LABORATORY EVALUATION OF COMMERCIAL FORMULATIONS OF BACILLUS THURINGIENSIS VAR. ISRAELENSIS AGAINST MOSQUITOI AND BLACK FLY' LARVAE'

Daniel Molloy, Stephen P. Wraight" Bruce Kaplan, Judith Gerardi, and Patricia Peterson

Biological Survey, New York State Museum, The State Education Department, Cultural Education Center, Albany, NY 12230

Abstract: Three commercial black fly and mosquito larvicidal fonnulations of Bacillus thuringicnsis Berliner vnr. israelensis de Barjac were bioassayed to determine their potencies relative to the international standard powder, IPS-78. The potencies of the wettable powder (WP) formulations of Bactimos'" and Vectobac'" and the water dispersible concentrate (\VDC) Teknar'" were detennincd to be 4530, 5723, and 336 International Toxic Units (ITU)/mg. respectively, against Simulium spp.; their respective ITV/mg values against Aedes aegypti (Linnaeus) were 3556, 2317, and 1373. Little correlation (r2 = 0.47) was evident between the potency rating (lTV/mg) of the fonnulations against mosquitoes and black flies; this indicated that t.he potency rating of a fonnulation against black flies cannot be simply predicted from the A. aegypti ITV/mg rating on its label. A moderate correlation between formulation potency and mean particle size was observed in the black fly assays (r2 = 0.77), but not against mosquitoes (r2 = 0.20). Aqueous suspensions of Teknar·WDC had u significantly smaller mean particle size and stayed significantly longer in suspension than other formulations. The relatively long duration of suspension of Teknar-WDC was viewed us important since the toxic crystals would be less likely to settle out in mosquito and black fly habitat-c;. A ratio based on the LC 50 values determined from standardized bioassay methods is suggested as it convenient way to express the relative potency of a fonnulation against mosquitoes and black flies.

Key Words: Teknar-WDC, Bactimos-\VP, Vectobac-WP,lPS-78, Aedes aegypli, Simulium, ITV/mg, particle size, settling rate, potency ratio.

J. Agric. Entomol. 1(2): 161~168 (April 1984)

Black flies and mosquitoes are major pests of both man and animals primarily because of the medical and economic consequences of the blood-sucking activities of the adult females. Although chemical insecticides have traditionally been the primary agent used for controlling these flies, their undesirable environmental effects have prompted the development of more environmentally-benign biological control methods. Research with one such biological agent, Bacillus thuringiensis Berliner var. israeLensis de Barjac, a bacterium highly specific for larval black flies and mosquitoes, has produced rapid and significant progress (World Health Organization 1982). This is evident from the present availability of EPA-registered formulations of this bacterium for black fly and mosquito control within 6 yr of its discovery (Goldberg and Margalit 1977). The filter-feeding larvae of black flies and

I D1PTERA: Culicidoe 2 DI~RA: Simuliidau 3 Publiohcd liS New York State M\l~el.lm Joumul Surica No. 352. Received for pl.lhliclltion 28 March 1983; accepted 8

November 1983. ,\ Present IIddreu; Boycll Thompson Institute for Plllnt Research at Cornell Univ., Tower Road. Ithllca. NY 14853.

161

162 J. Agric. Entomol. Vol. I, No.2 (1984)

mosquitoes die quickly following their ingestion of the toxic crystals (parasporal inclusion bodies) produced by the bacterium during sporulation.

This laboratory study was designed to evaluate the potency of three commercial formulations of B. thuringiensis vaT. israeiensis relative to the international standard powder, IPS-78. These commercial products were: the wettable powder (WP) formulations of Bactimos'" and Vectobac'" and the water dispersible concentrate (WDC) Teknar'w. The potencies of these fonnulations, expressed in International Toxic Units (ITU)jmg, were detennined in tests against both black flies and mosquitoes. An important objective of these tests was to detennine if the relative toxicities of the various formulations against black flies would correlate with those found against mosquitoes. Previous studies (Guillet and Escaffre 1979, Guillet et al. 1980) have suggested that particle size is a factor of considerable importance since it appears to influence the efficiency and rate of ingestion of the B. thuringiensis toxic crystals by black flies and also since it may affect the rate at which crystals settle out from suspension. As part of the evaluation of these three commercial formulations, we therefore examined the characteristics of particle size and crystal settling rate.

MATERIALS AND METHODS

Formulations used in these tests were: Bactimos-WP (lot no. LRB 676; Biochem Products, Monchanin, DE), Teknar-WDC (~402, lot no. BTl-I; Sandoz, Inc., San Diego, CAl, Vectobac-WP (=ABG-6108-IJ, lot no. 8278-55; Abbott Laboratories, Chicago, IL), and the international standard powder, IPS-78 (pasteur Institute, Paris, France).

Toxicity of Commercial Formulations Black Fly Assays. Stock inocula (0.2 mg/ml) were prepared by the following

procedure: 200 mg of the fonnulation were placed in a test tube (1.5 em Diarn containing 5 ml of deionized water and mixed on a vortex agitator for 3 min a maximum speed (liquid height ca. 9 em); this suspension then was added to a 2­liter glass flask with enough deionized water to produce a final volume of 1 liter and subsequently mixed by hand (20 circular shakes), Field-collected late-instar Simulium larvae were placed on the tray section of bioassay units (Gaugler et al. 1980) and allowed to attach and become acclimated in the flowing fresh water for a minimum of 48 hr pretreatment. Larvae used in the Teknar-WDC and Bactimos· WP assays (Test Series I) were all S. uittatum Zetterstedt, had a mean (95% CI) postgenal length of 378 (368-389) /-lm, and averaged 126/tray; the larvae used in the Vectobac-WP assay (Test Series II) were 32% S. uittatum and 68% a mixture of the species complex S. uenustum Say/So uerecundum Stone and Jamnback, had a mean (95% eI) postgenal length of 387(379-395) JLm, and averaged 33/tray5. All assays were conducted in the flow· through mode (Gaugler et al. 1980) with appropriate quantities of inoculum added every 30 sec to the fresh water entering each bioassay unit during a 15·min period. Exposures were conducted at l3°C in

5 Vert.ohl!c.WP 1I11B.llYS [felt Series 1I) could nOI be conducted simultBneously with the UllctimoR'WP and Teknar-WDC nasaYH [fest Series I); Veclobac·WP was received for lelliinc ca. 6 months after Test Serie" I WIlS completed. Pre"ious nperimentBtion (Molloy ctul. 1981) lIuCgCStll that hll'vlle of the 8. L'cnu.<tunt!S. ucrccumlum comlllClllnd S. u;Ualum thot lire of thc 88m" "i~e (liS they "'ere ill Test Series I and II) would he of similar SIJNCelllihility to II IhurillJ!iens~' VIIT.

ismell'llsis.

MOLLOY et al.: Bti Against Mosquito and Black Fly Larvae 163

the Teknar-WDC and Bactimos-WP assays with stream water temperatures ranging from 11 to 13°C during the posttreatment period; exposures in the Vectobac·WP assays were at 11°C with posttreatment range of 10-12°C. Mortalities recorded after 3 d of incubation were corrected by Abbott's formula and subjected to probit analysis (Finney 1971) to yield LC50 and LC95 values. Relative potency estimates were determined by the methods described in Finney (1971) for parallel line assays and expressed in ITV/mg relative to the LC 50 of IPS-78 (the international standard with a designated potency of 1000 ITV/mg).

Mosquito Assaysu. Procedures followed were similar to those described for the black fly assays except for the following: in preparation of stock inocula, 0.52 ml of the vortex-agitated suspensions were added to a 250·ml flask, and the final volume was brought up to 200 ml; exposures were conducted with 100 fourth-instar Aedes aegypti (Linnaeus) larvae (Rockefeller Institute strain) in trays containing 1 liter of deionized or distilled water at 26 (±ltC; larvae were fed ground rabbit chow as outlined in \Vraight et al. (1981); mortality was recorded after 48 hr.

Formulation Particle Size Two hundred and fifty rug of each formulation were placed in 250-ml flasks

containing 50 ml of deionized water and swirled by hand for 3 min. Approximately 400 particles were measured with a Zeiss compound microscope, and a mean value was determined 7• This procedure was repeated four times, and the average ±SE of the four means was calculated. The values obtained were tested for significance (5% level) using Tukey's w-procedure for comparison of means (Sokal and Rohlf 1969).

Formulation Settliflg Rate The relative settling rates of the crystals in aqueous suspensions of Bactimos­

WP, Teknar-WDC, and IPS-78 were measured in tests against both black flies and mosquitoes. A 5-g portion of each fonnulation was placed in i-liter flasks contain­ing 500 ml of deionized water and swirled by hand for 3 min. The content of each flask then was added to a separate plastic tub (40-cm Diam) containing 24.5 liters of deionized water at 26 (±ltC. The contents of each tub were stirred thoroughly to produce a uniform suspension (0.2 mg/ml, ca. 20 cm in depth). Samples were withdrawn slowly by pipette from the middle of each tub (ca. 10 cm from the bottom) at selected intervals over the next 36 hr. All samples were stored in glass flasks at 26 (±l)"C. To eliminate the possibility of crystal degradation affecting the experiment, black fly and mosquito tests (see below) were conducted simultan­eously in a single randomized experiment beginning shortly after the 36-hr sample was taken. Immediately prior to conducting the exposures, the material in each storage flask was resuspended by swirling with glass beads to remove material which had adhered to the flask walls.

Black Fly Tests. Bioassay procedures and experimental conditions were similar to those previously outlined (see Toxicity of Commercial Formulations), except for the following; mean (95% CI) postgenal length of the S. vitlatum larvae was 404 (397·412) ,urn; average number of larvae per bioassay tray was 136; I5-min

6 These lesw weI"(> conducted prior 10 the iHSIJAnCe of the standardized protocol for OllllRy of B. thurin~'iu~~L~ H-14 prellllf8' tions Ilg"in~1 mosquitoclI (World Henlth OrgllnizAtion 19RI) llnd thull differ to 1I limited degrc~ from them.

7 BeCAuse or the limitlllions of the light rnicro~cope, llarticlell <0.5 ,lint in Diam could not he mellSured 8ccltrlltely and thus ""ere not counted: thererore, the melln vllluell preHllnted are only n reltni"e inde~ or the pllrticle size of e8<:h fOmlu, lation.

164 J. Agric. Entomol. Vol. 1, No.2 (1984)

exposures (three replicates/sample) were conducted at 12°C and at dilutions which normally produce ca. 90% mortality (i.e., Baetimos-WP = 20 ppb, lPS-78 ~ 100 ppb, and Teknar-WDC = 320 ppb); mean (95% Cn mortalities were calculated from corrected (Abbott's formula) and arcsin-transformed data.

Mosquito Tests. Experimental procedures were similar to those just described except for the following: exposures (4 replicates/sample) were conducted with 40 early fourth·instar A. aegypti larvae (Rockefeller Institute strain); since Di were <50, means (95% el) were determined using the Freeman-Tukey transformation for the binomial distribution (Mosteller and Youtz 1961).

RESULTS AND DISCUSSION

The ITV/mg values calculated from the mosquito assays were very close to, and thus confirmed, the values stated on product labels (Table 1)8. Little correla­tion was evident in the potency ratings (lTU/mg) of the formulations against mosquitoes and black flies (r2 = 0.47; y ~ 1.775x - 756.66) (Table I). For example, although Bactimos-WP was 2.6 times more toxic than Teknar-WDC to mosquito larvae, it was 13.5 times more toxic than Teknar-WDC to black fly larvae. Guillet et a1. (1982), in tests of B. thuringiemis VSf. israelensis formulations against S. damnosum s.l. Theobald and A. aegypti also noted such a low potency correlation. Thus, the potency of a formulation to black fly larvae can not simply be predicted from the A. aegypti ITU/mg rating on its label.

The settling rate of the toxic components of Teknar-WDC was substantially slower than that of eitber lPS-78 or Bactimos-WP (Table 2). While the toxicity of samples of aqueous preparations of the latter u,'Vo powdered formulations decreased quickly (e!O% mortality after 5 hr of settling), only a moderate decrease in the activity of the Teknar-WDC samples was noted after 36 hr of settling in the mosquito tests and not at all in the black fly tests. The relatively rapid settling of Bactimos-WP and WS-78 was clearly demonstrated by the difference in A. aegypti mortality caused by the samples taken at 0,0 and 0.5 hr (Table 2).

The settling rates of the formulations corresponded directly with their particle sizes: those formulations with the larger particles (i.e., Bactimos-WP and IPS-78) had the fastest settling rates (Table 2)9. The relatively-rapid settling of Baetimos­WP and IPS-78 was visually apparent and could have been predicted from our data on mean and range of particle sizes: both latter formulations had particles ranging up to ca. 100 jJ.m (versus 28 J1.Ill for Teknar-WDC) (Table 2). Such results are in agreement with Stokes' law (Weast 1982) which indicates that the settling velocity of a particle, although closely related to its density, is also directly proportional to the square of the radius of the particle.

The potency (ITU/mg) of the fonnulations to black nies correlated moderately weU with particle size (r2 = 0.77; y ~ 0.0004x + 2.52) (Table I). These findings further support the work of Guillet and Eseaffre (1979) who first noted such a correlation in their assays with B. thuringiensis var. israelensis formulations against black nies. Such a correlation, however, \Vas not evident (rom our mosquito data (r2 = 0.20; y = 0.0005x + 2.73) (Table I).

8 A limillr complllriion could not be made (or the rru/mg valuu dctennined in the bllck f1)' llSA$YI linte luch values did

not IIllp1!lr on product labell. 9 Dilla on the leUling nile or vectobllc·WJ> lire not induded here becauae thil formulation became available 6 months

afu!r the .ettling leall '""tore conducted; """'ever, both our partide lize dlta lind our visull oblervltiolll or Vectobac·WP IIllpenaiollI IIlneAt thlt i4 aeltling I'lIte would be limilar to thlt of alnimOll·WI· and IPS·78.

Table 1. Toxicity of IPS-7S and commercial formulations of Bacillus thuringiensis var. israelen.<;is (on a total product weight basis) to black fly and mosquito larvae.

Mosquito larvae Black fly larvae

Particle Relative potency size <J..t-m)* LC values t OTU/mg) LC values Potency ratio

;s: Stated on Relative o

r­X ± S.E. product potency Le so mosQuitoes\ r­Fonnulation (range) 50 95 Calculated t label 50 95 OTUlmg) ( LCfjO black flies1 o

-< Test Series J

Teknar-WDC 2.1a ± 0.02 301 650 1373 1500 136 390 336 2_2 (0.;;-28.0) (282-321) (598-715) (1257-1501) (120-155) (336-461) (280-405)

IPS-78 3.7b ± 0.22 413 893 1000 1000 46 131 1000 8.2§ (0.5-103.7) (389-439) (818-986) (40-52) (111-159)

Bactimos-\VP 4.0bc ± 0.10 116 251 3556 3500 10 29 4530 11.6 (0.5-98.8) (109-123) (232-275) (3265-3881) (9-12) (25-35) (3762-5476)

Test Series II fPS-78 3.7b ± 0.22 614 1357 1000 1000 84 239 1000

(0.5-103.7) (574-660) (1201-1578) (68-103) (193-304)

Vectobac·WP 5.2d ± 0.30 265 586 2317 2000 15 42 5723 17.7 (0.5-122.0) (248-283) (530-661) (2109-2565) (12-18) (33-54) (4229-7735)

• Muns followed by the 5all1~ ItllU lll't not significantly differtnt (5~ le"el) u determintd from Tukc)··, w.procedufll for romparison of means (Sohl .nd RohlI 1969). t All dlltll upressed liS ppb 195';;, fiducilll limiu); I fl·min uposunos: aU o;onlml mortality S 1.0';:,. ; l'ot~nC}' expressed as ITli/ml (9:;'.;, fiduciAl limitll) relal;'·", 10 tht LeSO of IPS·i8 (duignllted potency'" 1000 lTU/mg). § Mean ratio of Ten Serie~ 1 .nd n.

Table 2. Comparative settling rates of the toxic components of Bactimos-WP, IPS-78, and Teknar-WDC as determined from the '" mortality to black flies and mosquitoes caused by samples from aqueous suspensions, '"

Particle size (p.m)* Percent mortality! (95% Cn after indicated hour

Formulation X ± S.E. (range) 0.01 0.5 1.5 5.0 12.0 36.0

Black fly laruae

B.ctimos-WP 4.0. ± 0.10 (0.5-98.8) 28(7-48)1 1(0-2) 2(1-4) IPS·78 3.7.b ± 0.22 (0.5-103.7) 84(80-87) 0 1(0-2) Teknar-WDC 2.1c ± 0.02 (0.5-28.0) 90(77-100) 88(85-92) 89(73-100) !'­

>',iMosquito larvae o

B.ctimos-WP 4.0. ± 0.10 (0.5-98.8) 97(85-100) 21(11-34) 10(2-25) 0 0 0 IPS-78 3.7.b ± 0.22 (0.5-103.7) 96(85-100) 42(29-55) 22(10-37) 0 0 0 Teknar-WDC 2.1c ± 0.02 (0.5-28.0) 87(82-92) 80(68-90) 72(62-81) 79(71-86) 79(68-88) 57(41-72) • MUIIlI foUOlI<ed by tbe aame letter IlJ"e DOt sis:"nificantly different (5'.t. level) .a detennined from Tulr.ey'a ""_procedure for comparison of muna (Solr.al &JId Rohlf 1969). t AU conlrOl morulity :S0.5'k, ~ lndiea~a the time post·mming when the umple of the aquKIUI auspellllion .... ",ithdnl'A'lt; aU lWa)'a were conducted simull.aneoualy. shortly after the 36.o-hr umple ... taken; the 0.0 hr doup lI"U

calculated 10 be the ca. LC90 of ueh formulation. with equal volum.. u.ed at other lime intervals. § Althoullh the doulte used in these aaetimos-WP trial5 (20 ppb/15 min) proved t.O be imuffieient to produce the desired (ca. 90%) IDOrtality at 0.0 hr, the Bsctimo.·WP data are ineludlld here

airn:e they ItiU lerve 1.0 indicate the relatNely I'lIIpid senling of thi. formulation (i.e.. I'l. mortality at 1.5 hr).

MOLLOY et a!.: Btl Against. Mosquito and Black Fly Larvae 167

If the relative potency of a formulation against mosquitoes and black flies is expressed by the ratio,

LC'iQ A. aegypti, LCso Simulium

then formulations which have relatively higher potencies against black flies would have higher ratio values. Such a ratio would appear to be a convenient way to express the relative potency of a fonnulation against mosquitoes and black flies. Ideally, such ratios would be generated using the standard B. thuringiensi.... bioassay methods recommended against black flies (Lacey et al. 1982) and mosquitoes ('''orld Health Organization 1981). The A. aegypti/Simu[£um LC50 ratios generated in the present tests correlated very well with the mean particle size of the formulations (1'2 = 0.98; y = 0.1948x + 1.82) (Table 1). This suggested that increasing the particle size of a fonnulation would have a higher probability of increasing its potency against black flies than against mosquitoes.

The ITU/mg values of Bactimos~WP and Vectobac-WP were considerably higher (>lOXj than that of Teknar-WDC against black flies (Table 1). However, the relatively low ITU/mg value recorded for Teknar- WDC against black nies did not indicate that it was an inferior formulation; it only meant that, on a weight basis, the liquid formulation Teknar-WDC was not as toxic to the black nies as the two powdered formulations. Thus, of the three commercial fonnulations tested, a greater quantity (i.e., mass) of Teknar-WDC would have to be added to stream water to achieve the same I}ercent black ny kill immediately below a treatment point. The lTU/mg rating of a fonnulation, although important, is only one characteristic to be considered in choosing a black fly larvicide; effective carry (the distance downstream that a larvicide will produce high black fly mortality) is another critical formulation aspect. Among the three commercial formulations considered here, Teknar-WDC would likely give the best effective carry. This was evident from the settling tests (Table 2) which indicated that Teknar-WDC would be less likely to settle out and thus would probably give good control for longer distances downstream in field applications against black flies. Also, since liquid formulations like Teknar-WDC'o are generally more convenient to work with in the field, this may be another reason for placing less emphasis on the potency of the product per se in choosing a formulation for a control program. In the final analysis, the choice of fonnulation is based primarily on the cost of effectively controlling black nies with each fonnulation. This information will become available only after anumber of large-scale field trials have been completed.

ACKNOWLEDGMENTS

The nssistance of t.he fotlowing in arranging supply of formulations is gratefully acknow­ledged; \"l. Beck (Sandoz, Inc.), R. Cibulsky and T. Couch (Abbott Laboratories), H. de Barjac (Pasteur Institute), and R. Rose (Biochem Products). This invest.igation received financial support in part from the UNDP/World Bank/WHO Special Programme for Research Ilnd Training in Tropical Diseases find the National Institutes of Health (AI15605). We express our appreciation for technical assistance to J. Clyne, T. Meyers, E. Petteys, and K. Ungeheuer, and to A. H. Ulldecn for critical review of the manuscript.

lO SUbHCll"cnt to thcMe tuts. both Bllctimos lind Vcctohllc hllve also heen offered ill liquid (ormulation.

168 J. Agric. EnLOmol. Vol. I, No.2 (1.984)

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