INTRODUCTIONThe cowpea, Vigna unguiculata (L.), is a legumeof the Fabaceae family. It is one of the most importantlegume crops in the world. The plant thrives intemperate climate and requires little agricultural inputsfor growth [1]. Cowpeas are popular and cheap in manydeveloping countries. In Nigeria, for instance, they area major staple crop. Small-holder farmers are the majorproducer of cowpea grains in Nigeria [2].Cowpeas serve as a rich and affordable source ofnutrients, especially protein, in sub-Saharan Africa andsome parts of America and Asia [3]. Cowpea beanscan be cooked, powdered, germinated, or even used aspart of a weaning formula. As a dish, they complementtubers and cereals. Consequently, cowpea grains arepresent in the diet of many developing countries wherepopulation suffers from malnutrition and proteindeficiencies.Cowpeas are mainly cultivated by the local farmersfor profits and satisfy the basic nutritional needs of thelocal population. However, farmers fail to meet thelocal demand as a result of drastic post-harvest lossescaused by insects and other pests. In fact, these lossesare considered as one of the underlying causes of foodscarcity and poverty [4].Insects damage cowpea grains by boring holes,thus causing weight loss, poor quality, and low marketvalue Cowpea aphids (Aphis craccivora L.), leafhoppers354Ajayi F.F. et al. Foods and Raw Materials. 2022;10(2):353–364(Empoasca spp.), cowpea weevils (Callosobruchusmaculatus), and witch-weed (Striga gesnerioides) arethe main insect species that feed on cowpea seeds [5, 6].Callosobruchus maculatus is the main pest thatcauses losses in cowpea grains. C. maculatus i s a fieldto-store pest that typically begins in the field, and thelevel of prior-harvest infestation determines the extentof damage to stored grains [7]. A range of insect pestcontrol measures have been adopted over decades toreduce the prevalence of cowpea grain loss in the fieldand during storage.According to Ogunfowokan et al., Nigerian farmersuse Lindane (Gammalin 20EC), Dichlorvosdichlorodiphenyltrichloroethane (DDT), Chlorpyrifos,Endosufan, and Aldrin to prevent pest infestation [8].Synthetic chemicals are very effective in preservinggrains and increasing their production yield. However,they have major drawbacks as improper use oftenresults in environmental pollution, pesticide residue infood, and toxic poisoning of the ecosystem [9]. Hence,several attempts have been made to test pesticides thatare environmentally friendly, harmless to people, andinexpensive.Resourceful African farmers tried to use suchnatural preservatives as plant powder, ashes, and cowdung. Several authors have documented the insecticidalefficacy of plant products on different types of pests [10].In fact, plant products with aromatic propertiesare known to prevent insect infestation of storedcowpeas [11]. Ikbal and Pavela made an extensiveresearch to assess the use of aromatic plants aspesticides during storage [12]. They reported thatessential oils of plant origin could serve as botanicalinsecticides as they contain a lot of bioactive compoundswith insecticidal, nematicidal, larvicidal, and antifeedantproperties that inhibit insect oviposition [13, 14].Essential oils are natural derivatives from aromaticplants which contain volatile and phenolic compoundswith unique flavors. Several works have reportedthe insecticidal effect of essential oils during grainstorage [15–19].Balanites aegyptiaca fruits have quite a numberof bioactive compounds with various medicinalproperties [20]. The essential oil extracted fromB. aegyptiaca possesses anticancer, antimicrobial,antioxidant, anticarcinogenic, antidiabetic, antifeedant,and antiviral activities [21, 22]. Natural fumigantsdeveloped from plants do not threaten the ecosystem.Insecticides based on essential oils are sold all over theworld, but their production does not exceed 5% [23]. InAsia, Europe, and North America, natural extracts havebeen used as insecticides for more than a century, muchlonger than any synthetic insecticides.Previous studies have investigated the insecticidalefficacy of oil extracted from B. aegyptiaca. Forinstance, Mokhtar et al. reported a strong effect ofB. aegyptiaca seed oil on the mortality rate of red flourbeetle (Tribolium castaneum Herbst) [17]. Similarly,Nwaogu and Yahaya investigated the insecticidalefficacy of oil extracted from B. aegyptiaca in storedcowpea seeds [18]. The studies provided evidence thatB. aegyptiaca seed oil could be used as an insecticideagainst storage pest. However, very few publicationsfeature the use of B. aegyptiaca essential oils incontrolling insect infestation of stored grains.Storage materials are also important for seedtreatments and grain quality. Buleti et al. conductedan experiment in the Northeast of Nigeria to assess theeffect of B. aegyptiaca oil on weevil growth in cowpeagrains stored in various packing materials [15]. Thepresent research provides some new data on using plantproducts as storage pesticides.STUDY OBJECTS AND METHODSPlant material. Fruits of Balanites aegyptiaca L.were acquired from the Gashua market in Yobe State,Nigeria. The fruits were authenticated at the Departmentof Agronomy, Faculty of Agriculture, Federal UniversityGashua, Nigeria.Preparing the seeds. The mesocarp of B. aegyptiacawas scraped with a clean sharp knife and driedin an oven at 45°C for 24 h to reduce stickiness. Theendocarp was broken down with a hammer to obtainseeds. Then, the seeds were dried to constant weight inan airtight oven (45°C, 72 h). Subsequently, the seedswere milled into fine particles using an electric blenderand stored in zip lock bags.Extracting B. aegyptiaca essential oil. Themethod developed by Nguefack et al. was used toextract essential oil from B. aegyptiaca seeds [24]. Theexperiment began by placing 500 g of pulverized seedsof B. aegyptiaca in a 5 L flask. After that, distilledwater was added to cover the sample. Essential oilwas obtained by hydrodistillation using a modifiedClevenger-type apparatus at normal atmosphericpressure and 96–97°C for 4 h. The resulting essential oilwas collected by drying it out with anhydrous sodiumsulfate and kept at 4°C in Eppendorf tubes until the gaschromatography-mass spectrometry (GC/MS) analysis.Evaluating the effect of B. aegyptiaca essential oilagainst Callosobruchus maculatus.Experimental site. The experiment was carriedout in the Agronomy Laboratory of the Departmentof Agriculture, Federal University, Gashua, after thegrowing season of 2021.Experimental design and treatment. Theresearch involved five storage materials (jute bags,polythene bags, sacks, plastic containers, and glassbottles), experimental samples (5, 10, and 15 mL ofB. aegyptiaca essential oil diluted in 1 mL of acetone),a check sample (with 0.125 g of aluminum phosphide),and control (acetone). The experiment was laid out in acompletely randomized design with three replications ofeach treatment.Insect culture: source and rearing. C. maculatuswas first cultured from a cowpea seed infested at thelocal market in Gashua. Weevils multiplied in fresh and355Ajayi F.F. et al. Foods and Raw Materials. 2022;10(2):353–364previously uninfested cowpea varieties in the laboratoryat an ambient temperature (27–30°C) and relativehumidity (70–75%).Treatment and maintenance of cowpea seeds. Fortykilograms of pristine cowpea seeds was purchaseddirectly from the local farmers in Gashua, immediatelyafter harvest. To destroy and/or prevent any initialinfection, they were placed in a plastic container andmaintained in the freezer below 0°C for five days. Afterthat, the seeds were taken out of the freezer and placedon a laboratory bench, covered with a screen, and left toequilibrate for 72 h [25].Adult mortality of C. maculatus. Mortality contacteffect of B. agyptiaca essential oil on adult C. maculatuswas determined using the method developed byObeng et al. [26]. According to the procedure, 200 g ofcowpea seeds was held in different storage materialsand then thoroughly mixed with: (a) 5, 10, and 15 mLof essential oil diluted in 1 mL of acetone; (b) 0.125 gof aluminum phosphide; and (с) acetone (control).After that, the storage materials were left open for 2 hat room temperature to disperse acetone. Thereafter,20 unsexed pairs (10 males and 10 females) of threeday-old C. maculatus beetles were introduced into thestorage materials. They were kept on laboratory benches.Dead insects were counted after 24, 48, and 72 h afterinfestation using Abbott’s equation [27]. To confirmmortality, insects were probed three times with a sharppin [28]. The data were subjected to Probit analysis [29].Oviposition. For this part of the experiment, 100 gof cowpea seeds was held in the varying storagematerials and thoroughly mixed with 5, 10, and 15 mLof essential oil diluted in 1 mL of acetone, 0.125 g ofaluminum phosphide, and control (acetone). After that,10 males and 10 females of three-day-old newly sexedC. maculatus beetles were introduced into the storagematerials, where they paired and laid eggs. Followingegg deposition, 100 seeds were randomly selected ondays 7, 30, 60, and 90, and the number of eggs depositedon the cowpea seeds was counted and recorded in eachtreatment and replicate [30].Egg hatchability. At this stage, 100 g of cowpeaseeds was infested with 20 (10 males and 10 females)sexed pairs of five-to-seven-day-old C. maculatusbeetles in a transparent plastic container. The insectspaired and laid eggs for six days. After oviposition,100 seeds (27 g) bearing eggs were chosen and placedin various storage materials that contained pure anduninfested cowpea seeds (73 g). They were thoroughlymixed with 5, 10, and 15 mL of essential oil diluted in1 mL of acetone, 0.125 g of aluminum phosphide, andcontrol (acetone).In each treatment and replicate, the cowpeaseeds were stored on the laboratory bench until adultbeetles emerged. The number of emerged adults wasrecorded on days 30, 60, and 90 after the exposure.The percentage of adult emergence was calculatedconversely from each of the treatments and replicatesaccording to the method developed by Adesina andOfuya [31], with a slight modification (1):(1)Seed perforation. This test included 100 g ofcowpea seeds held in the varying storage materials andthoroughly mixed with 5, 10, and 15 mL of essential oil,0.125 g of aluminum phosphide, and control (acetone).After that, 10 pairs of three-day-old newly sexed C.maculatus beetles were introduced into the storagematerials and kept in the laboratory for 90 days. Everyfour weeks for three months, the number of exit holeswas assessed by counting in each seed from a randomsample of 100 seeds.The weevil perforation index (WPI), which measuredthe protective ability of the storage material, wascalculated according to standard methods. If the weevilperforation index was ≥ 50%, it indicated an increase inweevil infestation or a low efficacy of the plant material.To obtain the percent protection ability (PPA), the weevilperforation index was subtracted from 100 using thefollowing equation (2):where WPI is the weevil perforation index; WPI > 50 isthe negative protectant of plant material, i.e., low antiweevilactivity; WPI < 50 is the positive protectant, i.e.,high anti-weevil activity.Seed weight loss. To calculate the seed weight loss,100 g of cowpea seeds was randomly selected after30, 60, and 90 days of storage. To obtain the final seedweight for the sample, all dead insects and other debrisin the cowpea seeds were removed, and the cowpeaseeds were weighed. As described by Sibakwe andDonga [32], the percentage of seed weight loss wascalculated using the following equation (3):(2)(3)Seed damage. After 30, 60, and 90 days of storage,100 g of cowpea seeds were randomly selected fromthe lots. We divided seeds into two groups, damagedand undamaged, and counted seeds with exitholes. Adenekan et al. [30] described how to quantifythe percentage of damaged seed using the followingequation (4):(4)Egg hatching =𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑝𝑝𝑝𝑝𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑝𝑝𝑝𝑝𝑁𝑁𝑁𝑁𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝 𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑒𝑒𝑒𝑒𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑒𝑒𝑒𝑒 𝑖𝑖𝑖𝑖𝑝𝑝𝑝𝑝 𝑡𝑡𝑡𝑡𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑡𝑡𝑡𝑡× 100WPI =𝑃𝑃𝑃𝑃𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑃𝑃𝑃𝑃𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑖𝑖𝑖𝑖𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑃𝑃𝑃𝑃𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑃𝑃𝑃𝑃𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑖𝑖𝑖𝑖𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 + 𝑃𝑃𝑃𝑃𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑃𝑃𝑃𝑃𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑖𝑖𝑖𝑖𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝 𝑆𝑆𝑆𝑆𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁 𝑖𝑖𝑖𝑖𝑝𝑝𝑝𝑝 𝐶𝐶𝐶𝐶𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝐶𝐶𝐶𝐶× 100Weight loss =𝐼𝐼𝐼𝐼𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 𝑤𝑤𝑤𝑤𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁ℎ𝑡𝑡𝑡𝑡 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 − 𝐹𝐹𝐹𝐹𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 𝑤𝑤𝑤𝑤𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁ℎ𝑡𝑡𝑡𝑡𝐼𝐼𝐼𝐼𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝 𝑤𝑤𝑤𝑤𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁ℎ𝑡𝑡𝑡𝑡 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒× 100Seed damage =𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑇𝑇𝑇𝑇𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒× 100Germination percentage =𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑒𝑒𝑒𝑒 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 𝑒𝑒𝑒𝑒 𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡 𝑡𝑡𝑡𝑡ℎ𝑁𝑁𝑁𝑁 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑇𝑇𝑇𝑇𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 𝑝𝑝𝑝𝑝𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝑝𝑝𝑝𝑝𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡× 100Egg hatching =𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑝𝑝𝑝𝑝𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑝𝑝𝑝𝑝𝑁𝑁𝑁𝑁𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝 𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑒𝑒𝑒𝑒𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑒𝑒𝑒𝑒 𝑖𝑖𝑖𝑖𝑝𝑝𝑝𝑝 𝑡𝑡𝑡𝑡𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑡𝑡𝑡𝑡× 100WPI =𝑃𝑃𝑃𝑃𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑃𝑃𝑃𝑃𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑖𝑖𝑖𝑖𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑃𝑃𝑃𝑃𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑃𝑃𝑃𝑃𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑖𝑖𝑖𝑖𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 + 𝑃𝑃𝑃𝑃𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑃𝑃𝑃𝑃𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑖𝑖𝑖𝑖𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝 𝑆𝑆𝑆𝑆𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁 𝑖𝑖𝑖𝑖𝑝𝑝𝑝𝑝 𝐶𝐶𝐶𝐶𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝐶𝐶𝐶𝐶× 100Weight loss =𝐼𝐼𝐼𝐼𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 𝑤𝑤𝑤𝑤𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁ℎ𝑡𝑡𝑡𝑡 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 − 𝐹𝐹𝐹𝐹𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 𝑤𝑤𝑤𝑤𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁ℎ𝑡𝑡𝑡𝑡𝐼𝐼𝐼𝐼𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝 𝑤𝑤𝑤𝑤𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁ℎ𝑡𝑡𝑡𝑡 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒× 100Seed damage =𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑇𝑇𝑇𝑇𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒× 100Germination percentage =𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑒𝑒𝑒𝑒 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 𝑒𝑒𝑒𝑒 𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡 𝑡𝑡𝑡𝑡ℎ𝑁𝑁𝑁𝑁 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑇𝑇𝑇𝑇𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 𝑝𝑝𝑝𝑝𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝑝𝑝𝑝𝑝𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡× 100Egg hatching =𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑝𝑝𝑝𝑝𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑝𝑝𝑝𝑝𝑁𝑁𝑁𝑁𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝 𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑒𝑒𝑒𝑒𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑒𝑒𝑒𝑒 𝑖𝑖𝑖𝑖𝑝𝑝𝑝𝑝 𝑡𝑡𝑡𝑡𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑡𝑡𝑡𝑡× 100WPI =𝑃𝑃𝑃𝑃𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑃𝑃𝑃𝑃𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑖𝑖𝑖𝑖𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑃𝑃𝑃𝑃𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑃𝑃𝑃𝑃𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑖𝑖𝑖𝑖𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 + 𝑃𝑃𝑃𝑃𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑃𝑃𝑃𝑃𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑖𝑖𝑖𝑖𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝 𝑆𝑆𝑆𝑆𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁 𝑖𝑖𝑖𝑖𝑝𝑝𝑝𝑝 𝐶𝐶𝐶𝐶𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝐶𝐶𝐶𝐶× 100Weight loss =𝐼𝐼𝐼𝐼𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 𝑤𝑤𝑤𝑤𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁ℎ𝑡𝑡𝑡𝑡 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 − 𝐹𝐹𝐹𝐹𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 𝑤𝑤𝑤𝑤𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁ℎ𝑡𝑡𝑡𝑡𝐼𝐼𝐼𝐼𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝 𝑤𝑤𝑤𝑤𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁ℎ𝑡𝑡𝑡𝑡 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒× 100Seed damage =𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑇𝑇𝑇𝑇𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒× 100Germination percentage =𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑒𝑒𝑒𝑒 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 𝑒𝑒𝑒𝑒 𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡 𝑡𝑡𝑡𝑡ℎ𝑁𝑁𝑁𝑁 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑇𝑇𝑇𝑇𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 𝑝𝑝𝑝𝑝𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝑝𝑝𝑝𝑝𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡× 100Egg hatching =𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑝𝑝𝑝𝑝𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑝𝑝𝑝𝑝𝑁𝑁𝑁𝑁𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝 𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑒𝑒𝑒𝑒𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑒𝑒𝑒𝑒 𝑖𝑖𝑖𝑖𝑝𝑝𝑝𝑝 𝑡𝑡𝑡𝑡𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑡𝑡𝑡𝑡× 100WPI =𝑃𝑃𝑃𝑃𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑃𝑃𝑃𝑃𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑖𝑖𝑖𝑖𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑃𝑃𝑃𝑃𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑃𝑃𝑃𝑃𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑖𝑖𝑖𝑖𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 + 𝑃𝑃𝑃𝑃𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑃𝑃𝑃𝑃𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑖𝑖𝑖𝑖𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝 𝑆𝑆𝑆𝑆𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁 𝑖𝑖𝑖𝑖𝑝𝑝𝑝𝑝 𝐶𝐶𝐶𝐶𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝐶𝐶𝐶𝐶× 100Weight loss =𝐼𝐼𝐼𝐼𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 𝑤𝑤𝑤𝑤𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁ℎ𝑡𝑡𝑡𝑡 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 − 𝐹𝐹𝐹𝐹𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 𝑤𝑤𝑤𝑤𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁ℎ𝑡𝑡𝑡𝑡𝐼𝐼𝐼𝐼𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝 𝑤𝑤𝑤𝑤𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁ℎ𝑡𝑡𝑡𝑡 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒× 100Seed damage =𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑇𝑇𝑇𝑇𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒× 100Germination percentage =𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑒𝑒𝑒𝑒 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 𝑒𝑒𝑒𝑒 𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡 𝑡𝑡𝑡𝑡ℎ𝑁𝑁𝑁𝑁 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑇𝑇𝑇𝑇𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 𝑝𝑝𝑝𝑝𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝑝𝑝𝑝𝑝𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡× 100356Ajayi F.F. et al. Foods and Raw Materials. 2022;10(2):353–364Seed germinability. After the storage period,15 seeds were randomly picked from the variousstorage materials to test the effect of the essentialoil concentrations on the germinability of cowpeaseeds. A seed from each treatment was placed in 9-cmPetri dishes with moistened Whatman filter paper onlaboratory benches at room temperature (27–30°C) andrelative humidity (70–75%) [33]. Each treatment wastriplicated. To avoid contamination, the seeds werewatered (23 mL) twice a day (morning and evening)with distilled water from a wash bottle. According toOlisa et al. [34], the germination percentage of cowpeaseeds was estimated from germination data on day 7after sowing according to the following equation (5):(5)Data analysis. Natural mortality in the controlsamples was corrected using Abbott’s formula [27]. Theacquired numerical data was square root transformed, and the adjusted mortality and other data inpercentages was transformed arc sine before beingsubjected to the analysis of variance using JMP 13Computer Software (2016). The Student Newman-Keuls (SNK) test was used to differentiate significanttreatment means at the 5% level of probability. Withthe SPSS statistical software (version 19), the data weresubjected to a two-way analysis of variance (ANOVA) atthe 5 % significance level, and Duncan’s Multiple RangeTest was used to separate the means.RESULTS AND DISCUSSIONMortality of Callosobruchus maculatus L. exposedto Balanites aegyptiaca L. essential oil stored indifferent storage materials after 24, 48, and 72 h.The treatment under discussion provided appropriateprotection to cowpea seeds against C. maculatus. Table1 shows the effect of B. aegyptiaca oil extract and suchstorage materials as jute bags, polythene bags, sacks,plastic containers, and glass bottles on cowpea weevilmortality.The experiment showed a statistically significantcorrelation between the effects of B. aegyptiacaessential oil and storage materials (F(16,75) = 41.813,P = 0.000) after 24 h of exposure. A simple main effectanalysis showed that B. aegyptiaca essential oil had astatistically significant effect on mortality of cowpeaweevils at 24, 48, and 72 h, respectively (P < 0.000).Glass bottles, plastic containers, and jute bagsproved to be the most effective storage material, whilesamples stored in polythene bags had the lowest beetlemortality rate at all the B. aegyptiaca essential oilconcentrations. Our results confirmed those obtainedby Buleti et al., who reported a higher weevil mortalityrate in grains stored in glass bottles compared to otherstorage materials [15]. On the other hand, the results canbe explained by the techno-functional properties of thestorage materials, e.g., water vapor permeability, theinteraction between the plant extracts and the material,etc. [35]. Thus, such vapor proof containers as glassbottles, plastic containers, and jars can provide goodinsulation against weevils, thereby inhibiting theirsurvival: insects suffocate as soon as they run out ofoxygen.In addition, the abrasive effect and contact toxicityof essential oils on the pest cuticle interferes with insectrespiratory mechanism, thereby causing a knock downeffect. This study is similar to the research conducted byKarimzadeh et al., who reported that the abrasive effectsof combined insecticides may cause abrasion of insectcuticle and dehydration of the insect body, thus leadingto insect mortality [36].Evidently, the B. aegyptiaca essential oil treatmentshad a noticeable effect on the population growth rate andmortality of the weevils. Low quantity of the essentialoil (5 mL) resulted in a lower mortality rate, while highquantities (15 mL) provided the highest mortality rate,irrespective of the storage material used.Similarly, the population of the weevils decreasedas the treatment intervals progressed from 24 to 72 h.For instance, at the same essential oil dose, plasticcontainers and glass bottles caused 62.5 and 70%mortality rate, respectively, after 24 h. Likewise, 80 and90% mortality rate were recorded after 48 h. However,100% mortality was recorded for glass bottles, plasticcontainers, and jute bags after 72 h. Most importantly,all the three doses and storage materials showed highmortality rates of C. maculatus after 72 h of exposure, ifcompared to the control samples.The insecticidal efficacy of B. aegyptiaca essentialoil could be attributed to such active compounds ashexadecanoic acid, (9Z,12Z)-octadeca-9,12-dienoic acid,(Z)-octadec-9-enoic acid, ethyl hexadecanoate, 3,3-dihydroxypropylhexadecanoate, and methyl hexadecanoate.All these compounds have been reported to possessrepellent and insecticide activities [17]. This resultconfirms the findings obtained by Mokhtar et al.,who observed 100% mortality of C. maculatus after24 h on cowpeas treated with chloroform extract ofB. aegyptiaca seeds at 1.131 mg·cm–2 [17]. Variousstudies have also demonstrated the insecticidal effectof B. aegyptiaca essential oil against such pests asC. maculatus, Tribolium casteneum, and khaprabeetle [17,18].In our research, various doses of B. aegyptiacaessential oil and storage materials provided an excellentprotection against C. maculatus, both independentlyand synergistically. B. aegyptiaca essential oil extracthad a substantial impact on the longevity and survivalof cowpea weevils. In addition, glass bottles, plasticcontainers, and jute bags caused 100% mortality at thehighest dose (15 mL) after 72 h of exposure.WPI =𝑃𝑃𝑃𝑃𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑃𝑃𝑃𝑃𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑖𝑖𝑖𝑖𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 + 𝑃𝑃𝑃𝑃𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑃𝑃𝑃𝑃𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑖𝑖𝑖𝑖𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝 𝑆𝑆𝑆𝑆𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁 𝑖𝑖𝑖𝑖𝑝𝑝𝑝𝑝 𝐶𝐶𝐶𝐶𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝐶𝐶𝐶𝐶× 100Weight loss =𝐼𝐼𝐼𝐼𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 𝑤𝑤𝑤𝑤𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁ℎ𝑡𝑡𝑡𝑡 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 − 𝐹𝐹𝐹𝐹𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 𝑤𝑤𝑤𝑤𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁ℎ𝑡𝑡𝑡𝑡𝐼𝐼𝐼𝐼𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝 𝑤𝑤𝑤𝑤𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁ℎ𝑡𝑡𝑡𝑡 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒× 100Seed damage =𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑇𝑇𝑇𝑇𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒× 100Germination percentage =𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑒𝑒𝑒𝑒 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 𝑒𝑒𝑒𝑒 𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡 𝑡𝑡𝑡𝑡ℎ𝑁𝑁𝑁𝑁 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑇𝑇𝑇𝑇𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 𝑝𝑝𝑝𝑝𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝑝𝑝𝑝𝑝𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡× 100357Ajayi F.F. et al. Foods and Raw Materials. 2022;10(2):353–364Table 1 Mortality of cowpea weevils exposed to Balanites aegyptiaca oil extract in different storage materials after 24, 48, and 72 hSamples Storage materials Mortality ± SE, %24 h 48 h 72 hBalanites aegyptiaca essential oil(5 mL)Glass bottle 20.00 ± 0.00 46.50 ± 4.79 90.00 ± 0.00Jute bag 12.50 ± 5.00 25.00 ± 4.08 70.00 ± 0.00Plastic container 20.00 ± 0.00 60.00 ± 0.00 61.20 ± 2.50Polythene bag 10.00 ± 0.00 25.00 ± 0.00 60.00 ± 0.00Sack bag 10.00 ± 0.00 30.00 ± 0.00 75.00 ± 0.00Balanites aegyptiaca essential oil(10 mL)Glass bottle 40.00 ± 0.00 65.00 ± 5.77 100.00 ± 0.00Jute bag 30.00 ± 0.00 60.00 ± 0.00 83.60 ± 2.50Plastic container 35.00 ± 0.00 75.00 ± 0.00 90.00 ± 0.00Polythene bag 23.70 ± 2.50 42.00 ± 5.00 70.00 ± 0.00Sack bag 32.50 ± 2.89 50.00 ± 0.00 90.00 ± 0.00Balanites aegyptiaca essential oil(15 mL)Glass bottle 70.00 ± 0.00 80.00 ± 0.00 100.00 ± 0.00Jute bag 50.00 ± 0.00 70.00 ± 0.00 100.00 ± 0.00Plastic container 62.50 ± 2.89 90.00 ± 0.00 100.00 ± 0.00Polythene bag 32.50 ± 2.89 55.00 ± 0.00 80.00 ± 0.00Sack bag 55.00 ± 0.00 70.00 ± 0.00 97.50 ± 5.00Aluminum phosphide Glass bottle 90.00 ± 0.00 100.00 ± 0.00 100.00 ± 0.00Jute bag 81.20 ± 2.50 90.00 ± 0.00 100.00 ± 0.00Plastic container 90.00 ± 0.00 100.00 ± 0.00 100.00 ± 0.00Polythene bag 85.00 ± 4.08 90.00 ± 0.00 95.00 ± 5.77Sack bag 90.00 ± 0.00 100.00 ± 0.00 100.00 ± 0.00Control (acetone) Glass bottle 0 0 20.00 ± 0.00Jute bag 0 0 0Plastic container 0 0 02.50 ± 2.89Polythene bag 0 0 0Sack bag 0 0 0A P < 0.05 P < 0.05 P < 0.05B P < 0.05 P < 0.05 P < 0.05AB P < 0.05 P < 0.05 P < 0.05A – essential oil; B – storage materialOviposition of C. maculatus exposed toB. aegyptiaca essential oil stored in differentstorage materials after 7, 30, and 90 days ofstorage. Figures 1–3 show the mean value ofoviposition in both treated and untreated cowpeas.The analysis of variance showed that a statisticallysignificant correlation between the effects ofB. aegyptiaca oil extracts and storage materialson the oviposition of cowpea weevils (F(16,75) =3346.73, P = 0.000). When compared to the control, thetreated samples showed a much lower oviposition. At aseven-day interval, all the storage materials showed thesame trend in the total oviposition of cowpea weevils asthe essential oil doses increased, except polythene bags(Fig. 1).B. aegyptiaca essential oil significantly reduced thenumber of eggs laid by weevils. The highest amountof eggs was recorded at 5 mL, and then it significantlydecreased at 10 mL. The lowest amount of eggs wasregistered at 15 mL. The drastic reduction in thenumber of eggs laid by cowpea weevils might havebeen caused by the toxicity of the plant material activecomponents to the weevils rather than by the preventionof oviposition.The previous section that the B. aegyptiaca essentialoil caused the highest mortality rate in the experimentalsamples, which was associated with its insecticidaleffect. The chemical composition of plant oils andtheir phytocompounds is known to produce a toxic andrepellent effect on insects that live in stored grain [37].Grains stored in glass bottles had the fewest eggs,while those stored in polythene bags had the maximalnumber of eggs. Even though polythene bags had thelowest effect on weevil oviposition, they also showeda significant reduction in the number of eggs laidcompared to the untreated samples. Buleti et al. alsoreported a reduction in the number of eggs laid in glassbottles [15]. Furthermore, the experimental samplesrevealed just a few eggs on day 90, with a meanfecundity of 1–4 eggs for glass bottles and 1–14 eggs forplastic containers. A slightly higher value was recordedfor polythene and sack bags with a mean of 18–45 and8–30 eggs, respectively. However, the values recorded358Ajayi F.F. et al. Foods and Raw Materials. 2022;10(2):353–364Figure 1 Effect of Balanites aegyptiaca on Callosobruchusmaculatus oviposition at various treatments in differentstorage materials after 7 days of storageFigure 3. Effect of Balanites aegyptiaca on Callosobruchusmaculatus oviposition at various treatments in differentstorage materials after 90 days of storageFigure 2. Effect of Balanites aegyptiaca on Callosobruchusmaculatus oviposition at various treatments in differentstorage materials after 30 days of storagein polythene and sack bags were lower than in thecontrol samples with a mean fecundity of 256–544 eggsper female after 90 days, which implied a significantdifference in the oviposition of treated cowpeas.The synergic treatment of B. aegyptiaca essentialoil and storage materials killed more than 50% of thetotal eggs laid at various stages of development from7 to 90 days. Likewise, the high numbers of eggs laidin polythene and sack bags could be explained by theporous surface of these materials that allowed moistureand air circulation. Such conditions encouraged weevilspresent in the seed lot to lay eggs and proliferatecontinuously.The lower oviposition rates observed in this studysuggested that B. aegyptiaca essential oil could beuseful as cowpea protectants. This finding confirms thatmade by Alves et al., who discovered that lemon grassessential oil extract reduced C. maculatus ovipositionsignificantly [38]. Previous studies by a Nwaoguand Yahaya and Aous et al. reported the effect ofessential oil of Cymbopogon schoenanthus (L.) on thedevelopment of freshly laid eggs and newborn larvae ofC. maculatus [18, 39]. The extract probably containeda powerful oviposition deterrent. Also, cowpea seedsthat are packaged in glass bottles and plastic containersshowed a low oviposition, which made them the optimalovipositional deterrents in this study.Egg hatchability of C. maculatus exposed toB. aegyptiaca essential oil stored in different storagematerials after 24, 48, and 72 h. Table 2 shows theeffect of B. aegyptiaca essential oil on C. maculatusegg development and hatchability. The main significanteffect of B. aegyptiaca essential oil and storagematerials (P < 0.05) was observed after 30, 60, and90 days. Similarly, the study revealed no significantNumber of eggs laid0Treatment80604020Packaging100120140Glass bottle Jute bagPlastic container Polythene bagSack bagNumber of eggs laid0Treatment10 mL 15 mL 5 mL AluminumphosphideControl(acetone)80604020PackagingGlass bottleJute bagPlastic containerPolythene bagSack bag10 mL 15 mL 5 mL AluminumphosphideControl(acetone)Number of eggs laid0Treatment400300200100600500Glass bottlePackagingJute bagPlastic container Polythene bagSack bag10 mL 15 mL 5 mL AluminumphosphideControl(acetone)359Ajayi F.F. et al. Foods and Raw Materials. 2022;10(2):353–364Table 2 Callosobruchus maculatus egg hatchability treated with Balanites aegyptiaca essential oil in different storage materialsafter 30, 60, and 90 days of storageSamples Storage materials Mean number of hatched eggs ± SE30 days 60 days 90 daysBalanites aegyptiaca essential oil(5 mL)Glass bottle 4.10 ± 0.17 2.40 ± 0.25 1.00 ± 0.00Jute bag 7.00 ± 0.00 4.70 ± 0.14 1.30 ± 0.08Plastic container 4.20 ± 0.68 3.10 ± 0.78 1.10 ± 0.03Polythene bag 8.60 ± 0.79 7.70 ± 0.17 5.50 ± 0.05Sack bag 6.40 ± 0.36 4.60 ± 0.09 1.60 ± 0.00Balanites aegyptiaca essential oil:(10 mL)Glass bottle 1.50 ± 0.78 1.00 ± 0.00 1.00 ± 0.00Jute bag 5.10 ± 0.15 3.60 ± 0.79 1.20 ± 0.02Plastic container 2.20 ± 0.15 1.20 ± 0.96 1.00 ± 0.01Polythene bag 5.40 ± 0.34 6.30 ± 0.24 3.30 ± 0.28Sack bag 3.20 ± 0.00 2.00 ± 0.02 1.00 ± 0.00Balanites aegyptiaca essential oil:(15 mL)Glass bottle 0.80 ± 0.09 1.00 ± 0.00 1.00 ± 0.00Jute bag 3.00 ± 0.00 2.20 ± 0.11 1.00 ± 0.00Plastic container 1.10 ± 0.05 1.10 ± 0.10 1.00 ± 0.00Polythene bag 2.90 ± 0.12 6.10 ± 0.13 1.90 ± 0.01Sack bag 2.90 ± 0.17 1.90 ± 0.07 1.00 ± 0.00Aluminum phosphide Glass bottle 0.20 ± 0.06 1.00 ± 0.00 1.00 ± 0.00Jute bag 1.00 ± 0.13 1.00 ± 0.00 1.00 ± 0.00Plastic container 1.00 ± 0.00 1.00 ± 0.00 1.00 ± 0.00Polythene bag 1.10 ± 0.00 1.00 ± 0.00 1.00 ± 0.00Sack bag 0.50 ± 0.04 1.00 ± 0.00 1.00 ± 0.00Control (acetone) Glass bottle 16.60 ± 0.00 20.40 ± 0.67 42.10 ± 1.14Jute bag 23.40 ± 0.84 27.30 ± 1.51 52.60 ± 31.1Plastic container 18.20 ± 0.56 21.90 ± 0.89 52.00 ± 0.04Polythene bag 31.30 ± 0.76 38.90 ± 0.93 72.40 ± 1.47Sack bag 26.80 ± 1.30 26.20 ± 0.46 66.40 ± 0.28A P < 0.05 P < 0.05 P < 0.05B P < 0.05 P < 0.05 P < 0.05AB P > 0.05 P > 0.05 P > 0.05A – essential oil; B – storage material(P > 0.05) interactive effect of B. aegyptiacaessential oil and storage materials on C. maculatusegg hatchability. The results demonstrated that theproductivity in the experimental samples was extremelylow at all intervals and doses, with the mean valuesranging from 1 to 8.6.Hence, over 65% of the total eggs laid in theexperimental samples died at different stages ofdevelopment in all the trials. B. aegyptiaca oilextracts obviously had a strong larvicidal effect on thedevelopment of immature weevils. Similarly, the highmortality of C. maculatus in the experimental samplesimplied that the plant had some phytochemical properties,which reduced egg production [40].Furthermore, the egg hatchability reduced as thestorage interval progressed from 30 to 90 days. Amongthe experimental samples, the highest mean values ofegg hatchability were observed in cowpeas stored inpolythene bags at all the essential oil concentrations.However, the values observed in polythene bags werelower in comparison to the control samples. Therefore,the low egg hatchability was caused by the effectivenessof B. aegyptiaca essential oil with its poisonous componentand physical properties, which affected thesurface and oxygen tension of eggs.The essential oils of Borago officinalis, Melissaofficinalis, Carapichea ipecacuanha, and Laurus nobilishave also been reported to reduce hatchability [41].Similarly, Piper gaudichaudianum essential oil showeda better insecticidal activity against Lucilia cuprinathird instar larvae under laboratory conditions [42].Weevil Perforation Index, weight loss, damage,and germinability of cowpea seeds treated with B.aegyptiaca essential oil stored in different storagematerials after 30, 60, and 90 days. These parametersof cowpea seeds indicate its suitability for consumptionand other aesthetic values because damaged seeds withholes and flour dust are not marketable. Figures 4 and5 show the weevil perforation index for cowpea seedstreated with different concentrations of B. aegyptiacaessential oil packaged in different storage materials ondays 30, 60, and 90.After 30 days, the highest weevil perforationindex was observed in polythene and sack bags. Whencompared to the control treatment, the cowpea seedtreated with B. aegyptiaca essential oil showed a360Ajayi F.F. et al. Foods and Raw Materials. 2022;10(2):353–364Figure 4 % Weevil perforation index of cowpea seeds treatedwith Balanites aegyptiaca in different storage materials after30 days of storageFigure 5 % Weevil perforation index of cowpea seeds treatedwith Balanites aegyptiaca in different storage materials after90 days of storagesubstantial reduction in seed damage. The analysisof variance showed a statistically significant effectof B. aegyptiaca oil extracts and storage materialson weevil perforation index (F(16, 75) = 176.150,P = 0.000). Exposure of weevil-infested cowpea seedsto B. aegytiaca essential oil in various storage materialsresulted in a significant reduction in seed weight(F(16,75 = 311.357, P = 0.000).The weevil perforation index decreased followingthe increase in B. aegytiaca essential oil concentrationfor all storage materials. The lowest weevil perforationindex was observed in the seeds packaged in glassbottles followed by those stored in plastic containers.The highest weevil perforation index was recorded in theseeds stored in polythene bags throughout the storageperiod (Figs. 4 and 5).Furthermore, exposure of weevil-infested cowpeaseeds to B. aegytiaca essential oil in various storagematerials resulted in a significant reduction in seedweight (F(16,75) = 311.357, P = 0.000). After 90 days ofexposure, the seeds treated with B. aegyptiaca essentialoil in all the storage materials showed a significantlylow weight loss at all doses (5, 10, and 15 mL), exceptthe cowpea seeds stored in polythene bags (Table 3).For these parameters, interactive treatment ofB. aegyptiaca essential oil and storage materialshad equally significant effects (P < 0.05) on cowpeaweevils.Taken together, B. aegyptiaca had a moredetrimental effect on the weevils in comparison tothe control. A similar research by Borzoui et al. alsoregistered a significantly low amount of seed damagebecause the oviposition rate was reduced by thesublethal doses of essential oil [43]. Among all thestorage materials, glass bottles and plastic bags showedthe lowest weevil perforation index, seed weight loss,and seed damage. The low seed weight loss and damageobserved in the seeds stored in these storage materialscould be attributed to the significant reduction ofweevils that could have caused seed damage.B. aegyptiaca essential oil and storage materialsdemonstrated both individual and interactive effects onthe germination rate of cowpea seeds. The germinationrate of the treated seeds ranged from 46.7 to 93.3%,which was lower than the values reported for cowpeaseed germination rate by Gad et al. [44].An increased germination rate of cowpea seedswas observed with increased B. aegyptiaca essentialoil concentration, which implied a strong relationshipbetween the treatment and germination rate. Similarresults were described by Bhavya et al. and Harshaniand Karunaratne, who indicated that essential oils andtheir principal components affected seed germination.Storage materials also affected seed germination [45, 46].In this study, glass bottles proved to be the optimalstorage material as indicated by the maximal seedgermination rate of 88.3, 91.6, and 93.3% after essentialoil treatment of 5, 10, and 15 mL, respectively. Storingseeds in appropriate storage material retained highergermination capacity rate.Number of seeds with holes0TreatmentNumber of seeds with holes0Treatment20.0040.0060.00Glass bottlePackagingJute bagPlastic container Polythene bagSack bag20.0040.0060.0080.00100.00Glass bottlePackagingJute bagPlastic container Polythene bagSack bag10 mL 15 mL 5 mL AluminumphosphideControl(acetone)10 mL 15 mL 5 mL AluminumphosphideControl(acetone)361Ajayi F.F. et al. Foods and Raw Materials. 2022;10(2):353–364Table 3 Effect of Balanites aegyptiaca essential oil and storage material on cowpea seed damage, weight loss, and germination ratecaused by Callosobruchus maculatus infestationSamples StoragematerialsSeed weight loss Seed damage Germination30 days 60 days 90 days 30 days 60 days 90 days rateBalanitesaegyptiacaessentialoil (5 mL)Glass bottle 8.30 ± 0.00 3.50 ± 0.03 1.00 ± 1.63 1.00 ± 0.00 1.00 ± 0.05 3.00 ± 0.00 88.30 ± 3.30Jute bag 13.40 ± 0.16 6.00 ± 0.19 1.50 ± 0.25 2.00 ± 1.63 4.50 ± 0.14 7.50 ± 0.21 64.60 ± 4.91Plasticcontainer12.50 ± 0.29 3.90 ± 0.00 1.20 ± 0.19 1.00 ± 0.00 1.90 ± 0.22 3.30 ± 0.23 80.00 ± 0.00Polythene bag 15.60 ± 0.17 8.50 ± 0.25 3.00 ± 0.16 5.90 ± 0.25 7.00 ± 0.17 11.40 ± 1.49 46.70 ± 0.00Sack bag 13.60 ±0.47 6.80 ± 0.14 1.40 ± 0.25 2.20 ± 0.00 5.00 ± 0.16 7.70 ± 0.81 60.00 ± 0.00Balanitesaegyptiacaessentialoil (10 mL)Glass bottle 7.80 ± 0.09 2.00 ± 0.08 0.80 ± 0.00 0 1.00 ± 0.00 1.90 ± 0.17 91.60 ± 3.30Jute bag 11.70 ± 0.04 5.40 ± 0.32 1.50 ± 0.00 2.00 ± 0.00 4.50 ± 0.10 7.00 ± 0.08 60.00 ± 0.00Plasticcontainer10.00 ± 0.13 2.30 ± 0.34 0.90 ± 0.17 0 1.00 ± 0.00 2.00 ± 0.00 80.00 ± 0.00Polythene bag 13.00 ± 0.17 11.50 ± 0.57 2.50 ± 0.21 3.90 ± 0.41 7.00 ± 0.00 9.10 ± 0.87 55.00 ± 2.01Sack bag 12.00 ± 0.74 5.70 ± 0.19 1.30 ± 0.28 2.00 ± 0.16 5.40 ± 0.18 6.90 ± 0.01 60.00 ± 0.00Balanitesaegyptiacaessentialoil (15 mL)Glass bottle 2.80 ± 0.16 1.60 ± 0.02 0.30 ± 0.23 0 1.00 ± 0.00 1.50 ± 0.00 93.30 ± 0.00Jute bag 10.80 ± 0.48 3.10 ± 0.00 0.70 ± 0.19 0 2.00 ± 0.07 5.00 ± 0.16 66.70 ± 0.00Plasticcontainer3.10 ± 0.24 1.90 ± 0.10 0.30 ± 0.19 0 1.00 ± 0.00 2.00 ± 0.71 93.00 ± 0.00Polythene bag 11.70 ± 0.84 9.70 ± 0.22 1.40 ± 0.16 2.00 ± 0.16 5.00 ± 0.38 6.00 ± 0.00 65.00 ± 1.88Sack bag 10.50 ± 0.38 3.40 ± 0.13 0.80 ± 0.00 0 4.00 ± 0.16 5.00 ± 0.00 60.00 ± 0.00AluminumphosphideGlass bottle 0 0 0.10 ± 0.00 0 0 0 100.00 ± 0.00Jute bag 0 0 0.10 ± 0.00 0 0 0 100.00 ± 0.00Plasticcontainer0 0 0.10 ± 0.00 0 0 0 100.00 ± 0.00Polythene bag 0 0 0.10 ± 0.00 0 0 0 100.00 ± 0.00Sack bag 0 0 0.10 ± 0.00 0 0 0 100.00 ± 0.00Control(acetone)Glass bottle 18.60 ± 0.26 37.40 ± 0.13 42.00 ± 0.08 15.70 ± 1.25 28.00 ± 0.16 71.80 ± 0.43 26.70 ± 0.00Jute bag 28.60 ± 0.36 42.40 ±1.77 48.50 ± 0.00 20.90 ± 0.68 30.80 ± 1.45 79.70 ± 1.39 20.00 ± 0.00Plasticcontainer20.80 ± 0.16 40.30 ± 1.57 45.70 ± 0.22 16.00 ± 0.16 29.20 ± 0.66 77.80 ± 0.44 26.70 ± 0.00Polythene bag 35.60 ± 0.77 56.00 ± 0.00 56.50 ± 0.25 23.70 ± 0.48 38.60 ± 0.41 91.50 ± 1.07 13.30 ± 0.00Sack bag 26.40 ± 0.16 44.30 ± 1.80 50.00 ± 1.63 18.90 ± 0.17 34.10 ± 1.57 84.10 ± 1.72 20.00 ± 0.00A P < 0.05 P < 0.05 P < 0.05 P < 0.05 P < 0.05 P < 0.05 P < 0.05B P < 0.05 P < 0.05 P < 0.05 P < 0.05 P < 0.05 P < 0.05 P < 0.05AB P < 0.05 P < 0.05 P < 0.05 P < 0.05 P < 0.05 P < 0.05 P < 0.05A – Balanites aegyptiaca essential oil; B – storage materialTherefore, the B. aegyptiaca essential oil could beused to protect cowpea seeds stored in glass bottlesbecause the values observed were related to theinternational germination threshold of 90% requiredby seed exportation. This result is similar that obtainedby Buleti et al., who also used glass bottles as storagecontainers [15].On the contrary, cowpea seeds stored in polythenebags displayed the lowest germination rate, whichindicated a strong relationship between weight loss,damage score, and germination rate. However, thegermination rates were not as highly related with seeddamage traits. The lowest germination rate observedcould be explained by weevil damage that occurreddue to minimal weevil mortality rate in polythenebags, which, in its turn, led to nutrient exhaustion [15].Storing seeds in inappropriate storage materials couldsignificantly decline their germination rate and resultedin a rapid loss of seed viability.Overall, even though B. aegyptiaca essential oilhad a stronger effect on cowpea weevils, they failed toremove cowpea weevils completely; rather, they onlylowered their numbers. Thus, hermetic storage couldenhance the quality of cowpea seeds during storage.CONCLUSIONThe combination of essential oil of Balanitesaegyptiaca L. and storage materials had significanteffects on weevil proliferation in cowpea seeds(P > 0.05) during storage. B. aegyptiaca essential oilproved to possess insecticide properties that can helpcontrol Callosobruchus maculatus L. in stored cowpeaseeds.362Ajayi F.F. et al. Foods and Raw Materials. 2022;10(2):353–364After 90 days of storage, mortality, oviposition, andegg hatchability fell down, following the increase inthe concentration of B. aegyptiaca essential oil. Inaddition, such storage materials as glass bottles, plasticcontainers, and jute bags also reduced the population ofcowpea weevils in cowpeas during storage. Hermeticstorage material – glass bottles – had the greatesteffecton weevil infestation and sustained the qualityof cowpeas under storage conditions. These findingssuggest that B. aegyptiaca essential oil could be usefulas a botanical insecticide against cowpea pests. A largescaletrial is required to perform a toxicity assay ofB. aegyptiaca essential oil.CONTRIBUTIONFeyisola Fisayo Ajayi obtained the funds, designedthe experiment, collected the data, and wrote themanuscript original draft, as well as performed theformal laboratory research. Akama Friday Ogoriconducted the formal analysis and research, wrotethe article, reviewed scientific publications, andedited the manuscript. Vivien O. Orede performed theformal analysis and research, reviewed and editingthe manuscript. Emmanuel Peters performed theformal analysis and research, as well as provided theexperimental design. All the authors were equallyinvolved in reading and approving of the final manuscriptbefore submission and are equally accountable for anypotential cases of plagiarism.CONFLICT OF INTERESTThe authors declare no conflict of interests regardingthe publication of this article.