1 d and 7 d after exposure, respectively (Dowdy, 1999). A similar exposure just to 50°C resulted in 1.3-28.8 and 51.3-65.0% when assessments were made 1 and 7 d after exposure, respectively. Adults that were fed or had access to food showed reduced mortalities that ranged from 0-1.3% and 0-56.3%, irrespective of whether observations were made 1 or 7 d after exposure. Protect-It® was the most efficacious dust producing 91.3-100% mortality of unfed adults after a 15-30 minute exposure to 34 and 50°C when mortality was assessed 1 d after exposure. The mortality with the other three DE dusts was greater at 50°C compared to 34°C, and the mortality of adults ranged from 8.9-76.3% based on mortality 1 d after exposure. However, all dusts produced 97.5-100% mortality at both temperatures when mortality of adults was assessed after 7 d. Mortality of adults that were unfed never reached 100%, irrespective of the temperature, exposure time, and post-mortality assessment time, except for adults exposed for 30 minutes to Protect-It® at 50°C. These results suggest that sanitation, in conjunction with heat and DE, is more effective than heat alone or DE plus heat. Additionally, this study also showed delayed mortality effects associated with heat alone and heat plus DE. Fields et al. (1997) reported that in an oat mill, completely mortality of adults of the confused flour beetle, Tribolium confusum Jacquelin du Val, occurred when temperatures reached 47°C after 32-38 h, but in the presence of DE complete mortality of adults occurred when temperatures reached 41ºC. Dowdy and Fields (2002) evaluated heat in combination with application of Protect-It® applied at 0.3 g/m2 to second and third floor surfaces of a pilot flour mill subjected to a heat treatment against T. confusum adults. The benefits of DE were only evident on the south side of the second floor where temperatures did not quickly reach 47°C. At the end of the heat treatment, adults exposed to partially and fully treated DE floor surfaces had 50 and 75% mortality, respectively, compared to 15% mortality of those exposed to heat alone. The combination of heat plus DE can kill insects quicker than heat or DE alone. Ebeling (1994) showed that the time required for 100% mortality of the German cockroach, Blatella germanica (L.) at a temperature of 43.3°C in the presence of a silica aerogel, a synthetic silica, was reduced from 147 to 41 minutes.
To our knowledge, there are no published studies that investigated the combined efficacy of DE and a range of temperatures on concrete surfaces, such as those found in empty bins. The combination of these treatment methods would involve lower energy inputs to obtain temperatures lethal to insects (Fields et al., 1997). Eliminating stored-product insects in empty bins prior to storage of newly-harvested grain, along with additional integrated pest management methods, such as bin sanitation, can increase the profitability and quality of stored grain in a more sustainable and environmentally friendly manner.
In the present investigation, laboratory experiments were designed to examine the influence of five temperatures below 50°C, two DE application rates to concrete arenas, and four exposure times on mortality of T. castaneum adults. Concrete arenas in 9-cm Petri dishes simulated the floor of empty bins.
The research determined the optimal temperature at which to apply DE, a sustainable method, to disinfest empty grain bins prior to storage of newly harvested grain. Farmers should plan to apply DE to the concrete floor of the storage bin when temperatures are greater than or equal to 42ºC inside the bin. Diatomaceous earth can be placed at a concentration of 5 g/m2 and a fan can be used to distribute it over the floor of the bin.
Cooperators
Research
2.1.Insects
Cultures of T. castaneum were reared in the Stored-Product Insect Research and Education Laboratory at Kansas State University in the Department of Grain Science and Industry, Kansas State University, Manhattan, Kansas, USA. Cultures were reared in 0.94-L glass jars filled with 250 g of a medium consisting of 95% organic, whole wheat flour (Heartland Mills, Marienthal, Kansas, USA) and 5% (by wt) brewer’s yeast in growth chambers at 28°C and 65% r.h. Jars were closed with metal lids fitted with filter papers and wire-mesh screens.
2.2.Concrete arenas
Ready-mix concrete (Rockite, Hartline Products Co., Inc., Cleveland, Ohio, USA) was mixed with tap water to make a slurry. The slurry was poured into plastic Petri dishes (Fisher Scientific, Denver, Colorado, USA) with a diameter of 9 cm, height of 1.5 cm, and surface area of 62 cm2. The ratio of grams of concrete mix to milliliters of water used to fill the Petri dishes was 2:1. The slurry was allowed to dry in the Petri dishes for 48 h before dishes were used in experiments.
2.3.Diatomaceous earth application
The DE formulation used for experiments was DiaFil610® (Imerys Minerals California, Inc., San Jose, California, USA) (Korunić, 1997). DiaFil® 610 is natural fresh water DE, white in color, and has an average particle size of 10 μm. It has a surface area of 26-28 m2/g. The DE moisture content is 3-5%. DE was applied at either at 2.5 or 5.0 g/m2 (the recommended rate for application to empty storage bins) directly onto the concrete arenas. After adding DE, the Petri dishes were gently shaken in a counter clockwise manner to evenly distribute DE on concrete arenas. Control treatment (0 g/m2) included concrete arenas that were not treated with DE.
2.4.Bioassays
Adults of T. castaneum used in experiments were separated from diet using a sieve with 841µm openings (Seedburo Equipment Co., Chicago, Illinois, USA). Ten adults of 1-4-weeks of age and of mixed sex were aspirated and placed on untreated and DE-treated concrete arenas. After insect introduction, concrete arenas were placed inside incubators with a volume of 0.14 m3 (Isotemp Standard Lab Incubator, Fisher Scientific, Denver, Colorado, USA) set at 28, 36, 42, 44, and 46°C. The temperature and humidity levels were measured using a HOBO® data logger (Onset Computer Corporation, Bourne, Massachusetts, USA). Humidity levels at 28, 36, 42, 44, and 46°C were an average of 65, 21, 20, 19, and 18% r.h., respectively. Generally, at elevated temperatures humidity levels are around 22-25% (Mahroof et al., 2003; Subramanyam et al., 2011). At each temperature, 20 untreated concrete arenas and 20 arenas each treated with DE at 2.5 and 5.0 g/m2 were placed in incubators. At each of the temperatures, a replication consisted of five arenas representing a DE application rate of 0, 2.5, or 5.0 g/m2 that were sampled after 4, 8, 12, and 24 h of exposure. At each exposure time, all adults from five arenas (50 adults) were pooled and placed in 150 ml round plastic containers holding 30 g of T. castaneum diet. Containers were closed with perforated lids covered with a fine mesh to prevent insect escape but to allow air diffusion. Care was taken when picking adults from DE-treated concrete arenas so as to not transfer any DE to insect diet in containers. Containers were then held at 28°C and 65% r.h. for an additional 24 h before mortality assessments were made. To determine mortality, adults were separated from the flour in containers using an 841-µm sieve. Live and dead adults were counted, and percentage mortality was calculated based on number of dead adults out of the total. Each temperature, DE rate, and exposure time combination was replicated six times.
- Data analysis
Mortality in DE treatments was corrected for control mortality using Abbott’s formula (1925). Corrected mortality data were transformed to angular values and analyzed using the general linear model procedure in SAS (SAS Institute, 2008). A three-way analysis of variance (ANOVA) was run to determine significant differences (P < 0.05) in mortality due to the main and interactive effects of temperature, DE rate, and exposure time. Corrected mortality data by temperature and DE rate were analyzed using one-way ANOVA to determine significant differences (P < 0.05) among exposure times. If ANOVA was significant, the Ryan-Einot-Gabriel-Welsch multiple comparison test was used for mean separation (SAS Institute, 2008).
The mortality of T. castaneum adults on untreated arenas at 28, 32, and 36°C was <4% after 4-24 h of exposure (Table 1). At 44°C, the mortality of adults was 1% at a 4 h exposure, but was between 26-63% at exposures of 8-24 h. Similarly, at 46°C, mortality of adults was 19% at 4 h but was 48, 76, and 100% at 8, 12, and 24 h exposures, respectively. There were no significant differences among exposure times in control mortality at 28 and 36°C (Frange = 0.95-1.00; df = 3, 20; P≥0.4133). However, significant differences were observed among exposure times at temperatures of 42, 44, and 46°C (Frange = 4.04-7.34; df = 3, 20; Prange = 0.0017-0.0212).
The corrected mortality of T. castaneum adults exposed to 2.5 and 5.0 g/m2 increased with an increase in temperature and exposure time. Three-way ANOVA showed that temperature, DE rate, and exposure time were significant (P< 0.05) (Table 2). In general, more adults died at 5.0 g/m2 than at 2.5 g/m2. At 28, 36, 42, 44, and 46°C mortality across the exposure times at 5.0 g/m2 was 2.2-13.9%, 0.4-10.6%, 2.0-6.2%, and 6.1-27.2%, respectively. At 44°C, at 8 and 24 h, mortality in 2.5 g/m2 treatment was 0.3-1.5% greater than in 5.0 g/m2 treatment. Similarly, at 46°C a 24 h exposure at both DE rates resulted in 100% mortality of adults. Except for the temperature and exposure time interaction, all two and three way interactions were not significant. The significant temperature and exposure time interaction indicated that the mortality responses over time at the different temperatures were not consistent.
One-way ANOVA of T. castaneum corrected mortality over time at 2.5 g/m2 DE treatment at 28, 36, 42, 44, and 46ºC was significant (Frange = 4.32-35.82; df = 3, 20; Prange = <0.0001-0.0168). Similarly, T. castaneum corrected mortality at 5.0 g/m2 DE treatment over time at five temperatures was significant (Frange = 3.56-38.78; df = 3, 20; Prange<0.0001-0.0326). The trends observed in mortality of adults at each of the five temperatures at 2.5 g/m2 and 5.0 g/m2 were similar. Only the 24 h exposure at both DE rates produced significantly greater (P < 0.05) mortality at 28, 36, and 42°C. Adult mortality at 44 and 46°C at both the DE rates reached 99-100% after a 24 h exposure. However, the high control mortality at 44 and 46°C in DE exposures, especially at 12 and 24 h confounded from truly gauging the effect of DE at these temperatures. Nevertheless, the laboratory results support that a combination of heat plus DE can increase mortality of T. castaneum adults at temperatures below 50°C.
- Discussion
DE dusts are typically composed of 80-93% silicon dioxide, as well as varying amounts of organic matter, clay minerals, magnesium carbonate, among others (Antonides, 1998; Subramanyam and Roesli, 2000; Shah and Khan, 2014). DE works by abrading the insect’s cuticle, interfering with the water retention ability of the insect, and results in death through desiccation (Korunić, 1998; Dowdy and Fields, 2002).
The use of elevated temperatures (50-60°C) is a long-standing technology that is a safe and proven method to manage stored-product insects in empty bins and grain-processing facilities (Dosland et al., 2006; Subramanyam et al., 2011). Lethality in insects at high temperatures depends on both the temperature and exposure time (Evans and Dermott, 1981; Fields, 1992; Denlinger and Yocum, 1999; Mahroof et al., 2003). Death in insects exposed to elevated temperatures is due to quicker formation of lethal lesions, where the healing process that counters the lesions become less operative (Denlinger and Yocum, 1999). At elevated temperatures, insects’ cuticular wax becomes more fluid, allowing loss of water, leading to death by desiccation (Hepburn, 1985). Additionally, insect’s respiration, an indicator of overall metabolic rate, is adversely affected at elevated temperatures (Neven, 1998). At the cellular level, exposure of insects to elevated temperatures decreases hemolymph pH and ion concentration, denatures lipids, carbohydrates, proteins and nucleic acids, inactivates major glycolysis enzymes, and disrupts plasma membrane (Hochachka and Somero, 1984; Denlinger and Yocum, 1999; Neven, 2000).
In our study, the mortality of T. castaneum adults increased with increasing temperatures and exposure times. At temperatures of 28, 36, 42, and 44°C the combined effect of DE and heat was better than heat alone. The use of DE at temperatures between 36 and 46ºC was shown to increase mortality of T. castaneum adults on concrete arenas compared to heat alone or DE treatment at 28°C and 65% r.h. A DE treatment of 5.0 g/m2 produced slightly greater mortality that varied with temperature and exposure time, than at 2.5 g/m2, but the differences observed were not large enough to justify using the higher DE rate.
At 44°C after a 24 h exposure, the DE treatments contributed to an additional 35-36% mortality of adults, because temperature alone provided 63% mortality. Unlike observations made by Tilley et al. (2007) at 50°C, we observed 100% mortality of T. castaneum adults after a 24 h exposure to 46°C alone on untreated arenas. Insects succumb to elevated temperatures greater than 35°C, and at more extreme temperatures, the death of the insect occurs quicker (Fields, 1992). Our results support previous studies conducted at a few constant temperatures that the insecticidal effects of DE increase as the temperature increases (Arthur, 2000; Dowdy and Fields, 2002). The addition of DE to heated environments can result in a more rapid water loss from insects, especially at low humidities, resulting in faster death of insects (Mahroof et al., 2003). Furthermore, increased activity of insects at higher temperatures may result in greater pick-up of DE particles, leading to rapid desiccation effects due to the combined effect of DE and heat. Our results suggest that it is possible to obtain effective control of T. castaneum adults by combining DE with temperatures of 44 or 46°C. In practical heat treatments of empty bins (Tilley et al., 2007), unlike the laboratory experiments where temperatures remain constant, temperatures are dynamically changing over time. However, temperatures in the range of 44-46°C can be maintained for several hours to obtain an effective kill of stored-product insects in the presence of DE when disinfesting empty bins. Additional studies are warranted in empty bins to determine minimum temperature-time combinations, with and without food, to show how the combined effect of DE and heat can be used for effective disinfestation under practical field conditions.
Educational & Outreach Activities
Participation Summary:
Refereed Journal Article:
Frederick, J. L. and B. Subramanyam. 2016. Influence of temperature and application rate on efficacy of a diatomaceous earth formulation against Tribolium castaneum adults. Journal of Stored Products Research 69:86-90.
Oral Presentations:
Frederick, J. L., Bh. Subramanyam, H. Dogan. Influence of temperature and dosage on the efficacy of a diatomaceous earth formulation on Tribolium castaneum adults. ASABE Annual International Meeting, 16 July 2015, New Orleans, LA.
Frederick, J. L., Bh. Subramanyam. Diatomaceous earth efficacy against Tribolium castaneum is influenced by dosage and exposure temperature. North Central Branch of Entomological Society of America, 1 June 2015, Manhattan KS. 1st place winner in PhD 10 minute paper for Medical, Urban, Veterinary Entomology session.
Frederick, J. L., Bh. Subramanyam, H. Dogan. Diatomaceous earth efficacy against Tribolium castaneum is influenced by dosage and exposure temperature. Kansas State University Research Forum, 31 March 2015, Manhattan KS.
Frederick, J. L., Bh. Subramanyam, Casada, M., Tilley, D. Heat treatments for managing pests in empty storage bins. Graduate Student Symposium, 2014, Manhattan, KS.
Project Outcomes
This research study is timely due to the interest on the part of researchers and food producers to adapt more sustainable technologies to reduce insects in grain. Not only is diatomaceous earth with heat treatment effective in empty storage bins, but it is also effective in larger grain processing facilities, such as flour mills, feed mills, and other food processing facilities. Dr. Subramanyam is world-renowned for his studies in stored products, and when he travels to give talks in the US and outside the US, heat treatment is always a topic of interest. This research has been discussed to audiences in Thailand, Ethiopia, Vietnam, Malaysia, Pakistan, as well as in Kansas, Iowa, Minnesota, and Kentucky, among others.
Economic Analysis
Life Cycle Analysis of Different Pest Management Methods for Storage Bins and Stored Grain
Under proper conditions, grain can be stored and maintained for several years. If proper conditions are not maintained, however, spoilage can begin to occur after a matter of hours.
Conditions that effect quality of stored grain include:
- Moisture
- Temperature
- Oxygen supply
- pH
- grain condition
The greatest concern with regards to stored grain is damage due to microorganisms and other pests. The aim of this life cycle analysis is to compare different methods of managing stored product insects including ambient aeration of stored grain, fumigation, chilled aeration, and heat treatment of the empty storage bin with diatomaceous earth.
A life cycle analysis was conducted comparing the above mentioned methods.
FUMIGATION
Phosphine is a fumigant that is widely used on stored grains. By law, it must be applied by a certified technician and the following points are important to note:
- Good sealing is critical
- Remove or break up any crust on grain surface
- Fumigation should occur when grain temperature is between 70-90ºF
- EPA exposure limit is 0.3 ppm
The biggest concern with fumigation is toxicity. If incorrectly done, death of animals or humans could occur. Fumigation typically occurs 2 or 3 times: once before the bin is filled, and once or twice after and throughout the storage time.
AERATION
Fumigation is not the only step taken to reduce or eliminate the growth and reproduction of storage pests. Aeration must also occur. With ambient aeration, large volumes of air pass through the grain mass using a fan and an air delivery system. The design of the aeration system must provide adequate volumes and pressures of air for both the quantity and depth of the stored grain. The idea behind grain aeration is to maintain the grain at a low enough moisture so that pests do not flourish.
CHILLED AERATION
Chilled aeration systems contain a refrigerated unit that allows chilled air to pass through the mass of grain. Compressor capacities range from 107-130 kW. Fans provide airflow rates around 16,500 m3/h at 2000 Pa of static counter pressure, and the average grain chilling capacity is 350 t per day (Maier et al. 1996).
HEAT TREATMENT
Heat treatment, a more than 100-year-old technology, involves raising the ambient temperature of a an empty bins/storage space or a clean gran-processing facility to 50-60°C for 24 h or less to kill stored-product insects. Heat treatment is an environmentally benign and a safer alternative to chemical insecticides.
LIFE CYCLE ANALYSIS
Figure 1 shows the system boundary for the LCA. A functional unit of 1 ton of grain, specifically popcorn, was used due to the availability of data collected. Raw material acquisition was analyzed for three systems: fumigation with ambient aeration, chilled aeration (no fumigation), and heat treatment.
|
|
Figure 1 System Boundary
Data
The data used to conduct this LCA was taken from research by Maier et al. and current research conducted at Kansas State University and the USDA in Manhattan, Kansas.
- Bin yield: 133.9 tons
- Ambient aeration fans used were 5.6 kW axial-flow
- Grain chiller connected load: 9.3 kW
- Cost of fumigation + application: $0.66/ton of grain
- Cost of heat treatment + DE: $1.00/ton of grain
- Propane fuel used for 100,000 Btu heaters
Assumptions
- Coal used for electricity
- Can ignore grain harvest and downstream use of grain
- Phosphine applied with no toxic releases
- Ambient aeration fans: $5,000
- Chilled aeration unit: $25,000
- Heaters for heat treatment: $5,00/unit (2 units total = $1,000)
Total Emissions
Raw material emissions were calculated based on the cost of the aeration fans, a chilled aeration unit, two propane fueled heaters, plus the cost of phosphine and its application, and the cost of diatomaceous earth and its application, atmospheric emissions were obtained from the Economic Input-Output Life Cycle Analysis Tool by Carnegie Mellon.
Operation emissions were calculated based on the time of operation of aeration fans, the chilled aeration unit, and heat treatment time. Aeration fans ran for 100 hours totaling 560 kWh and the chilled aeration unit ran for 280 hours totaling 2604 kWh. Heaters for heat treatment run for approximately 8 h.
|
Method |
CO2 Emissions (lb/ton of grain) |
|
Ambient aeration with fumigation |
64 |
|
Chilled aeration |
279 |
|
Heat treatment |
2 |
Because of the intensive use of electricity required to operate the chilled aeration system and ambient aeration, CO2 releases are significantly higher than from the use of heat treatment. The major difference between the use of fans for ambient aeration and chilled aeration versus heat treatment, is that heat treatment is conducted when the storage bin is empty. The prior two methods are conducted with grain in the storage bin..
REFERENCES
- Carnegie Mellon University Green Design Institute. (2012) Economic Input-Output Life Cycle Assessment (EIO-LCA) US 2002 (428) model [Internet], Available from: <http://www.eiolca.net/> [Accessed 25 Apr, 2012]
- Maier, D.E., R.A. Rulon, L.J. Mason. Chilled versus ambient aeration and fumigation of stored popcorn. Parts 1 and 2. 1997. J. Stored Products. 33(1):39-58.
- Maier, D.E., R.A. Rulon. Evaluation and optimization of a new commercial grain chiller. 1996. Applied Engineering in Agriculture. 12(6):725-730.
Areas needing additional study
More studies are needed to determine whether or not there is an influence of gender or age of stored product insects to DE.