Toxicology of diatomaceous earth, phyto oils and their admixed emulsions against adults of Tribolium castaneum (Herbst)

Tribolium castaneum (Herbst), one of the invasive stored pest, is resistant to the most of synthetic insecticides used against this it. Moreover, synthetic insecticides are a major threat to human health, the environment, and the ecosystem. The development of some smart tools is needed to minimize the use of hazardous chemicals. As an alternative, nano-insecticides are on the horizon. Emulsions are expressed as sustain release of insecticidal components to achieve maximum efficacy and low residual toxicity. In this study, some essential oils (Cymbopogon citratus (DC.) Stapf, Ocimum basilicum L., Curcuma longa L., and Trachyspermum ammi L.), diatomaceous earth (DE), and their nano-emulsions are evaluated against T. castaneum. Essential oils and DE were tested at four (60 ppm, 30 ppm, 15 ppm, 7.5 ppm) different concentrations with and without emulsions, and data was recorded after 6, 12, 24, 48, and 72 h of exposure respectively. The mortality observed in essential oils of C. citratus, O. basilicum, and C. longa without emulsion after 72 h of treatment at the highest concentration was 98%, 95%, and 85%, respectively. While, at the highest concentration the lowest mortalities were observed with DE and T. ammi i.e. 65%. Insecticidal activity of emulsion essential oils increased to 100%, 98%, 90%, and 68.3% for C. longa, C. citratus, O. basilicum, and T. ammi, respectively. The results support that these admixed emulsions could be used as an alternative to synthetic insecticides in conventional formulations.


Introduction
Food production must be increased to feed the ever-increasing population. Post-harvest losses by insect pests account for 25-50% [1,2]. Tribolium castaneum (Herbst) is one of the most common pest of stored grains and is known to infests about 233 different types of crops [3]. The T. castaneum is responsible for considerable losses in stored wheat [4][5][6]. The most applied method for its control is fumigation [7]. Among fumigants, methyl bromide is discontinued due to ozone depletion effects [8]. The T. castaneum has developed resistance against malathion, cypermethrin, primiphos-methyl, and bifenthrin [9,10]. Alternate to the pesticides are biopesticides, which can be used safely against the insect pests to reduce the limitations of synthetic pesticides [11].
Diatoms are the most abundant algae [41]. They are advantageous over chemical pesticides being non-hazardous to non-target organisms [42]. The DE (diatomaceous earth) absorbs all liquid content in insect cuticles and leads them to death [43][44][45][46]. Nano and microencapsulation technologies are employed widely to increase the availability and stability of natural substances in essential oils [47]. The nano formulated biopesticides have a controlled release mechanism to exhibit maximum efficacy [48]. Nanofabrication of insecticidal substances can increase the insecticidal effect by improving the delivery of active ingredients to the target site because of ultra-small particles which can even pass through extracellular spaces [49].
The study was designed to estimate the insecticidal efficacy of the above-stated oils and DE against T. castaneum. The present study consequently focused on the development of new formulations by using technology to improve the efficacy of these biopesticides which could be an important contribution to agriculture.

Insect culture
Infested wheat flour was collected from the local market of Multan. Adults of T. castaneum were sieved and transferred to rearing jars supplied with a mixture of flour (sterilized) and 5% yeast in Ecotoxicology Laboratory, Department of Entomology, Bahauddin Zakariya University, Multan. The rearing jars were kept at 28 ± 2 ºC and 70% R.H. for insect multiplication [50]. Pupae were separated and kept in batches to get homogenous population. One week old adults of T. castaneum were collected through pit fall traps and starved for 24 h before treatment.

Plant materials
Leaves of C. citratus (lemongrass), rhizomes of C. longa (turmeric), seeds of O. basilicum (basil), and T. ammi (ajwain) were purchased from the local market of Cantt, Multan, Pakistan. Leaves, rhizomes and seeds of respective plants were washed, and shade dried for 2 days and crushed by a grinding mill to a fine powder. (Table 1).

Oil extraction
Dried lemongrass leaves powder, turmeric rhizomes, ajwain and basil seed powders (20 g) were suspended in 350 ml acetone at 56 ºC for 12, 6, 8 and 8 h respectively, in the Soxhlet apparatus. Excess solvent was removed by placing the mixture (in a flask) over the rotary evaporator at 56 ºC and 25 rpm to dry excess solvent The concentrate obtained was stored in glass vials at 4 ºC [51-53].

Preparation of admixed emulsions
The oils extracted were then utilized to make admixed emulsions. They were made in two phases because being inorganic DE could not be mixed directly with test oils. The oil phase constituted 14% oil, 3% ethanol, and 3% Tween 20 representing 20% of the emulsion. The oil phase was stirred at 86 ºC and 750 rpm of 1 h. While the aqueous phase was prepared by mixing of 2 g of DE in 78 ml of water and was stirred at 86 ºC and 75 rpm for 1 h. Both aqueous and oil phases were intermixed at the same temperature for 3 min. The emulsion thus prepared was centrifuged at 10,000 xg for 30 min and the emulsion was stored in dark bottles [54,55].

Bioassays
The efficiency of DE, plant essential oils (lemongrass, turmeric, basil, ajwain), and their emulsions was tested at four serially diluted concentration i.e., 60 ppm, 30 ppm, 15 ppm and 7.5 ppm against adults of T. castaneum. Control was treated with distilled water designated as 0 ppm. The experiment was performed using a completely randomized design with 6 replications of each treatment. Filter papers of 5 cm diameter were treated and air-dried for 1 h. Dried filter papers were kept in already sanitized same diameter petri dishes and 10 insects were placed in each petri dish. The petri dishes were kept at room temperature and the mortality data was taken at intervals of 6, 12, 24, 48, and 72 h, respectively [56].

Statistical analysis
Data were analyzed through Two-Way ANOVA in Minitab 19 Software. Mean mortalities were compared by LSD to find homogenous groups [57]. The toxicity of different treatments (LC 50 ) was determined by Probit analysis.

Table 1
List of plant species and their major insecticidal compound used against T. castaneum.

Efficacy of turmeric oil and emulsion
Percentage mortality of T. castaneum after treatment with turmeric essential oil significantly increased with increase in concentration (P < 0.00001) and time Percentage mortality of T. castaneum observed after exposure to turmeric oil was 56%, 63.3%, 70%, 83.3% and 98.3% at 6, 12, 24, 48 and 72 h of treatment at highest concentration.
The emulsions of turmeric oil expressed comparatively high mortality (P < 0.0001) than turmeric oil, while the pattern of mortality percentage was dependent on time lapse after treatment and concentration. At 60 ppm percentage mortality of turmeric emulsion was increased at 17.33% i.e., 73.33% after 6 h of treatment as compared to 56% mortality of turmeric oil at same concentration and time interval. Other concentrations and time intervals expressed subsequent pattern of observation statistically significant from each other and control (P < 0.0001) (Fig. 2).

Efficacy of basil oil and emulsion
Basil essential oil was found significantly effective (P < 0.00001) against T. castaneum at tested concentrations (60, 30, 15 and 7.5 ppm) and time interval. The percentage mortality increased with increase in concentration and time after treated with basil oil. Basil emulsions treatments were recorded to have higher percentage mortality than basil emulsion treated units. Basil oil expressed 53.3% mortality after 6 h of treatment at 60 ppm while emulsion exhibited 58.3% at same conditions. Other concentrations of basil oil and emulsion showed similar  pattern of mortality. All the treatments were statistically different from each other and control (P < 0.00001) (Fig. 3).

Efficacy of ajwain oil and emulsion
Percentage mortality of T. castaneum after treatment with ajwain oil and emulsion significantly increased with increase in concentration and time (P < 0.00001). Emulsion of ajwain oil resulted 3.4% increased mortality i.e., 30% as compared to ajwain oil (26.67%) at highest concentration after 6 h of treatment. All treatments exhibited similar trend of percentage mortality while being significantly different from each other (P < 0.00001) (Fig. 4).

Efficacy of diatomaceous earth
Percentage mortality of T. castaneum after treatment with diatomaceous earth significantly increased with an increase in concentration and time (P < 0.0001). The 60 ppm concentration of DE resulted in 20% mortality at 6 h interval. Percentage mortality was recorded as 23.33%, 56.67%, and 65% in DE at 60 ppm concentration after 24, 48 and 72 h of treatment, respectively. All the tested concentrations expressed parallel trend of mortality. Percentage mortality of T. castaneum in control was 1.67% after 72 h of treatment (Fig. 5).

Toxicity of plant-based oils and their emulsions against T. castaneum
The results of the study indicated that LC 50 of T. castaneum was dependent on post treatment exposure time (

Discussion
The study was designed to evaluate the insecticidal efficacy of plant based oils and their admixed emulsion as a bio-pesticide to replace the harmful effects of synthetic insecticides from the environment [58]. Results confirmed the effectiveness of nano emulsions by controlling the population of T. castaneum. Toxicity effect was found increased with increase in concentration and exposure time. Essential oils of C. citratus, T. ammi, O. basilicum, and C. longa were found toxic to the adults of T. castaneum. Emulsion formation increased the toxicity of turmeric oil by 2% as mortality of T. castaneum was observed 100% after being exposed to turmeric emulsion as compared to turmeric oil (98%) at highest concentration. Reports of similar studies confirm the same toxicity pattern of phyto oils and their emulsions [26,48,55]. The increased insecticidal activity of emulsions is explained as increased insect cuticle penetration and lowering the αamylases, and acetylcholine esterase in T. castaneum [59]. Similar results were obtained when turmeric nanoparticle was used on microbes [60]. The opposite phenomenon is observed when turmeric was tested on aphids (Aphis gosypii) [21]. Three per cent increase in the pesticidal efficacy of lemongrass oil was observed when its emulsion was used. Lemongrass oil resulted in up to 95% mortality in T. castaneum similar to Sitophilus zeamasis along with feeding repellency [2,61]. Lemon grass emulsion exhibited 3% increased insecticidal activity (i.e., 98%) as compared to lemon grass oil which is a similar to nano application of Hugona mystax against mosquito [62]. Basil oil was comparatively less toxic to T. castaneum as compared to lemongrass oil and turmeric oil. The pesticidal activity of basil oil was enhanced by 5% when converted to emulsion. Significantly high adult mortality of 85% was noticed with basil oil. These results are quite similar to the study conducted on Sitophilus oryzae and Culex pipiens larvae [37,63]. The well pronounced adulticidal and larvicidal effect of basil oil could be due to its neurotoxic activity of inhibiting acetylcholine esterase [37]. Basil oil is known to exhibit high repellence effects against Aphis gosypii and Phenacoccus solenopsis [64]. The mortality percentage increased to 90% when used as nano-emulsion against T.castaneum parallel to similar studies on Aedes Table 2 Analysis of insecticidal effect of plant oils and diatomaceous earth with their respective lethal concentrations, fiducial limits (95%), chi-square (χ 2 ) and slope ( ± standard error) after an exposure of 6, 12, 24, 48 and 72 h.   [65]. Ajwain oil has a moderate effect on T. castaneum and resulted in 65% mortality. The efficacy increased in ajwain emulsion by 15%. The results of ajwain oil tested alone were equal to previously studied Trogoderma granarium and Prostephanus truncatus [66] and Culex quinquefasciatus [67]. Ajwain oil exhibit significant acetylcholine esterase enzyme inhibition due to thymol against larvae of Tuta absoluta. The comparatively low mortality in ajwain oil could be due to its relatively less effectiveness on adults [68]. Ajwain oil also exhibited significant results against Fusarium oxysporum, Meloidogyne incognita, and Odontotermes obesus [1]. Ajwain emulsion enhanced mortality by 68.33%. Similar effects were seen when ajwain microemulsion was used against Culex quinquefasciatus [69]. Diatomaceous earth was also used to test its effect on T. castaneum where it resulted 65% mortality. A slight contrast to these results is observed on Sitophilus zeamais where it caused 69% mortality [44]. The DE is reported effective in management of Sitophilus oryzae, Tribolium confusum, and Rhyzopertha dominica [70].

Conclusions
Plant based oils could serve as best possible economical alternative to synthetic pesticides usually employed for pest management. In the current study, essential oils from four plant species were evaluated against the adults of T. castaneum. Results from the study strongly support the use of turmeric, lemongrass, basil and ajwain as potential candidate in insecticidal formulations. Future need is to improve bioavailability of their major insecticidal compounds by developing them as micro and nano-carriers. The present study enlightens the fact that nano-formulations are more efficacious than oils and DE used alone. Development of plant oils as nano-emulsions could significantly enhance their penetration in insect cuticle. They can be utilized for sustainable management of storage pests on commercial grounds. The need of the hour is to further investigate phyto oils based nano emulsions for their mode of action, target site and safety towards non targets to develop new commercial formulation.