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1 REPORT The Comparative Effects of Hermetic and Traditional Storage Devices on Grain: Key Findings from AflaSTOP s Off-Farm Controlled Tests in Eastern Kenya AflaSTOP: Storage and Drying For Aflatoxin Prevention December

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3 This report was written by AflaSTOP Program Director Sophie Walker, ACDI/VOCA; Dr. Claudia Probst, Independent Consultant to AflaSTOP; and Dr. Ramon Jaime, Independent Consultant to AflaSTOP. It was prepared in February 2015 and finalized in December 2015 for USAID and the Bill and Melinda Gates Foundation, who jointly fund the AflaSTOP program through Meridian Institute and co-implementers ACDI/VOCA and Agribusiness Systems International (ASI). For more information on the program, visit

4 TABLE OF CONTENTS I. BACKGROUND... 5 II. KEY FINDINGS... 7 A. Aflatoxin... 7 B. Fumonisin... 9 C. Insect infestation D. Grain quality factors III. DETAILED STATISTICAL OUTPUT A. Aflatoxin analysis and conclusions Aflatoxin development as a function of grain moisture Aflatoxin development as a function of storage devices Aflatoxin development as a function of disturbing the storage devices monthly B. Fumonisin analysis and conclusions Fumonisin development as a function of grain moisture Fumonisin development as a function of storage devices Fumonisin development in relation to regulatory limits Key Findings from the Fumonisin Analysis C. Insect presence and grading analysis and conclusions Insect development as a function of storage devices Distribution of insect infestation Grain weight loss as a function of storage devices Conclusion IV. OTHER FINDINGS A. Presence of odors in the storage devices B. Moisture loss over time V. DATA ANALYSIS SUMMARY... 40

5 I. BACKGROUND The AflaSTOP: Storage and Drying for Aflatoxin Prevention (AflaSTOP) project is identifying the most promising storage options to arrest the growth of aflatoxin during storage and designing viable drying options that will allow smallholder farmers to dry their grain to safe storage levels. The project works to ensure that businesses operating in Africa are able to provide these devices to smallholder farmers. It is jointly implemented by ACDI/VOCA and its affiliate Agribusiness Systems International under the direction of Meridian Institute. For more information on AflaSTOP and other key reports and resources, visit: AflaSTOP is testing new and existing hermetic storage devices suitable for smallholder farmers to store maize at moisture content up to 15 percent. Use of these hermetic storage devices should not adversely impact grain quality, increase mycotoxin content (e.g. aflatoxin and fumonisin), or increase insect infestation. This document contains a summary of the key findings (Section 1) and the initial detailed statistical output (Section 2) from AflaSTOP s Off-Farm Storage Device Testing. This research, now complete, investigated performance of hermetic (i.e. air-tight) storage devices in a controlled environment (e.g. small, enclosed, village / small town-based store rooms), compared to the control - the traditional, non-hermetic polypropylene bag used by the majority of smallscale farmers in East Africa. Table 1, below, provides an overview of each of these devices. Methodology summary: a randomized complete block design with six replications was implemented in two districts of the Eastern Province, Kenya, namely Makueni and Meru. Locally grown maize was purchased in each district and thoroughly homogenized prior to storage in any device. Baseline levels of grain moisture and mycotoxin levels were established. The performance of each device was monitored and data collected monthly for a total of six months, following set up in April and May Block samples were collected at set up (T0), and 5 months after the set up (T5), monthly samples were taken up to 6 months (T6). A comprehensive statistical analysis was performed by an independently contracted consultant to investigate the influence of storage device, grain moisture content, and location on the above mentioned factors. For a full description of AflaSTOP s methodology, see 5

6 Table 1: Storage Device Tested Storage technology Characteristics Storage Capacity and Estimated Price Metal silo Produced by local artisans; made out of aluminum; placed on a pallet; size ranges from 200-1,000 kg ~312 kg Price ~$144 Plastic silo Produced by local, large scale, commercial water tank manufacturer Kentainers; made out of heavy duty reinforced plastic, placed on a pallet ~350 kg Costs ~$92 Photo: Kentainers Bulk bag Photo: GrainPro PICS Bag Produced by Grain Pro and imported into the country duty free, made out of patented plastic technology, placed on custom made frame, capacity ranges from 800-1,300 kg (they are introducing a new 500kg bag) Triple layer plastic bag introduced to W Africa by Purdue University, kg now manufactured in Kenya by Bell Industries. Needs to be placed on a pallet, capacity ranges kg Up to 1,300 kgs Bulk bag costs ~$190 Frame can be made locally ~90 kg Costs ~$2.50 per bag Grain Pro Super Bag (GP) Produced by Grain Pro and imported into the country duty free, made out of patented plastic technology, needs to be placed inside another bag and placed on a pallet capacity ranges kg ~90kg Costs ~$2.50 each (plus an additional PP bag at about $0.50) Polypropylene (PP) bag Control: Traditional storage bag made of polypropylene. These are non-hermetic and widely available and used. $0.50 6

7 II. KEY FINDINGS Five different hermetic devices listed in table 1 and the control polypropylene bags were set up as wet and dry treatments in 12 stores in Eastern Kenya. Each storage devices was sampled every month for a six month period following on the short rains harvest in Eastern Kenya in These samples have been analyzed for aflatoxin, fumonisin, and moisture content. A series of samples were graded for insect damage, weight loss from insect damage, discoloration, mold, and different species of insects. Key findings from this analysis are summarized in the following sections. A high-level snapshot of major takeaway s is presented below. Hermetic storage significantly arrests aflatoxin growth during the storage period. 3 hermetic devices reduced aflatoxin increases in storage to <5% Prevents insect infestation, moisture loss, and grain discoloration No device (hermetic or non-hermetic) arrested fumonisin growth Bulk devices with higher moisture levels (13.7 and 14.2% MC) developed foul odor in later months. Hermetic storage makes a significant contribution to reducing household aflatoxin contamination, eliminating insects, and reducing household exposure to insecticides. Based on superior performance in the off-farm tests, the following devices will be tested on-farm with smallholder farmers in Eastern Kenya; PICS bag Metal Silo Grain Pro Grain Safe A. Aflatoxin Aflatoxin is a byproduct of fungal activities of toxic strains of aspergillus. Maize is particularly susceptible to aflatoxin contamination especially in tropical climatic conditions. Contamination starts in the soil but continued fungal growth during storage results in increasing aflatoxin levels in the maize over time. Over 74% of the maize AflaSTOP tested in Meru and Makueni was infected with aflatoxin levels above the national limit of 10ppb and 65% and 35% respectively were above 150ppb. Aflatoxin has been extensively linked as a primary cause of liver cancer and has been classified as a human carcinogen (group 1 carcinogen) by the International Agency for Research on Cancer. There are numerous studies which demonstrate animals exposed to aflatoxin also experience stunted growth development, and there are an increasing number of studies primarily from West Africa which are demonstrating that underweight and immune function are directly affected by aflatoxin exposure. Aflatoxin development as a function of grain moisture AflaSTOP found no statistical differences for maize stored at 13.72% and 14.22% average moisture content ( wet ) compared to grain stored at 12.20% and 13.12% moisture levels ( dry ) in regard to aflatoxin increase over time in both Makueni and Meru, Kenya, respectively. The 7

8 same result was obtained for all devices in both investigated districts. This implies that varying moisture levels up to 15% do not have any effect on aflatoxin development in stored maize. However, in the present work, moisture levels above 15% were not investigated; hence the importance of moisture cannot be ruled out completely 1. While the increase rates in aflatoxin levels was reduced when maize was stored in hermetic devices, compared to the traditional PP bags, maize in all devices stored at higher moisture levels developed a strong odor. The odor became apparent when opening the devices and indicates additional microbial activity and a likely occurring fermentation, which might make the grain unacceptable to farmers. The project is exploring how to investigate these issues. Aflatoxin development as a function of storage devices A linear regression analyses was conducted based on aflatoxin increase over time as compared to the traditional PP bags. Using the results from the region with the most significant differences (Makueni) the new storage devices were ranked according to their performance ability, presented in the table below, with (1) performing the best and (6) performing the worst. Table 2: Ranking of Device Performance in Makueni* Performance Ranking Storage Device Percent (%) Increase 1 (best) Grain Pro Bulk bag No increase 2 PICS Bags 1.8% increase in total aflatoxin per month 3 Metal silo 3.1% increase total aflatoxin per month 4 Grain Pro Super Bag 10% increase total aflatoxin per month 5 Plastic silo 13% increase total aflatoxin per month 6 (worst) Control: PP bags 92% increase total aflatoxin per month *Percent increase is based on a 500ppb baseline contamination level from devices tested in Makueni (see Table 8). Results for Meru are presented in Table 6. Additional information is provided in Table 9. Over the six month period aflatoxin levels increased in traditional PP bags between % in Meru and between % in Makueni. Performance evaluation per location In Meru and Makueni, all tested hermetic devices showed a significantly reduced rate of increase in total aflatoxin content, compared to the traditional PP bags. However, there were significant differences among devices in Makueni for both the aflatoxin content (ppb) and the average percentage of increase at 5 months. The Plastic silo and the GP Super Bag performed significantly worse than the Bulk Bag, PICS bag and Metal silo, but still significantly better than the traditional PP bag. For the top three devices (Bulk bag, PICS bag, and Metal Silo) no significant differences between devices were detected. This reinforces the ranking order in Table 2. The tested storage devices work on hermetic principles, which refer to an airtight storage of the grains. Limiting oxygen is thought to prevent both insect and (most) microbial activity, hence leading to improved storage quality. Airtight storage of the devices was repeatedly disturbed by our monthly sampling. The periodic opening and closing of the devices vaguely simulates the 1 Scoot W., Baributsa D., Woloshuk C Assessing Purdue Improved Crop Storage (PICS) bags to mitigate fungal growth and aflatoxin contamination 8

9 disturbance of the storage devices by farmers. Farmers are likely to open and close the storage device more frequently, which consequently allows oxygen to build up again. The periodic opening of the hermetic devices had no significant effect on the ability of the device to reduce aflatoxin increase, however the repeated disturbance in the polypropylene bags did significantly increase the aflatoxin levels. As the section above confirms, the reduced increase in aflatoxins in the tested hermetic devices was not due to moisture content. In all likelihood it is due to the reduced oxygen content, even with the repeated opening of the bags. The influence of a daily handling of the bags, as seen by smallholder farmers, will be tested by AflaSTOP in a subsequent phase of on-farm testing with smallholders directly, which will utilize the three best-performing devices from the off-farm, controlled tests. The real world manipulation and use of the storage devices is a very important aspect of its overall effectiveness and acceptability for smallholder farmers. Results from the On- Farm tests will be available for publication in early 2017, at Performance evaluation across locations The effectiveness of the Bulk Bag, PICS bag and Metal Silo to contain aflatoxin increase were not affected in the different locations within and across regions. They were very consistent with low variation among locations, treatments (dry and 13.12% MC and wet and 14.22% MC) and regions. Key Result: hermetic storage devices significantly reduced the increase in aflatoxin over time compared to current farmer storage practice. The rate of aflatoxin increase remained approximately the same regardless what moisture level the grain was below 15% moisture content. B. Fumonisin Fumonisin is a byproduct of fungal activities of toxic strains of fuarium. Maize is potentially susceptible to fumonisin contamination especially in tropical climatic conditions. Contamination starts in the soil but continued fungal growth during storage results in increasing fumonisin levels in the maize over time. Over 75% of the samples taken at storage set up (a month after the farmers had shelled their maize) were infected with fumonisin levels above the Kenyan national limit of 1ppm (part per million). Fumonisin has been extensively linked with esophageal cancer in humans, and liver cancer in animal studies. It is also implicated in neural tube defects in new born babies. Fumonisin contamination was detected in the stored maize and generally increased over time. This highlights the presence and activity of fumonisin producing fungi in local production areas. Potential health consequences of fumonisin consumption have not been rigorously established in Kenya since the focus is laid on aflatoxins. Generally, analysis indicates no significant differences among devices, treatments and regions in terms of fumonisin levels or increases over time. There were, however, some localized results; in Makueni where fumonisin levels were significantly lower in the PICS bags 9

10 compared to the GP bag and Metal Silo; in Mitheru, Meru all wet devices experienced significant increases in fumonisin, and all dry devices except the plastic and metal silos experienced significant increases in fumonisin. While aflatoxin levels were reduced when maize was stored in hermetic devices, fumonisin levels appear to continue increasing. Key Result: hermetic storage devices did not control the increase in fumonisin over time C. Insect infestation All hermetic devices significantly controlled infestation of a range of live insects, and no hermetic devices experienced any significant grain damage over time. All the bulk devices - Bulk Bag, Metal Silo and Plastic Silo - had some level of live insects at the device s outlet. A proportion of the Metal Silos and the Plastic Silos had insects at the inlet as well. A proportion of the GP bags and the PICS bags had some level of visible insect infestation. However the insect load was not sufficient during the storage period to cause significant damage to the bulk of the grain stored. Key Result: hermetic storage devices kill insect infestation present at the beginning of storage and prevent further infestation over time D. Grain quality factors Hermetic devices slowed the discoloration of the grain which occurs over time in storage. Key Result: hermetic storage reduces the increase in discoloration of grain over time compared to current farmer storage practice 10

11 III. DETAILED STATISTICAL OUTPUT A. Aflatoxin analysis and conclusions Aflatoxin development as a function of grain moisture The influence of grain moisture content (wet treatments and 14.22% MC compared to dry treatments and 13.12% MC in Makueni and Meru, respectively) on the rate of toxin increase was investigated by AflaSTOP. Each store represents one block or replicate (n = 6). Each store had two of each device, one for grain with higher moisture (wet) and one for grain with lower moisture (dry). Devices were randomly placed in each store to achieve a complete randomized block experimental design. Table 3 and 4 summarize the combined aflatoxin rate of increase per store. Table 3: Aflatoxin content (ppb) in different stores and treatments (dry and wet) in Meru at Month 0 (start) and Month 5 and total increase (%) at Month 5 Meru Month 0 Month 5 Total aflatoxin Aflatoxin (ppb) Aflatoxin (ppb) increase (%) Store Location Wet Dry Wet Dry Wet Dry 1 Marima 2084 a 2073 a PolePole 2061 a 1980 a Giampampo 2131 a 1868 a Mitheru 1510 b 1370 b Kariene 1935 a 1851 a Mwichiue 1951 a 2046 a Note: Locations with same letter are not significantly different. Columns with no letter mean that there are no significance differences among locations. Treatments (wet, dry) with asterisks are significantly different at the indicated location at that period of time. Table 4: Aflatoxin content (ppb) in different stores and treatments (dry and wet) in Makueni at Month 0 (start) and Month 5 and percent of increase at Month 5 Makueni Month 0 Month 5 Total aflatoxin Aflatoxin (ppb) Aflatoxin (ppb) increase (%) Store Location Wet Dry Wet Dry Wet Dry 1 Kola 431 * b 505 * ab Katuua 465 ab 503 ab 717 * 1211 * 42 * 140 * 3 Mukuyni 458 ab 455 b Mukutano 441 * b 572 * ab Mumandu 570 a 611 a Kiva 574 * a 468 * b Note: Locations with same letter are not significantly different. Columns with no letter mean that there are no significance differences among locations. Treatments (wet, dry) with asterisks are significantly different at the indicated location at that period of time. 11

12 Result: There were some differences among stores at set up, but not significantly, except for Mitheru. This is likely due to homogenization procedures, which were not completely accurate. Mixing large amount of grains is not always perfect and some variation in aflatoxin content is expected. While Store 4 in Mitheru had significantly lower aflatoxin levels at set up consistently across all devices, this did not significantly affect the overall analysis of the results. There was no difference between stores at the five month period in Meru. This means statistically aflatoxin increase in all the Meru stores behaved in the same way - thus different conditions in different stores did not affect the increase in aflatoxin levels. In Makueni, there were some differences among stores at set up but not at month 5. However a significant difference was observed between the wet and dry grain in one store (store 2) in which the dry grains had significantly more aflatoxin than the wet grains (again, all devices combined). However, the actual overall aflatoxin level for the wet or dry grain in that store was not significantly different compared to the other stores, and considering that each store represents one replicate (out of six), it does not affect the overall analysis. Key result: different environmental and geographic conditions did not influence the growth of aflatoxin in the different storage devices Impact of moisture on aflatoxin levels in PP bags over time The impact of moisture level on aflatoxin increases can be easily shown in a graphic form when comparing wet and dry treatment of PP bags in Meru and Makeuni. In Meru the wet maize was 13.72% moisture content (MC) and the dry maize was 12.22% MC. In Makeuni the wet maize was 14.22% MC and the dry maize was 13.12% MC. The wet maize and the dry maize aflatoxin levels over time match very closely. 12

13 Graph 2: Changes in aflatoxin levels in wet and dry maize stored in PP bags in Meru over time (weeks) Graph 1: Change in aflatoxin levels in wet and dry maize stored in PP bags in Makueni over time The same pattern was seen in Makueni, as seen in Graph 2. 13

14 Key result: Grain moisture (up to 15%) does not influence aflatoxin development in any device or location Aflatoxin development as a function of storage devices AflaSTOP investigated whether or not the storage devices significantly prevented the increase of aflatoxin over time. Tables 5 and 6 summarize the aflatoxin levels at set-up (Month 0) and (Month 5) in Meru (Table 5) and Makueni (Table 6). Note: Devices designated with the letter B functioned as control bags in which grains were stored from set-up to end (Month 6) without opening the bags. (i.e. PICS indicates a PICS bag that was opened each month for sampling. PICS B was sampled at set up, and then again in Month 6). This allowed AflaSTOP to investigate how much the disturbance (repeated re-opening of bags) of the hermetic conditions influenced the outcome. Table 5: Aflatoxin content (ppb) in different devices and treatments (dry and wet) in Meru at Month 0 (start) and Month 5 and percent of increase at Month 5 Meru Month 0 Month 5 Total aflatoxin increase Aflatoxin (ppb) Aflatoxin (ppb) (%) Device Wet Dry Wet Dry Wet Dry PP 1867 B 1919 B 4121 aa 4134 aa 116 a 117 a PICS 1997 B 1729 B 2410 cda 2226 ca 27 cd 17 c Bulk bag 1895 B da 2122 c 14 d 11 c GP bag 1949 B 1883 B 2349 cda 2165 ca 23 cd 14 c Plastic d 2285 c 15 d 20 c Metal B 2208 d 2383 ca 16 d 25 c Note: Devices with same lowercase letter (columns) are not significantly different). Columns with no letter mean that there are no significantly differences among devices. Treatments (wet, dry) with asterisks are significantly different at the indicated location and time (i.e. a significant difference between wet and dry treatment at that site / device). Different capital letter indicates significantly differences between month 0 and month 5 for the indicated device. Results: There were no statistically significant differences in aflatoxin content among devices at set-up (month 0) in Meru. The difference seen in aflatoxin content between wet and dry grain in GP bag B is due to initial mixing and not to storage. Changes in B bags will be discussed below. In general there were no significant differences among hermetic devices with dry grain. However, among devices with wet grain aflatoxin increases were significantly lower (below 16%) in metal silos, plastic silos and bulk bags compared to GP B bags (35%), and GP A, PICS A and PICS B (23 to 31%) 14

15 As established above, there were no significant differences in the increase of aflatoxin levels between grain moisture levels. Table 6: Aflatoxin content (ppb) in different devices and treatments (dry and wet) in Makueni at Month 0 (start) and Month 5, and percent of increase at Month 5 Device Month 0 Month 5# Total aflatoxin Makueni Aflatoxin (ppb) Aflatoxin (ppb) increase (%) Wet Dry Wet Dry Wet Dry PP 440 B 515 B 2645 aa 2981 aa 424a * 491a * PICS d 519 f 15 d 3 f Bulk bag d 524 f 0 (-8) d* 4 f* GP bag 480 B 541 B 610 cda 874 dea 21 cd* 73 de* Plastic B 463 d 1013 cda 0 (-8) d* 102 cd* Metal d 551 ef 14 d 9 ef Note: Devices with same letter are not significantly different. Columns with no letter mean that there are no significance differences among devices. Treatments (wet, dry) with asterisks are significantly different at the indicated device. Different capital letters indicate significant between month 0 and month 5. Results: There were no significant differences in aflatoxin content among devices at set-up (month 0) in Makueni. At the end PP bags (month 5) had significantly higher aflatoxin content than PP B bags (Month 6) in both wet and dry treatments implying that the monthly disturbance of the grain increased the levels of aflatoxin in PP bags see below. When comparing data presented in tables 5 and 6 from only Month 0 and Month 5 (B bags month 6) by a paired t-test there was a significant increase in aflatoxin levels in the wet and dry PICS bags, the wet bulk bag, the wet and dry GP bag and the dry metal silo in Meru. While these increases were significant within devices, that increase was not significant when compared to other hermetic devices. However, when data from all months was included in an analysis using a Tukey HSD multiple comparison test, there were no significant differences between month 0 and month 5. In Makueni there was a significant increase in aflatoxin levels in the wet and dry GP bags and the dry plastic silo. Further exploring plastic silo devices containing dry grain, the device in one store failed from the very beginning and showed linear increases in aflatoxin similar to the PP bags. Two devices started failing by month 3, another one by month 5, and the final device between month 5 and 6 leapt over 700% in aflatoxin levels. Wet devices. In the wet maize all other devices were significantly different from PPA and PPB bags. GPA bag has a significantly higher level of aflatoxin than bulk bag. There were no significantly differences among the Bulk Bag, PICA, PICB, GPB There were no significantly differences among the PICA, PICB, GPB and GPA 15

16 Dry devices PP A bags were significantly different compared to all other devices with the most significant increases in aflatoxin levels. PP B bags were significantly different compared to all other devices with the second highest most significant increase in aflatoxin levels. PP B bags and the plastic silo were not significantly different indicating that a plastic silo did not performed better than an undisturbed traditional PP bag. The Bulk Bag, PICA, PICB, Metal Silo all performed in a similar manner with the Bulk bag and the PIC A bags performing significantly better than all other devices, The plastic silo and the GPB Bags had significant increases in aflatoxin levels compared to the Bulk bag, PICS A, PICS B and metal silo. Aflatoxin development as a function of disturbing the storage devices monthly To be able to sample the hermetic devices they had to be opened. AflaSTOP reasoned that any farmer who had a larger device would open it regularly to remove maize for consumption, therefore opening the device monthly and removing between 2 to 8kgs was similar to expected use of the device by smallholder farmers. However the bagged devices were more likely to be left unopened, as a farmer would open and finish one bag before moving onto the next. Therefore at set up two of each bag devices (PP, PICS and GP Bag) were set up and sampled. One of each bag was marked B and was left un-sampled until month, whereas the other bags were sampled once a month. Table 7: Aflatoxin content (ppb) in different bagged devices and treatments (dry and wet) in Makueni at Month 5 and Month 6 Makueni Aflatoxin (ppb) A bags T5 Total aflatoxin increase (%) T0 - T5 B bags T6 Total aflatoxin increase (%) T0 - T6 Device Wet Dry Wet Dry Wet Dry Wet Dry PP 2645 Aa 2981 Aa 424 Aa* 49 A1a* 2075 Ba 1732 Ba 311 Bb 243 Bb PICS 579 b 519 b 15d 3f 697 b 618 c 38cd 21ef GP bag 610 b 874 Bb 21cd* 73de* 890 b 1221 Ab 77c 142c Note: Devices with same lowercase letter (columns) are not significantly different. Columns with no letter mean that there are no significantly differences among devices. Treatments (wet, dry) with asterisks are significantly different at the indicated location. Different capital letter indicates significantly differences between month 0 and month 5 for the indicated device. 16

17 Table 8: Aflatoxin content (ppb) in different bagged devices and treatments (dry and wet) in Meru at Month 5 and Month 6 Meru A bags T5 Aflatoxin (ppb) B bags T6 Total aflatoxin increase (%) T0 - T5 Total aflatoxin increase (%) T0 - T6 Device Wet Dry Wet Dry Wet Dry Wet Dry PP 4121 aa 4134 aa 116 a 117 a 3457 b 3126 b 81 b 65 b PICS 2410 cda 2226 ca 27 cd 17 c 2488 cd 2197 c 31 cd 15 c GP bag 2349 cda 2165 ca 23 cd 14 c 2581 c 2107 c 35 c 11 c Note: Devices with same lowercase letter (columns) are not significantly different. Columns with no letter mean that there are no significantly differences among devices. Treatments (wet, dry) with asterisks are significantly different at the indicated location. Different capital letter indicates significantly differences between month 0 and month 5 for the indicated device. Results: Sampling the bags every month significantly increased the levels of aflatoxin in the PP bags, compared to the PPB bags but it did not have a significant effect on the hermetic bags. Opening the hermetic bagged devices once a month did not increase aflatoxin levels. There was a significant increase in aflatoxin levels in the GPB B dry bags in Makueni these bags at the same time the GP B bags had more holes in them than the GP bags (holes were accessed at the end of storage). It is possible that disturbing the GP bags monthly knocked off insects and therefore they did not have as much success penetrating the bags. The higher level of holes would allow more oxygen into the bags, which in turn allowed the aspergillus to start propagating and the aflatoxin levels start increasing. Since the GP bag which was disturbed did not have an increased aflatoxin level it can be concluded that disturbing the hermetic devices once a month did not reduce their effectiveness at controlling aflatoxin increases. Summary: The devices can be ranked in order of performance in terms of reducing aflatoxin increase (most effective at the top of the list); 1. Bulk Bag 2. PICA, PICB, Metal Silo 3. GPA, GPB, Plastic Silo 4. PP While at times the performance of the devices overlaps (in terms of statistical significance) it should be remembered that there is a large difference in the % increase in aflatoxin between the worse performing devices (GP bags 77%, GP bags 213% and the Plastic Silos 249%) and the better performing devices (Bulk 4%, PICS 38% and Metal 26%). This further supports the selection of testing the Bulk Bag, PICS bag and Metal Silo in Phase 2, AflaSTOP s On-Farm testing with small-scale farmers. 17

18 Disturbing the PP bags significantly increased aflatoxin levels by month 5 above the significant increased aflatoxin levels seen in the undisturbed PPB bags, however disturbing the grain in PICS bag and the GP bag had no significant effect. Using linear regression analysis (Table 7 and 8) confirm that the slowing the increase of aflatoxin levels is due to the impact of the storage devices as well as more accurately determine the increase in aflatoxin levels each month by device. Since there is no moisture effect and to increase replicates, all treatments are combined - i.e. wet and dry devices are analyzed together. Table 9: Regression Models for aflatoxin content and increase by devices in Meru Variable Device Intercept Slope (ppb) Meru Slope % increase r 2 P>F DF Aflatoxin ppb Bulk bag Aflatoxin ppb GP bag < Aflatoxin ppb PICS < Aflatoxin ppb PP < Aflatoxin ppb Metal Aflatoxin ppb Plastic Note: The intercept is where the regression line crosses time 0 and is considered the estimated quantity at the start (month 0). The slope indicates the amount of toxin increasing per month, the smaller the slope the lower the rate of increase. Only models with r2 > 0.7 and a P>F below 0.05 are considered significant. Aflatoxin increase in Meru is significantly higher in PP compared to all other devices. While Bulk bag and plastic were significantly lower compared to PICS. Table 10: Regression Models for aflatoxin content and increase by devices in Makueni. Variable Device Intercept Slope Slope % increase Makueni r 2 P>F DF Aflatoxin ppb Bulk bag Aflatoxin ppb GP bag < Aflatoxin ppb PICS Aflatoxin ppb PP < Aflatoxin ppb Metal Aflatoxin ppb Plastic < Note: The intercept is where the regression line crosses time 0 and is considered the estimated quantity at the start (month 0). The slope indicates the amount of toxin increasing per month, the smaller the slope the lower the rate of increase. Only models with r2 > 0.7 and a P>F below 0.05 are considered significant. In Meru and Makueni only the PP bag shows a significant increase in aflatoxin levels. The other devices are all working within the same statically significance level. However it is possible to 18

19 differentiate performance using either the percentage increase in aflatoxin levels each device exhibits or the r 2 value. Any r 2 value below 0.05 shows a device which is really effective and reducing the increase in aflatoxin levels. Table 11: Performance of devices expressed as % increase per month r 2 Device Meru % increase in aflatoxin levels per month Device Makueni % increase in aflatoxin levels per month Bulk bag Bulk bag Plastic PICS Metal Metal GP bag GP bag PICS Plastic PP PP Results: Table 9 clearly demonstrates that maize stored in the bulk bag experiences minimal increases in aflatoxin. In Meru all the devices (other than traditional PP bags) performed within 5% of each other. In Makueni the failures of the Plastic Silo and the GP bag in the wet devices can clearly be seen to have changed their positions in the table. Both devices in Makueni experienced aflatoxin increases of above 10% per month. B. Fumonisin analysis and conclusions Fumonisin development as a function of grain moisture AflaSTOP investigated fumonisin increase as a function of moisture content (wet and 14.22% MC versus dry at and 13.12%MC, in Makueni and Meru, respectively) to determine the effect of different moisture level in the grain on the rate of fumonisin increase. Each store represents one block or replicate (n = 6). Each store had two of each device, one for grain with higher moisture (wet) and one for grain with lower moisture (dry). Devices were randomly placed in each store to achieve a complete randomized block experimental design. Table 12 and 13 summarize the combined fumonisin increase per store. r 2 19

20 Table 12: Fumonisin content (ppm) in different stores and treatments (dry and wet) in Meru at Month 0 (start) and Month 5 and total increase (%) at Month 5 Meru Month 0 Month 5 Total Fumonisin Fumonisin (ppm) Fumonisin (ppm) increase (%) Store Location Wet Dry Wet Dry Wet Dry 1 Marima 1.71 a 1.89 a 1.24 c c 1.10 c c 2 PolePole 1.78 a 1.78 a 1.19 c 1.46 bc c bc 3 Giampampo 1.89 *a 0.91 *b 1.59 *bc 2.32 * b 6 *bc 55 * b 4 Mitheru 0.97 *b 1.48 *a 5.07 *a 3.9 *a 238 * a 162 * a 5 Kariene 1.59 a 1.47 a 2.12 b 1.98 b 42 b 32 b 6 Mwichiue 0.98 *b 1.52 *a 1.93 bc 2.06 b 29 bc 38 b Note: Locations with same letter are not significantly different. Columns with no letter mean that there are no significance differences among locations. Treatments (wet, dry) with asterisks are significantly different at the indicated location. Table 13: Fumonisin content (ppm) in different stores and treatments (dry and wet) in Makueni at Month 0 (start) and Month 5 and percent of increase at Month 5 Makueni Month 0 Month 5 Total Fumonisin Fumonisin (ppm) Fumonisin (ppm) increase (%) Location Wet Dry Wet Dry Wet Dry Stor e 1 Kola 1.64 a 2.03 a 1.56 * 2.15 * ab 5 * 46 * ab 2 Katuua 1.14 b 0.92 c ab ab 3 Mukuyni 1.02 * b 1.45 * b b 0 (11) 0 (-5) b 4 Mukutano 1.34 ab 1.47 b a a 5 Mumandu 1.55 a 1.75 ab ab 8 12 ab 6 Kiva 1.67 a 1.74 ab ab ab Note: Locations with same letter are not significantly different. Columns with no letter mean that there are no significance differences among locations. Treatments (wet, dry) with asterisks are significantly different at the indicated location. Result: There were some differences among the stores at set-up. This is likely due to homogenization procedures. Mixing large amount of grains always results in some variation in fumonisin content. In Meru there were significant differences among stores and between treatments (although there had also been the same significant difference between treatments at baseline). Two stores maintained the same Fumonisin levels, Fumonisin levels at 3 stores increased for wet treatment between 6-42% and for dry treatment between 32-55% by month 5. One store increased 238% wet and 162% dry. In Mitheru fumonisin, levels increased equally in all devices with no significant differences at month 5 in the wet treatment, while in the in the dry treatment fumonisin levels increased significantly in 4 of the devices, although in the metal and plastic silo, fumonisin levels did not significantly change. 20

21 Relative humidity data and temperature data was collected hourly at all locations during the storage period. A brief examination of this data does not show any apparent deviation on the ranges of either temperature or relative humidity in Mitheru compared to the other stores. This could potentially indicate a reason for the higher fumonisin levels in all the devices in this store. In Makueni, there were no significant differences among stores for the wet treatment. However there were significant differences among stores for the dry treatment; one store remained the same, 4 stores fumonisin levels increased between 12-50%, and one store, Kola, increased at a significant 46% compared to Kola s wet treatment. There is a significant difference in the increase of fumonisin levels between the Meru and Makueni regions, with Meru being significantly higher than Makueni, However, when Mitheru store in Meru region is excluded from the analysis, there is no significant difference in the change of fumonisin levels between regions. Key conclusion: different environmental and geographic conditions did influence the growth of fumonisin and the different hermetic devices did not restrict the growth of fumonisin. Therefore, the external conditions were able to influence the internal conditions within the devices regardless of the hermetic conditions. Fumonisin development as a function of storage devices AflaSTOP investigated whether or not the storage devices significantly prevented the increase of fumonisin over time. Tables 14 and 15 summarize the fumonisin levels at set-up (Month 0) and end (Month 5) in Meru (Table 14) and Makueni (Table 15). Note: devices designated with the letter B and highlighted in grey were hermetic control bags in which grains were stored from set-up to end without opening the bags and were sampled in month 6. This allowed the research team to investigate how much the disturbance (repeated re-opening of bags) of the hermetic conditions influenced the outcome. 21

22 Table 14: Fumonisin content (ppm) in different devices and treatments (dry and wet) in Meru at Month 0 (start) and Month 5 and percent of increase at Month 5 Meru Month 0 Month 5# Total Fumonisin Fumonisin (ppm) Fumonisin (ppm) increase (%) Device Wet Dry Wet Dry Wet Dry PP 1.37B A PP B# (-3) PICS B A PICS B# Bulk bag B A GP bag B A GP bag B# Plastic Metal 1.55* 1.25*B A Note: Devices with same lowercase letter are not significantly different. Columns with no letter mean that there are no significance differences among devices. Treatments (wet, dry) with asterisks are significantly different at the indicated location. Different capital letter indicates significantly differences between month 0 and month 5 for the indicated device. # Data for unopened devices (B) correspond to month 6. Table 15: Fumonisin content (ppm) in different devices and treatments (dry and wet) in Meru at Month 0 (start) and Month 5 and percent of increase at Month 5 excluding data from Mitheru Meru (excluding Mitheru) Month 0 Month 5# Total Fumonisin Fumonisin (ppm) Fumonisin (ppm) increase (%) Device Wet Dry Wet Dry Wet Dry PP PP B# (-27) 0 (-23) PICS PICS B# Bulk bag GP bag (-5) GP bag B# Plastic (-7)* 21* Metal 1.66* 1.21* (-4) Note: Devices with same letter are not significantly different. Columns with no letter mean that there are no significance differences among devices. Treatments (wet, dry) with asterisks are significantly different at the indicated location. Different capital letters indicate significant differences between month 0 and month 5 at the indicated device. # Data for unopened devices (B) correspond to month 6. Results: There were no significant differences in fumonisin content between devices at set-up (month 0). The difference seen in fumonisin content between wet and dry grains in the metal silo is due to initial mixing and not to storage. In month 5 there was no significant difference in fumonisin levels in devices at set up in Meru. As discussed earlier, when the Mitheru store is removed from the data, no significant increases in 22

23 fumonisin levels were detected. In the plastic silo, the dry treatment was significantly higher compared to the wet treatment. Table 16: Fumonisin content (ppm) in different devices and treatments (dry and wet) in Makueni at Month 0 (start) and Month 5, and percent of increase at Month 0 (start) and Month 5 and percent of increase at Month 5 Makueni Month 0 Month 5 Total Fumonisin Fumonisin (ppm) Fumonisin (ppm) increase (%) Device Wet Dry Wet Dry Wet Dry PP PICS 1.35 * 1.99 * Bulk bag GP bag B A Plastic B 1.60 * 2.13 *A 8.44 * * Metal 1.23 B A Note: Devices with same letter are not significantly different. Columns with no letter mean that there are no significance differences among devices. Treatments (wet, dry) with asterisks are significantly different at the indicated device. Different capital letters indicate significant differences between month 0 and month 5 at the indicated device. Results: There were no significant differences in fumonisin content between devices at set-up (month 0). The difference seen in fumonisin content between wet and dry grains in the PICS bag is due to initial mixing and not to storage. There was a significant increase in fumonisin levels between month 0 and month 5 in both the Plastic silo and GP Bag dry treatments. In the Plastic silo dry treatment fumonisin, increase levels were significantly higher compared to the wet treatment. Currently there is no explanation, particularly when considering no significant increases were seen in either of the PP bags. Table 17: Comparison of Fumonisin content (ppm) in Monthly sampled bags in Month 5 and B bags in Month 6 bagged devices and treatments (dry and wet) Makueni Month 5 Sampled bags Month 6# B Bags Fumonisin (ppm) Fumonisin (ppm) Device Wet Dry Wet Dry PP PICS GP bag Note: No significant differences were noted 23

24 Table 18: Comparison of Fumonisin content (ppm) in monthly sampled bags in Month 5 and B bags in Month 6 bagged devices and treatments (dry and wet) Meru Month 5 Sampled bags Month 6# B Bags Fumonisin (ppm) Fumonisin (ppm) Device Wet Dry Wet Dry PP PICS GP bag Note: No significant differences were noted There were no significant differences between the undisturbed devices (B bags) and the normal devices for fumonisin, indicating that monthly sampling and opening of the bags did not influence fumonisin levels. The use of a linear regression analysis (Table 19 and 20) confirms that Mitheru in Meru was an outlier and that there was no significant increase in fumonisin in any one device. Table 19: Regression Models for fumonisin content and increase by devices in Meru as a function of time Variable Device Intercept slope Slope r 2 P>F DF Meru ppm % ppm % Fumonisin ppm Bulk bag Fumonisin ppm GP bag Fumonisin ppm PICS < Fumonisin ppm PP Fumonisin ppm Metal Fumonisin ppm Plastic Note: The intercept is where the regression line crosses time 0 and is considered the estimated quantity at the start (month 0). The slope indicates the level of toxin increase per month. The smaller the slope, the lower the rate of increase. Only models with r2 > 0.7 and a P>F below 0.05 are considered significant. 24

25 Table 20: Regression Models for fumonisin content and increase by devices in Meru as a function of time excluding Mitheru Variable Device Intercept slope Slope r 2 P>F DF Meru ppm % Ppm % Fumonisin ppm Bulk bag Fumonisin ppm GP bag Fumonisin ppm PICS Fumonisin ppm PP Fumonisin ppm Metal Fumonisin ppm Plastic Note: The intercept is where the regression line crosses time 0 and is considered the estimated quantity at the start (month 0). The slope indicates the level of toxin increase per month. The smaller the slope, the lower the rate of increase. Only models with r2 > 0.7 and a P>F below 0.05 are considered significant. The regression analysis is consistent with the ANOVA analysis showing that the fumonisin levels in the Mitheru store were uncharacteristically high. As such, Mitheru can be considered an outlier and therefore data excluding Mitheru may be more consistent and closer to reality. At this point, AflaSTOP cannot account for the reasons for the high fumonisin levels in the Mitheru store. Table 21: Regression Models for fumonisin content and increase by devices in Makueni as a function of time Variable Device Intercept slope Slope r 2 P>F DF Makueni ppm % ppm % Fumonisin ppm Bulk bag Fumonisin ppm GP bag Fumonisin ppm PICS Fumonisin ppm PP Fumonisin ppm Metal Fumonisin ppm Plastic Note: The intercept is where the regression line crosses time 0 and is considered the estimated quantity at the start (month 0). The slope indicates the amount of toxin increasing (or decreasing when slope is negative) per month. The smaller the slope the lower the rate of increase. Only models with r2 > 0.7 and a P>F below 0.05 are considered significant. Results: Fumonisin increases were not significant in any device. 25

26 Table 22: Performance of devices expressed as % increase per month r 2 Device Meru % increase in fumonisin levels per month Device Makueni % increase in fumonisin levels per month Bulk bag Bulk bag Plastic PICS Metal Metal GP bag GP bag PICS Plastic PP PP In the fumonisin regression model tables, the slope indicates the monthly percentage increase. It should be noted that the models are not significant (r 2 extremely low) which would indicate that there is no significant increase in fumonisin for any of the devices or locations, except for Mitheru, where there is a significant regression model (P>F < ) and a good coefficient of determination (r 2 = 0.628). Furthermore, most devices have good r 2 (~0.65 to 0.77) except for PP, which has an r 2 = Fumonisin development in relation to regulatory limits AflaSTOP investigated whether or not the storage devices significantly prevented the increase of fumonisin Table 23: change in the percentage of samples at specific regulatory fumonisin levels Makueni Below 1ppm 1-1.9ppm >2 3.9 > ppm T * T * Meru* T T Mitheru T T * 56.3 * 8.3 * Note. Meru data does not include Mitheru Kenya's legal limit for fumonisin is 1ppm in food and feed (in the US the limit is 2ppm in flour, and 4ppm in grain). Whereas we were able to demonstrate that at least 75% of the farmers we collected the grain from had significant aflatoxin contamination, we did not test fumonisin at the farm level. Therefore, the percentage of samples with fumonisin at start up is based on the homogenized grain. While the average increase in fumonisin levels is not as dramatic as the increase in aflatoxins, in Mitheru the levels increase 238% in the wet treatment and 162% in the dry treatment across all devices, except for the metal silo and plastic silo in the dry treatment and all samples by month 5 were above 2ppm. In Makueni the percentage of samples at the level between 2 and 3.9 was significantly higher (15%) in month 5 compared to month 0. r 2 26

27 Key Findings from the Fumonisin Analysis Fumonisin increases were not significantly different in hermetic devices compared to PP bags. Significant increases in fumonisin occurred only at a specific location (Mitheru). Hermetic devices did not significantly control increases in fumonisin levels. Moisture levels of the grain did not significantly affect fumonisin levels since sometimes the greater significant increase was within the wet devices (Mitheru) and at other times in the dry devices (Kola). C. Insect presence and grading analysis and conclusions Insect development as a function of storage devices While there was evidence of insects at the opening and the outlets (table 24), often it would only be between 1-10 insects. It is interesting to note that it was more often the dry devices which had the presence of insects. The insects most commonly present were Sitophilus spp (weevil) and Tribolium castaneum (red dust beetle), with occasional Oryzaephilus surinamensis (saw tooth beetle) or Rhyzopertha dominica (lesser grain borers) in the bag devices. The PP bags had a massive load of insects including weevil, red dust beetle, saw tooth beetle, and lesser grain borers, among others. At set-up, particularly in Makueni, Sitotroga cerealella (Anjoumois grain moth) was seen along with small numbers of Trogodema granarium (Khapra beetle). Live Prostephanus truncatus (Larger grain borer) was only seen once. Table 24: Percentage of devices which had observable insects present when opened in month 6 Storage Device Location in Device Top / inlet Outlet PP* PICS* Bulk Bag GP Bags* Plastic Silo Metal silo Note: Devices marked with asterisk do not have outlets. AflaSTOP investigated whether or not the storage devices significantly prevented the increase of insects over time. Table 25 summarizes whether significant changes were seen in insect numbers by species and at which month these significant changes were first observed. 27

28 Table 25: Significant increase in devices experience of specific insect numbers and the month at which this significance was first seen Meru PP bag Makueni PP bag Insect species Hermetic devices Month change Hermetic devices Month change Weevil NS S 4 NS S 2 Large Grain Borer NS NS NS NS Red Flour Beetle NS S 4 NS S 3 Moth NS NS NS S 5 Khapra Beetle NS NS NS NS Lesser Grain Borer NS NS NS S 4 Saw Tooth Beetle NS S 4 NS S 3 All insects mentioned in the table were present at baseline sampling. Significant levels are indicated S=significant NS = not significant Table 26: Percentage of samples with live insects at the start (month 0) of the storage in Meru and Makueni for Hermetic, PP and all devices. Insects All devices Makueni Meru Alive insects (Percent) 100 * 18 * Weevil (no outliers) 72 * (69 * ) 5 * Larger Grain Borer 0.15 * 0 * Red Dust Beetle 4.1 * 0.5 * Moths 1.8 * 0 * Khapra Lesser Grain Borer 0 0 Saw Tooth Beetle All insects (no outliers) 78 * (75 * ) 6 * indicate significant differences between regions At baseline sampling, there was a significantly higher presence of insects in maize sourced from Makueni than from Meru. Results: All hermetic devices significantly controlled insect infestations with no significant increase in any insect numbers between set up and month 6. Distribution of insect infestation In Meru, the most common insect was Weevil, followed by Red Flour Beetle and to a lesser extent Saw Tooth Beetle. Significant changes in their numbers were seen in month 4. At month 6, an average of 636 insects (all) were counted in a ~ 2kg sample. Graph 3: Proportions of main insects seen at T6 in PP bags, Meru Weevil Red Flour Beetle Saw Tooth Beetle 28

29 In Makueni, the most common insect was also Weevil, followed by the Red Dust Beetle, the Lesser Grain Borer, and the Saw Tooth Beetle. Significant changes in weevil numbers were seen in month 2, Red Dust Beetle and Saw tooth Beetle in month 3, Lesser Grain Borer in month 4, and moth in month 5. On average at month 6 in a ~ 2kg sample, 1,947 insects were counted (including other insects whose numbers did not significantly change). While AflaSTOP found small numbers of Large Grain Borer and Khrapa Beetle at set up, there was no significant increase in their numbers over time in the PP bags. Makueni had a significantly higher level of Lesser Grain Borer which first appeared in month 1, and Saw Tooth Beetle which first appeared in month 2 and increased between month 2 and month 3 by over 550%. During certain months, AflaSTOP found over 400% increase in specific insect numbers: in Meru, Weevils in month 2, and Saw Tooth Beetle in month 4, in Makueni Saw Tooth Beetle in month 3, and Lesser Grain Borer in month 4. During several months, AflaSTOP found that the common insect presence increased by more than 100%. Graph 4: Proportions of main insects seen at T6 in PP bags, Makueni Weevil Red Flour Beetle Moth Lesser Grain Borer 29

30 Table 27: Average insect numbers in approximately 2 kg samples at start up and after 6 months of storage and monthly percentage change in insect numbers in PP bags Meru Makueni Number of insects T0 - T6 Monthly % increase Number of insects T0 - T6 Weevil 4 * * * * 76 Red Flour Beetle * * 321 Lesser Grain Borer * * 183 Saw Tooth Beetle Moth Note: Percentages with * are significant when compared to the other region. Monthly % increase Grain weight loss as a function of storage devices Grain weight loss in storage is caused by moisture content loss either as a loss through evaporation when moisture content of the grain comes into equilibrium with the relative humidity in the surrounding environment or from the actions of insects. Insects feeding on the grain can cause the grain to break (insect damage grain), and make holes in the grain (insectholed grain) causing mass loss of the grain. AflaSTOP investigated whether or not a storage device significantly prevented the increase of damage of the grain over time by insects. In Meru, there was a significant change in the percentage of damaged grain by insect in the PP bags increasing from 1.2% at set up to 2.9% in month 6, but there was no significant change in insect damage grain in the hermetic devices. In Makueni, there was no significant change in insect-damaged grain in any device, either PP or hermetic, regardless of treatment - dry (1.5% to 1.5%) or wet (1.8% to 1.7%). AflaSTOP investigated whether or not the storage devices significantly prevented the increase of insect-holed grain to whole grain over time. To estimate the damage inflicted by insects, AflaSTOP calculated the approximate weight of the grain lost by comparing the weight of the grains with holes with the same number of whole unblemished grains. 30

31 Table 28: Investigating the percent of loss in weight based on 100 grams of grain in PP bags only in Makueni and Meru (there was no significant change in hermetic bags from start up) Month PP Makueni PP Meru Dry Wet Dry Wet c 1.12 b 0.11 b 0.43 c bc 2.16 b 0.45 ab 0.51 c abc 2.63 b 0.34 ab 1.07 c abc a 0.48 ab 3.33 bc abc ab 1.72 ab 7.05 bc ab ab 1.32 ab ab a a 3.43 a a Note: Months with same letter are not significantly different. There was no significant difference between treatments in either location Results: Hermetic devices did not experience significant percentage increase in percent of loss in weight based 100 grams of grain, nor was there a significant difference between the wet and dry treatment in any of the hermetic devices. In combined data from Meru and Makueni, there were no significant differences among devices, including PP bags, from the initial month through month 3 for percent of loss in weight based 100 grams of grain. However, from month 4 through month 6, there were significant differences between PP and the rest of the devices. The percent of loss in weight based 100 grams of grain increases significantly after each month in the PP, but not in the rest of the devices. There were no significant differences among the rest of the devices at any time of sampling (Tables 5). Likewise, there were no significant differences among time of sampling (effect of time) for percent of loss in weight based 100 grams of grain in the hermetic devices regardless of region or dry or wet treatments. When PP is analyzed by region and treatment, there is a constant increase in the percent of weight loss with time in both regions and both treatments, resulting in a significantly higher percentage in month 6 compared to month 0 in both regions and treatments, except for dry treatment in Makueni. In PP, bags there were significant increases in loss of weight over time, but there were no significant differences in weight loss between the dry and the wet treatments in either region. The low percentage in weight loss figures in Meru Dry is actually due to the fact that in a number of PP devices in Meru there were virtually no grains remaining with just holes. Therefore, the data from the wet treatments is more representative. There was no significant change in the estimated weight of the grain with insect holes in any of the hermetic devices. Table 29 summarizes data in weight lost through insect action in the PP bags over a 6 month period. 31

32 Table 29: Percentage increase in weight loss of grains damaged by insects in PP bags over time Month Meru Makueni Makueni - no outlier b 1.1 d 1.1 d b 2.1 cd 2.1 d b 5.3 bcd 3.2 cd 3 b abc 11.4 bcd 4 b abc 13.6 bc ab 16.8 ab 16.8 ab 6 a a 25.6 a A significant loss in weight occurred in month 5 in Meru, whereas in Makueni, a significant loss in weight occurred in month 4. This is consistent with the much higher insect numbers in Makueni (particularly in the dry treatments) and therefore their impact on the grain. Months with the same lower case letter (a, b ) among rows are not significantly different. Note: weight was adjusted to account for moisture loss, i.e. weight in later months was standardized to month 0 for accurate comparison. Result: placing grain in hermetic storage stops insects damaging the grain. The significant difference in weight loss in Makueni compared to Meru can be explained by the significantly higher number of insects present in the samples. Insect development as a function of grain moisture Using Variance Components Analysis and Analysis of Variance, AflaSTOP investigated insect development as a function of grain moisture based on the two different treatments in each region. Analysis indicates that moisture content of grain at the levels from over 12% to below 15% do not have a significant effect on the development of insects. Visible mold development as a function of storage devices AflaSTOP investigated whether the storage devices significantly prevented the increase of visible mold over time. In terms of the composite samples containing grain from all the devices, there was no significant increase of visible mold in any device. At sampling, AflaSTOP noticed visible mold on the grain surface (either at the top of the bag or the inlet of the device). For the most part it was only a small numbers of grains with spots of green mold, or threads of white mold on the surface level of the grain. More mold was seen in month 5 than in month 6. The different molds were seen mostly in the wet treatments (13.7% and 14.12% MC). The Bulk Bag wet and the GPA wet bag were the least effected with over 75% of all other devices showing some presence when they were opened. In the case of the GPA bags, the lack of mold could be related to the fact that over 90% of the wet bags had insect holes. However, this mold was not noticeable in grading and was either due to only a limited number of grains being affected so that they did not show up as a significant difference at grading, or the visible molds degraded over time and were no longer visible at grading. 32

33 Discolored grain development as a function of storage devices Discolored grains is an important grading factor used both by the formal and informal trading sectors in Kenya. Maize with higher degrees of discoloration will be discounted in price in the market. It was important to check whether hermetic storage had an effect on the coloration of the grains. Every sampled collected was tested for aflatoxin and fumonisin. For grading, however, only one sample per device per treatment per month was graded. AflaSTOP graded the T0 sample first and then graded T6, T5, T4, etc. When there was no change and the current month was similar to T0 we stopped grading samples from that device and treatment. Hence the following tables will not have data for some months. Table 30: Weight of post-harvest discolored grain in different devices (among rows) and sampling month (among columns) in Meru region Meru Bulk GP PICS Metal Plastic PP Month Dry Wet Dry Wet Dry Wet Dry Wet Dry Wet Dry Wet ab ab 7.7 c b bc b bc 3 9.2B 9.9 AB 8.9 B 7.5 * b 10.3 * AB 10.4 AB 9.5 * ab 15.5 * abc,a ab ab 17.0 ab B B B 12.6 ab 11.1 B B 14.6 * ab 21.6 * a,a * a 9.0 * a 19.2 ab Note: Devices with same capital letter (A, B) among columns are not significantly different at the month indicated. Months with the same lower case letter (a, b ) among rows are not significantly different at the device indicated. Asterisks indicate significant differences between treatments dry grain and wet grain at the indicated device and month. Result: AflaSTOP investigated whether the storage devices significantly prevented the increase of discolored grains over time. In a paired comparison T-test between month 0 and month 6 (data from all hermetic devices are combined) there are significant differences between month 0 and month 6 in the increase in discolored grain in both treatments (wet and dry grain) and regions. In devices with dry grain in Meru, the percentage of discolored grain increased 2.3% from month 0 (10.7 %) to month 6 (13.0 %), while in devices with wet grain, it increased 1.7 % from month 0 (7.8%) to month 6 (9.5%). All devices experienced a slow but significant increase in discoloration over time Wet hermetic devices on average discolored 1.7% (Metal below 1%, and bulk bag above 3% compared to PP bag around 14%) Dry hermetic devices on average discolored 2.3% (GP around 1% and PICS over 6% (probably closer to 4% the month 0 level was surprisingly low)) and PP bags around 3% 33

34 In PP bags, that increase is significantly faster in wet bags than in dry in months 3 and 5, respectively for wet and dry. However, in month 4 and month 6, the increase is no longer significant (dry lag behind but catch up) PP wet had significantly more discolored grain than all hermetic wet devices Inexplicably, dry grain in metal silos was discolored significantly higher than for wet, both in month 3 and 6. Table 31: Weight of post-harvest discolored grain in different devices (among rows) and sampling month (among columns) in Makueni region Bulk GP PICS Metal Plastic PP Month Dry Wet Dry Wet Dry Wet Dry Wet Dry Wet Dry Wet b b,b 5.5 * 10.9 *A ab * 11.4 * ab a Note: Devices with same capital letter (A, B) among columns are not significantly different at the month indicated. Months with the same lower case letter (a, b ) among rows are no significantly different at the device indicated. Asterisks indicate significant differences between treatments dry grain and wet grain at the indicated device and month. In a paired comparison T-test between month 0 and month 6 (data from all hermetic are combined) there is a significant difference between month 0 and month 6 in the increase of discolored grain in both wet and dry grain. In devices with dry grain in Makueni, the percentage of discolored grain increased 1.7 % from month 0 (4.8 %) to month 6 (6.5), while in devices with wet grain, it increase 3.4 % from month 0 (6.2 %) to month 6 (9.6 %) All devices experienced a slow increase in discoloration: Wet devices averaged 1.7% - ranging from around 2% (PICS, Metal, plastic and PP) to over 5% (Bulk bag and GP bag) In dry devices, the average was 2.3% ranging from below 1% (PICS) to around 4% (PP, plastic, and metal). In PP bags, that increase is significantly faster in bags with wet grain than in bags with dry grain in months 3 and 4. However, the increase after month 4 was not significant. In the field, farmers relate discoloration with aflatoxin content. Pearson correlation analysis between discoloration and aflatoxin within the PP bags indicates a weak correlation, but not conclusive. The correlation was strongest in Meru wet with a correlation coefficient (r = 0.65 compared to Makueni wet r = 0.4. In dry devices, Meru has an r = 0.55 compared to Makueni, where r = 0.3. When all data was combined from all regions, the correlation coefficient r was Aflatoxin as a function of insect population, moisture and time Using correlation and multiple linear regressions analyses, AflaSTOP investigated whether the aflatoxin contamination in the PP bags was a function of insect population, moisture, or time. 34

35 Correlation analyses of aflatoxin with insects and moisture on PP data in Meru indicate that there were correlation coefficients higher than 0.5 only with weevil (0.7) and red beetle (0.56). Correlation analyses of aflatoxin with insects and moisture on PP data in Makueni indicates that there were correlation coefficients higher than 0.5 only with moisture (-0.72) (discussed in the aflatoxin section of this report), weevil (0.71), and red beetle (0.7). There is a strong correlation between moisture, time and aflatoxin in PP bags. However, the multiple regression analysis using these variables (data on the fungi Aspergillus was not available) indicates that aflatoxin increase is only a function of time, and not related to moisture content. Furthermore, negative correlation between moisture content of the grain and aflatoxin indicates aflatoxin continued to increase at the same rate that moisture content decreased. There is no correlation coefficient over 0.5 between aflatoxin and any variable in the combined data set of all hermetic devices (excluding PP) in either Makueni or Meru. Multiple regression models also show a relationship with aflatoxin of the same variables as the correlation analysis, which were included in most multiple regression equations. Result: Correlation between insect populations and aflatoxin doesn t imply causation by the insects. The PP bags provided an equally supportive breeding ground for aspergillus, weevil, and red dust beetle. Since the environment was conducive for both the fungi growth, and consequently aflatoxin increase as well as insect reproduction, there is a positive correlation between the levels of aflatoxin and insect numbers. However, this experiment does not provide any conclusive evidence of causal relationship between increase in insect numbers and increase in aflatoxin. In AflaSTOP, we successfully homogenized the maize, which meant that aflatoxin (and therefore aspergillus spores) was evenly distributed throughout the grain. Where the fungus is restricted to small points of infection, it is possible that the movement of insects through the grain spreads secondary infections. In places such as the U.S., these isolated pockets of aflatoxin are what drive the need for vigorous sampling methodologies. However, when AflaSTOP went to the field to identify contaminated maize in Kenya, a high incidence of contaminated maize was readily identified, which indicates that aspergillus is already widespread throughout the grain. This is the case even before insect actions. One way to prove the role of insects as vectors for spreading aspergillus spores, and therefore aflatoxin, would be to run experiments comparing the increases of aflatoxin levels in grain with different levels of insect infestation, including treatments with no insects. Conclusion Hermetic bags controlled all insect infestations and even though at certain times some live insects were observed in the hermetic devices, they were not at levels which caused significant damage to the grains or loss in weight. Therefore, farmers can save on insecticide cost by using 35

36 hermetic devices and can store their grain in hermetic devices for at least 6 months without significant insect damage if the devices are set up correctly. In more humid conditions (Meru) all hermetic devices slowed down discoloration of the grain with the increase being 5% less than in the PP bags, and although some were better than others, there was only a 1% differential in the weight of discolored grains between the different hermetic devices. 36

37 IV. OTHER FINDINGS A. Presence of odors in the storage devices The rains occurred in October and November. During the sampling period project staff noticed, particularly in devices with wet grain (13.7% and 14.12% MC), a distinct and unpleasant smell which was possibly related to fermentation. It was not possible to scientifically quantify the smell - but qualitative analysis was carried out in Makueni in Month 6, ranking the odors from 0 - no unusual smell, 1 - slight smell which disappeared during sampling, 2 - distinct smell (but not catch the back of the throat), decreasing while sampling to 3 - very bad, no change at the end of sampling. Table 32: Average smell score across devices in Makueni T6 Storage Device Grain Type Average Smell Metal silo Wet 2.50 Metal silo Dry 1.25 Plastic silo Wet 2.67 Plastic silo Dry 2.00 Bulk bag Wet 1.00 GP A Wet 1.50 GP B # Wet 2.00 PICS A Wet 1.25 PICS B# Wet 1.25 Note: Devices marked with B were not opened until month 6. There was no unpleasant odor in bulk bag dry, GP dry, PICS dry, and PP wet and dry bags. Further investigation suggests that the odor indicates the activity of other microorganisms in these devices related to anaerobic microorganisms also seen in silaging. The unpleasant odors are probably indicative of the presence of butyric acid which occurs most often when silaging material is too wet. One possibility is that in wet devices, there was more condensation along the inside surface of the device; this could pool along the sides of the device, increasing the moisture content along the sides, which in turn led to the formation of butyric acid and the bad smell which comes with it. B. Moisture loss over time Farmers dry their maize to about 15% moisture content, then pack it in their stores (mostly an outside store or a dedicated room in their home) in PP bags. Moisture is lost over time to the atmosphere. This was very clear in the PP bags. In Meru, where the average moisture level of the dry bags was 12.20% at set up - the moisture remained between 12 and 12.8% during the six months. However the maize at 13.72% moisture dropped down to 12.5% within about 3 months. 37

38 Graph 7: Moisture levels over time in dry maize in all devices in Meru Graph 5: Change in moisture levels in wet maize in all devices in Meru 38