Fly ash-based water dispersible powder formulation of Bacillus thuringiensis var. israelensis: Development & laboratory evaluation against mosquito immatures

Mosquitoes play a predominant role in the transmission of malaria, dengue fever, yellow fever, filariasis and several other diseases1. Effective control of aquatic mosquito larvae has been reported to be achieved using Bacillus thuringiensis var. israelensis (Bti)2. Although Bti has been in use for more than two decades, no resistance has been detected in target insects exposed to this biolarvicide. Bti is highly selective for use against members belonging to the family Culicidae and Simuliidae. It offers an additional advantage of not affecting non-target species of vertebrates and invertebrates, thereby ensuring the safety of its prolonged use on a large scale, without damaging the environment3. The effectiveness of Bti is dependent on the bioavailability of the material in treated areas, which in turn depends on the design of the formulation. An ideal mosquito larvicidal formulation comprises the active ingredient (Bti), additives and carrier material. A number of additives such as carboxymethyl cellulose (CMC), gum Acacia, Xanthan gum, guar gum, pectin, starch, polyethylene glycol (PEG), sodium alginate, paraffin, gelatin and lignin have been used in biopesticidal formulations456. In India, coal and lignite are the most economic and easily available raw materials for power generation, but its utilization is faced with several environmental constraints, the main being production of fly ash (FA) in enormous quantities (120 million tonnes per annum)7 which needs to be disposed of in an appropriate manner.

The present study reports the development of water dispersible powder (WDP) formulations using FA generated from the neighbouring Neyveli Lignite Corporation, Neyveli, Tamil Nadu, India, and an indigenous isolate of Bti (VCRC B17). The best formulation was taken up for the evaluation under laboratory conditions against the larval stages of Culex quinquefasciatus, Anopheles stephensi and Aedes aegypti, vectors of filariasis, malaria, chikungunya and dengue, respectively.

Material & Methods

Bacterial strain: Bti (VCRC B17), an indigenous isolate8, was obtained from the microbial culture collection of ICMR-Vector Control Research Centre, Puducherry, India. The strain was revived from the lyophilized spores and streaked onto modified nutrient yeast salt medium (NYSM) agar slants9. The slants were incubated at 30°C for 48 h and then stored at 4°C for further use.

Inoculum preparation: The seed inoculum was produced using shake flasks. The first-stage seed was prepared by inoculating 10 ml of NYSM broth with one loop full of a slant culture and incubating at 30°C on a rotary shaker (200 rpm) for 6 h. The second-stage seed was prepared by inoculating 2 per cent v/v of first-stage seed into 600 ml of NYSM medium in a 2 l Erlenmeyer flask and incubating on a rotary shaker as done earlier.

Pilot sale fermentation: The second-stage seed in log phase was used to inoculate a 100 l bioreactor at 2 per cent (v/v). Fermentation experiments were conducted in a pilot scale bioreactor (Bioengineering, Wald, Switzerland) filled with 60 l production medium (2.0% soya powder) as described earlier10, except for the stirrer speed which was adjusted to 200 rpm. Fermentation was terminated after completion of spore-crystal complex formation as confirmed by microscope (Motic Microscope Model DM143, Germany). The fermentation was repeated on three different days.

Continuous flow centrifugation: The sporulated culture broth was harvested by centrifugation using a continuous flow centrifuge (CEPA Z41, Germany) as described elsewhere11. The bacterial biomass deposited in the form of a cake on the rotor was scooped out and used for the preparation of WDP formulation.

Processing of carrier material: The lignite FA was obtained from Neyveli Lignite Corporation Limited, Neyveli, Tamil Nadu, India. It was powdered using a ball mill and the FA powder was sieved through 25-μ mesh, to obtain particles of size ≤25 μ to suit the feeding size range of mosquito larvae. This FA material was sterilized and used in the preparation of formulations. The elemental analysis of FA was done using scanning electron microscope (SEM) (Model JSM-6510LV, JEOL, USA) and energy dispersive X-ray spectroscopy (EDS) (INCAPentaFETx3 Model 8129, Oxford Instruments, England).

Development of formulation: Sixteen types of WDP formulations were prepared using a mixture of bacterial biomass: FA (4:5) and various quantities of organic, plant-based and synthetic polymer additives, namely CMC or Acacia gum or soluble starch or PEG or Xanthan gum purchased from HiMedia, India, respectively (Table I). The formulations were dried at 40°C, ground to a fine powder, sieved to a size of ≤25 μ and stored after confirming the final moisture content to be 5 per cent. The final product of these formulations was greyish fine-sized powder that dispersed readily when mixed with water.

Table I Composition of fly ash-based water dispersible powder formulations

Laboratory evaluation: The WHO standard procedure12 was followed to determine the efficacy of the WDP formulations against late third instars of Cx. quinquefasciatus, obtained from the rearing and colonization facilities of our institute. Suspensions of the WDP formulations were made by suspending 10 mg of each formulation in 10 ml of sterile distilled water and mixed well using glass homogenizer. Range-finding bioassays were performed using a wide range of concentrations ranging from 1 to 10 μg. Each concentration had four replicates each, along with appropriate number of controls which contained only plain water. Larval mortality was scored after 24 h. The experiment was done at least three times on different days with freshly prepared suspensions.

Statistical analysis: Larval mortality in control (5-20%) was corrected according to the Abbott's formula13. The corrected mortality was subjected to mortality-concentration regression analysis14 to calculate 50 and 90 per cent lethal concentration (LC₅₀ and LC₉₀) values as well as their 95 per cent fiducial limits (95% FL) using log-probit analysis software (SPSS Statistics ver. 21, IBM Corporation, NY, USA). The LC₅₀ and LC₉₀ values obtained for WDP formulations were subjected to one-way ANOVA followed by Tukey's honest significant difference (HSD) multiple comparison test to determine the differences in formulations.

Susceptibility of Anopheles stephensi and Aedes aegypti: The most effective WDP formulation from the test with Cx. quinquefasciatus was selected and the dose required for inciting LC₅₀ and LC₉₀ for the larvae of other two major mosquito vectors, namely, An. stephensi and Ae. aegypti, were determined using the WHO procedure12. The LC₅₀ values obtained for different mosquito species were compared using one-way ANOVA and post hoc tests (Tukey HSD) were performed to determine the difference in the susceptibility among species.

Toxicity against non-target organisms: The safety of the selected WDP formulation to non-target organisms that are commonly found in association with mosquito larvae in aquatic habitats, namely Ostracods, Cyclops, Daphnia sp., Notonecta sp., Diplonychus sp. and fish, was studied15. These organisms were collected from the aquatic environments where the biopesticides are applied and tested in laboratory at the dose of 1.54 mg/l (10 times the LC₉₀ value obtained for An. stephensi which was the species found to be most tolerant to the formulation) in quadruplicate. The fauna was observed for one week for mortality if any.

Mammalian toxicological studies: The safety of this WDP formulation on mammalian systems was done at the International Institute of Biotechnology and Toxicology (IIBAT), Padappai, Tamil Nadu, India. The tests done were acute oral toxicity/pathogenicity in Wistar rats and acute dermal toxicity/pathogenicity and primary skin irritation in New Zealand white rabbits, respectively.

Results

Nature and properties of fly ash (FA): The SEM observation of FA sample revealed greater number of hollow glass spheres called cenospheres (Fig. 1), which are hard, rigid, lightweight and inert largely made of silica and alumina. This property prompted us to use FA as carrier in our formulation. The size of the FA particles used in the formulation ranged between 1 and 25 μ. The SEM-EDS analysis of FA revealed the presence of only macro- and micro-nutrients such as Si, Al, Ca, Fe together with Mg, S, Na and Cu. It did not contain any toxic heavy metals such as Pb, Cd, Ni, As, Hg, Se and Cr and radionuclides such as U, Ra and Th. (Fig. 2).

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Fig. 1

Scanning electron micrograph of fly ash showing cenospheres in different sizes (×2000).

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Fig. 2

(A) Energy dispersive X-ray spectroscopy image showing the major elements in the fly ash. (B) Scanning Electron Micrograph (Energy dispersive X-ray spectroscopy) showing the peaks of major and minor elements and its actual proportion in fly ash.

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Evaluation of the water dispersible powder (WDP) formulations: Larvicidal efficacy of the various WDP formulations is given in Table II. Among the 16 formulations tested against Cx. quinquefasciatus, WDPA2 was found to be the most active, with an LC₅₀ value of 0.0419 mg/l and LC₉₀ of 0.0753 mg/l. This formulation contained FA and technical grade Bti in the ratio 4:5 with one per cent CMC as a binding agent. The ANOVA test showed that the LC₅₀ and LC₉₀ values among the WDP formulations were significantly different at P<0.001. The post hoc tests indicated that the activity of WDPA2 formulation was significantly different and required lesser concentration when comparing its LC₅₀ and LC₉₀ values with that of all the other formulations (P<0.01). The formulation WDPAA without binder showed less activity than WDPA2 with the LC₅₀ and LC₉₀ of 0.0646 mg/l (0.0606-0.0686) and 0.1062 mg/l (0.0999-0.1142), respectively (Table II). The formulation which showed the least activity was WDPB3 with LC₅₀ of 0.0830 mg/l (0.0779-0.0883) and LC₉₀ of 0.1378 mg/l (0.1287-0.1496).

Table II Toxicity of fly ash-based water dispersible powder formulations against late third instar Culex quinquefasciatus larvae

Susceptibility of different species of mosquito larva: Among the three species of mosquito larvae tested, the LC₅₀ and LC₉₀ values of the WDPA2 formulation against Cx. quinquefasciatus, Ae. aegypti and An. stephensi larvae were 0.0417, 0.0462 and 0.1091 mg/l and 0.0755, 0.0928 and 0.1541 mg/l, respectively (Table III). The ANOVA test showed that the LC₅₀ and LC₉₀ values among the mosquito larvae of different species were significantly different at P<0.05. The post hoc tests indicated that the LC₅₀ and LC₉₀ values were significantly lower at P<0.06 and P<0.05 for Cx. quinquefasciatus compared to that of Ae. aegypti and An. stephensi (P<0.05), respectively. The test further indicated that the LC₅₀ and LC₉₀ values for Ae. aegypti larvae were significantly (P<0.05) lower than that for An. stephensi.

Table III Susceptibility of III instar larvae of different mosquito species to water dispersible powder A2 formulation

Effect of ingredients (individual/combined) of the WDPA2 formulation on mosquito-larvicidal activity: Among the components tested, the Bti with FA+CMC resulted in mortality rate of 51.7 per cent. The percentage mortality due to FA and CMC was only 1.3 and 2.3 per cent, which was well below the allowed mortality levels in control experiments. The ANOVA results showed that there was significant difference among percentage mortality due to ingredients (P<0.001). The post hoc tests further indicated that the percentage mortality in Bti+ FA+CMC (WDPA2) was significantly different from FA and CMC tested alone (P<0.001). However, the mortality in FA alone did not differ significantly from CMC. Hence, FA and CMC as carrier and additive did not have any significant effect on the larval population and the larvicidal activity of the WDPA2 formulation was only due to the presence of Bti.

Tests against non-target organisms and mammalian systems: When tested at 10 times the concentration of WDPA2 formulation used for obtaining 90 per cent kill in the least susceptible mosquito larvae of species An. stephensi, the WDPA2 formulation was found to be safe to Crustaceans, namely Ostrocods, Cyclops and Daphnia, insects, namely Notonectids and Diplonychus, and fish, namely Poecilia (Table IV). Acute oral toxicity conducted on rats and acute dermal toxicity and primary skin irritation tests conducted on rabbits showed that the WDPA2 formulation was non-toxic and non-irritant on mammalian systems.

Table IV Toxicity of water dispersible powder A2 formulation to non-target organisms

Discussion

The trend to use biological control agents in mosquito control programmes has gained widespread importance in recent years due to detrimental effects of chemical insecticides on the environment and human health. One of our indigenously isolated mosquito-larvicidal agents Bti (VCRC B17) was found to be highly effective against larvae of different mosquito species in various aquatic habitats1617. Mosquito larvae (especially late instars) being filter feeders are reported to selectively feed on particles of colloidal to 50 μ in size181920. Hence, incorporation of FA as a carrier material has enhanced the chances of ingestion of this formulation by the mosquito larvae. The SEM analysis of FA used for making WDP formulations contains only macro- and micro-nutrients which is beneficial when applied in freshwater bodies such as paddy fields. Dutta et al21 revealed that the leaching of toxic elements/heavy metals from FA was negligible when the pH of the water body was alkaline or nearly neutral and mosquito larval-breeding habitats are always reported to be alkaline in nature.

This study showed that the LC₅₀ values were lower for Culicines than for Anophelines. The susceptibility pattern of the larval stages of the three vector species to this biopesticide was of the following order: Cx. quinquefasciatus<Ae. aegypti<An. stephensi. This was in agreement with many other reports with this bacterial species2223. The relative lower efficacy of Bti formulation against Anopheles species might be due to their reduced filtration rates as has been reported by Aly et al24. Further, this variation in the susceptibility has been attributed to variation in the gut pH of these insect species which is known to play a major role in the activation of the endotoxins of this bacterium25. Differences in the feeding behaviour of these three species are also known to be responsible for this varied susceptibility26.

The formulation was found to be safe to non-target organisms found in association with mosquito larvae in the aquatic habitats. This observation reinforces the results of several earlier studies conducted with Bti152728. This study showed that the carrier material and additive, namely FA and CMC used in this formulation, as well as the active ingredient, Bti, were safe on mammalian systems. Hence, FA can be effectively used to replace commercially available carrier materials in the preparation of biopesticidal formulations. FA has earlier been successfully used as such or as carriers for the production of biofertilizers and biopesticides for agriculture729. Furthermore, powder formulations are reputed for their long shelf life, miscibility in water compared to technical grade materials, microgels and aqueous suspensions30.

In conclusion, with biopesticides gaining wide importance in recent times in the wake of maintaining a safe environment, use of FA will help not only in adding to its utilization as almost half of the formulated material contains FA but also in ensuring safety to the environment where it is applied as it has proved to be safe to non-target organisms and mammalian systems.