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

Methods: An indigenous isolate Bti (Vector Control Research Centre B17) was mass produced using a 100 l fermentor in soya-based medium. The bacterial biomass was mixed with lignite FA and made into WDP formulations. The most effective formulation was used for determining 50 per cent lethal concentration (LC50) against the larval stages of major mosquito vector species, effect on non-target organisms and mammalian systems using standard protocols.

(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 environment 3 . 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 formulations [4][5][6] . 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 isolate 8 , 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 slants 9 . 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 earlier 10 , 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 elsewhere 11 . 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.
Laboratory evaluation: The WHO standard procedure 12 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 formula 13 . The corrected mortality was subjected to mortality-concentration regression analysis 14 to calculate 50 and 90 per cent lethal concentration (LC 50 and LC 90 ) 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 LC 50 and LC 90 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 LC 50 and LC 90 for the larvae of other two major mosquito vectors, namely, An. stephensi and Ae. aegypti, were determined using the WHO procedure 12 . The LC 50 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 studied 15 . 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 LC 90 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

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).

Evaluation of the water dispersible powder (WDP)
formulations: Larvicidal efficacy of the various WDP formulations is given in

Effect of ingredients (individual/combined) of the WDPA2 formulation on mosquito-larvicidal activity:
Among the components tested, the Bti with FA+CMC    (Table IV). * Means within a given column followed by the same alphabet letter are not significantly different, Tukey's multiple range test (α=0.05), P<0.001; + Pearson χ 2 goodness of fit test; # If the significance level is <0.150, a heterogeneity factor is used in the calculation of confidence limits. LC 50 , lethal concentration that kills 50% of the exposed larvae; LC 90 , lethal concentration that kills 90% of the exposed larvae; 95% FL, 95% upper and lower fiducial limits; WDP, water dispersible powder formulation Pearson Chi-square goodness of fit test; # If the significance level is less than 0.150, a heterogeneity factor is used in the calculation of confidence limits; LC 50 , lethal concentration that kills 50% of the exposed larvae; LC 90 , lethal concentration that kills 90% of the exposed larvae; 95% FL, 95% upper and lower fiducial limits 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.

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 habitats 16,17 . Mosquito larvae (especially late instars) being filter feeders are reported to selectively feed on particles of colloidal to 50 µ in size [18][19][20] . 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 al 21 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 LC 50 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 species 22,23 . 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 al 24 . 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 bacterium 25 . Differences in the feeding behaviour of these three species are also known to be responsible for this varied susceptibility 26 .
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 Bti 15,27,28 . 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 agriculture 7,29 . Furthermore, powder formulations are reputed for their long shelf life, miscibility in water compared to technical grade materials, microgels and aqueous suspensions 30 .
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.  Diplonychus spp. + 10 10 0

Acknowledgment
Poecilia spp. 10 10 10 10 10 10 0 * 10 times (1.54 mg/l) the concentration of LC 90 value of Anopheles stephensi larvae; + As there was cannibalism among Diplonychus spp., safety testing was done having one individual in each testing cup. Ten replicates each were kept for control and treatment. R, replicate; LC 90 , lethal concentration that kills 90% of the exposed larvae