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Deposit characteristics and toxicity of fipronil formulations for tobacco budworm (Lepidoptera: Noctuidae) control on cotton
Crop Protection 18 (1999) 493}499
Deposit characteristics and toxicity of "pronil formulations for tobacco budworm (Lepidoptera: Noctuidae) control on cotton Venkat K. Pedibhotla *, Franklin R. Hall, Je! Holmsen The Scotts Company, 14111 Scottslawn Rd, Marysville, OH 43041, USA Laboratory for Pest Control Application Technology (LPCAT), Ohio State University, 1680 Madison Ave., Wooster, OH 44691, USA Rhone Poulenc Ag. Company, Research Triangle Park, NC 27709, USA Received 22 December 1998; accepted 22 June 1999
Abstract Three water-dispersible "pronil formulations have been tested for toxicity against second instar tobacco budworm, Heliothis virescens (F.). Topically, EC is the most potent of the three assays followed by WG and SC. However, pick-up assays on para"lm did not show signi"cant di!erences in toxicity between formulations. Droplet spread and deposit morphology of the formulations were studied on cotton. Although the initial droplet spread was observed to be highest for EC, the "nal dried deposits of formulations were not signi"cantly di!erent from each other. The deposit patterns of all three formulations on cotton are di!erent. For EC, the formulation/AI is associated with an annulus (outer ring) whereas the WG and SC are particulate in appearance and dispersed across the deposit. From the rain-washing studies on cotton, SEM photographs indicate that SC has greater retention than EC or WG. 1999 Elsevier Science Ltd. All rights reserved. Keywords: Heliothis virescens; Deposit morphology; Droplet spread; Deposit weathering
1. Introduction Among the major pests attacking cotton, heliothines account for about 91% of the yield loss when no control measures are undertaken (Duck and Evola, 1997). In the United States alone, the total production of cotton was worth $ 25.7 billion against an annual loss of 15.4% due to pests (Oreke et al., 1994). Through the proli"c use of insecticides, heliothines have developed resistance to many classes of insecticides including, cyclodienes, organophosphates, carbamates, and pyrethroids (Sparks, 1981; McCa!ery et al., 1989; Kanga and Plapp, 1995). Despite the growing resistance problem worldwide, synthetic organic chemicals continue to dominate the market for the control of pests. Currently, among the new crop of insecticides developed with unique modes of action, "pronil, a phenylpyrazole, has potential for the management of lepidopterous pests of cotton. Fipronil is primarily
* Corresponding author. Tel.: #1-937-645-2669; fax: #1-937-6447104.
a stomach poison with some contact activity that can be e!ectively used against both chewing and piercing}sucking pests (Colliot et al., 1992). Fipronil exerts its action through blocking the c-aminobutyric acid (GABA)-gated chloride channel in insects (Hainzl and Casida, 1996). Recent US EPA policies (IPM, Risk-reduction, etc.) aim for a reduction in pesticide usage (Hall, 1997). Northern European countries have passed a legislation that a 50% reduction in pesticide use should be mandated by the year 2000 (Matteson, 1995). In general, application of pesticides is an ine$cient process leading to signi"cant losses in the environment and only low amounts of agrochemical reaching the intending target (Adams and Hall, 1989; Graham-Bryce, 1983). In order to enhance the application e$ciency of insecticides, various parameters of the dose-transfer process have to be understood. The dose-transfer process can be broadly classi"ed into spray application and bioavailability. Bioavailability is a biological phenomenon generally used to describe the availability of an applied toxicant on a plant canopy or crop foliage and its transfer to a target pest resulting in a biological e!ect. Formulation/AI properties and their
0261-2194/99/$ - see front matter 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 2 6 1 - 2 1 9 4 ( 9 9 ) 0 0 0 5 0 - 2
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V.K. Pedibhotla et al. / Crop Protection 18 (1999) 493}499
interactions, surface morphology of the leaf, and pest behavior are some of the features that in#uence bioavailability, consequently a!ecting the pick up toxicant from the target surface (Ford and Salt, 1987). The objectives of this study were two-fold. First, to evaluate and compare the toxicity of three "pronil formulations, an emulsi"able concentrate (EC), a suspension concentrate (SC), and a wettable granule (WG), by pick-up and topical assays against tobacco budworm (TBW), Heliothis virescens. Second, to conduct various studies on physical characteristics of the formulations including droplet spread, deposit morphology, and rainwashing for a better understanding of the bioavailability mechanisms on cotton.
2. Materials and methods 2.1. Insects Tobacco budworm eggs were shipped bimonthly to the Laboratory for Pest Control Application Technology (LPCAT) by Rhone Poulenc Ag. Company, Research Triangle Park, NC. TBW were obtained from a continuously maintained culture, with no known resistance to any insecticides. All experiments were performed using II instar larvae. Larvae were reared on arti"cial diet and were contained in a constant-environment incubator (253C, 70% RH, a photoperiod of 14 : 10 [L : D] h). Arti"cial diet for TBW was prepared and shipped to LPCAT by Rhone-Poulenc in powder form. The diet was stored in freezer at 43C and was periodically used for experimentation. 2.2. Chemicals Three water-dispersible "pronil formulations 800WG (wettable granules), 200SC (suspension concentrate), and 300EC (emulsifyable concentrate), provided by Rhone Poulenc Ag. Company, Research Triangle Park, NC, were evaluated in all experiments. Distilled water was used to prepare appropriate concentrations of assay solutions for all formulations. 2.3. Topical application This test estimates the lethal dose of each formulation following direct application to the dorsal surface of the larva. To each larva, 0.25 ll of solution was applied using a micro-applicator (Isco., Inc., Lincoln, NE). Ortho X-77 at 0.1% v/v was added to formulations to aid wetting. After treatment, each larva was placed in a plastic diet cup along with arti"cial diet (Lepidoptera diet provided by Rhone-Poulenc) and stored in an incubator (273C incubator, 70% RH, a photoperiod of 14 : 10 [L : D] h). To each control larva 0.25 ll of acetone was applied.
Mortality was assessed after 24 and 48 h. Five concentrations were used and 20 larvae were tested per treatment which was replicated three times. 2.4. Pick-up assay on paraxlm This experiment is useful to estimate the lethal dose of the formulation from toxin pick-up alone. Wet "lter paper (9 cm diameter, grade 1, Whatman, Hillsboro, OR) was placed under para"lm disks, in the bottom of a Petri dish (9 cm diameter). Droplets were applied to the para"lm using an Isco (model M) micro-applicator. Each droplet was 0.5 ll and a total of 24 droplets were placed in a circular pattern with a 2 cm diameter untreated region in the center of the dish and a 5 cm untreated ring along the outside margin of the dish. Furthermore, droplets were placed such that droplets in one row were approximately centered in the gaps between droplets in the other row. Five concentrations, 7.2, 3.6, 1.8, 0.9, and 0.45 g a.i./l, of each formulation were used. Arti"cial diet prepared in the laboratory was placed in the center and three larvae per Petri dish were released in the outside untreated region after the deposits dried. Mortality was assessed after 24 and 48 h. For each concentration, "ve Petri dishes were used and each treatment was replicated three times. 2.5. Spread behavior Rate of spreading measurements were performed on cotton leaves. Cotton (Gossypium hirsutum L.) was seeded in plastic pots (12 cm diameter, 4 seeds per pot) containing a commercial growing medium. Upon emergence plants were selected for uniformity and thinned to two plants per pot. The temperature in the green house was maintained at 28$33C day during the day and 24$33C at night. All formulations were applied at 5 g/l and all assay solutions included Nigrosin (0.4%, w/v), a standard dye, to aid detection of the deposit boundary. One microliter droplets were placed on cotton leaves and the area of the droplets were measured upon contact with the leaf surface and after 0.5, 1, 5, 10, and 15 min. Spread measurements were conducted using an image analysis system consisting of a 18}108 mm 6x zoom lens (Fujinon Inc. Wayne, NJ) "tted to a Kodak megaplus camera (CKOD839-4900, Eastman Kodak Co., San Diego CA). All leaf samples were backlit (Fostec system, Fostec Inc. Auburn NY 13021) and the data was captured using a Data Raptor-PCI capture board (Bit#ow Inc. Woburn, MA) installed in a pentium computer. 2.6. Deposit morphology Fipronil formulations at 5 g a.i./l were applied to cotton to examine the droplet structure. Various droplet sizes (200 & 500 lm) were applied to leaves using a
V.K. Pedibhotla et al. / Crop Protection 18 (1999) 493}499
droplet generator. The droplet generator produces uniformly sized droplets with a pulse generator which is activated by a piezo-electric crystal (Reynolds et al., 1987b). The size of droplets was con"rmed by capturing them in corning #uid and measuring under a microscope "tted with an eyepiece graticule. The dried deposit was marked and the leaf area excised for imaging. Scanning electron microscopy (SEM) was used to examine the resulting deposit after drying. 2.7. Deposit weathering A rain simulator was used to determine the in#uence of rain washing on retention of "pronil on cotton (Reynolds et al., 1987a). An upward-pointing Tee-Jet 8002 nozzle (Tee-Jet Spraying Systems, Wheaton, IL) "tted to a rotating bar was used to produce a spray pattern of uniform rain. Distance of the drop fall was 4 m with a water pressure of 25 psi at the nozzle ori"ce. The rainfall amount over a period of time was measured with a rain gauge. The mono-size droplet generator was used to apply droplets with a diameter of 400 lm on the abaxial surfaces of cotton leaves (potted plants). After 24 h, rain washing experiments were carried out on treated plants which were placed under the rain simulator for 15 min in a 130 mm/h rain. Upon drying, leaf portions were excised and viewed under SEM. Each experiment was replicated three times and had control plants which were treated but not rain washed. 2.8. Statistical analysis Probit analysis were performed using PROC PROBIT (SAS Institute, 1985) to estimate the lethal dose (LD ) for topical and lethal concentration (LC ) for pick-up assays. LD and LC s were expressed as micrograms a.i. per larva. LD s and LC s were considered signi" cantly di!erent if their 95% FL did not overlap, which is p(0.01.
3. Results and discussion By comparing the LD s and LC s from topical and pick-up assays, we observed variations in toxicity to the three formulations, WG, EC, and SC (Table 1). In the topical assay, EC (0.07 lg AI) was the most toxic followed by WG (0.54 lg AI) and SC (1.75 lg AI). From pick-up assays, we noted that the LC of all three formulations, WG (1.7 lg AI), EC (1.4 lg AI), and SC (1.2 lg AI) were within the same range. Interestingly, the high toxicity of EC in the topical assay was not re#ected in pick-up assay. This could be attributed to the solventAI interactions with the insect cuticle in#uencing the penetration and binding properties of the EC formulation. For pick-up assays, para"lm has been used as it is
495
Table 1 Topical and pick-up toxicity of "pronil formulations to II instar H. virescens after 48 h Assay
Formulation
LD (lg a.i.)
95% FL
Slope$SE
Topical
WG SC EC
0.54 1.75 0.07
0.51}0.56 1.33}1.90 0.06}0.09
1.69$0.3 1.91$0.35 1.24$0.27
Pick-up
WG SC EC
2.0 1.4 1.7
1.6}2.2 1.2}1.7 1.4}1.9
2.4 $0.6 1.25$0.28 1.1 $0.24
an conventional substitute for leaves. Additionally, this assay was designed speci"cally to determine the pick-up toxicity only by contact and not through feeding behavior. This is accomplished as larvae move through toxin treated deposits for reaching the diet placed in the center of the petri dish. Work on adhesion and uptake of contact insecticides to the insect cuticle has been conducted by Welling (1977). It was observed that the toxicity of such chemicals depend largely on the partition coe$cients between the surface waxes of insect cuticle and water, for uptake and penetration of toxicant. Sato (1992) showed that parathion is 20 times more soluble in the cuticular wax of Spodoptera litura and Mamestra brassicae than carbaryl. Although "pronil is primarily a stomach poison it also exhibits contact activity. Unlike certain lepidopterous larvae, such as the cabagge looper which consumes large amounts of foliage, TBW wander on the leaf surface to reach and feed on the bolls. For this reason, the pick-up assay is crucial to determine the amount of toxicant that is transferred to the TBW larvae during its activity on a surface. Studies on the spread and deposit patterns were conducted on a representative cotton leaf surface because, deposit physiology is often a!ected by the morphological characteristics of the leaf surface (Hall et al., 1995). Baker et al. (1983) observed that spread of oil droplets were restricted on relatively wax-free leaves as compared with waxy leaf surfaces. However, the reverse was found true for non-oil droplets. For oil formulations, deposits will spread over time on plant surface and may penetrate into the underlying cuticle (Crease et al., 1987). For certain pests, this penetrated insecticide may be unavailable resulting in loss of transfer e$ciency via pick-up mechanisms. Although the mechanism of action is unclear, chemical agents in a formulation are known to enhance cuticular permeability through increased spreading consequently providing better crop protection (Mcwhorter and Ouzts, 1994; Schreiber et al., 1996). Foliar uptake of toxicants are dependent on the intrinsic and extrinsic properties of the plant cuticle (Schonherr and Riederer, 1989).
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Fig. 1 shows the spread of 1 ll droplets of the three formulations. The initial deposit sizes of WG and SC were smaller than their "nal deposits. However, for EC formulation the initial deposit size was greater than its "nal deposit size. Correlation between toxicant presentation on the target surface and toxicant pick-up by an insect can be made by spread behavior studies. For example, Crease et al. (1987) from their spread behavior
Fig. 1. Spread of 1 ll droplet of WG, EC, and SC formulations on cotton.
studies demonstrated that the addition of high viscosity carrier oils increased the pick-up of cypermethrin ULV formulations. This is especially true for lepidopterous larvae which have greater contact area with the target compared with other insects (Crease et al., 1987). Our droplet spread studies indicate that the areas of the "nal deposits of all three water-dispersible formulations were not di!erent from each other. This result can be correlated to pick-up assays which showed that all three formulations exhibited similar toxicity to TBW larvae. The deposit morphology of WG, EC, and SC as observed from SEM studies are shown in Figs. 2(A), (B) and (C), respectively. From SEM photographs of the EC deposit (Fig. 2(A)), it appears that the formulation is associated with an annulus (ring-like boundary). However, with SC and WG there were similarities between deposits and it appears that the formulation within the deposit is spread out as particulates on the leaf. For WG, presence of the annulus imparts a &crater-like' appearance to the deposit. It was di$cult to detect WG deposit at smaller droplet sizes (200 lm) on the leaf surface. However, at 500 lm the deposit was visible (Fig. 2(C)). This
Fig. 2. SEM photographs of "pronil formulation deposits on cotton leaves: (A) EC formulation, 200 lm; (B) SC formulation, 200 lm; (C) WG formulation, 500 lm.
V.K. Pedibhotla et al. / Crop Protection 18 (1999) 493}499
study has shown that SEM methodology can be used to explore and understand the deposit patterns of formulations on plant surfaces. The optimum droplet size for an insecticide is based on the nature of crop foliage, type of the insect pest, and formulation being applied. As early as 1969, Himel determined that (50 lm was the optimum droplet size for insect control. But small-sized droplets ((100 lm) are prone to cause drift problems and if they are too large ('300 lm) then it might result in poor coverage of the target (Dorr and Pannell, 1992). Therefore, the spray droplet sizes for various formulations have to be optimized because a uniformly similar droplet size for all foliar applications cannot be translated into optimal biological e$ciency. Deposit weathering studies, through visualization of the deposit, make an attempt to address the retention properties of formulations to cotton after simulated rain-washing. Although the match between natural rains and the simulated rains may not be exact, there is su$cient data to treat the simulated rains as representative of natural ones (Reynolds et al., 1987a). Figs. 3(A), (B) and
497
(C), show deposit images of EC, SC, and WG formulations, respectively, after the rain tests and illustrate the physical nature of the deposit on the target surface. These SEM images suggest that the rainfall impacted the EC and WG deposits more than SC deposits. It is important to note that rain-washing tests were conducted 24 h after the application of the insecticide on cotton and these tests do not take into account the amount of AI that has been adsorbed and/or translocated into the leaf. Although "pronil is not a systemic insecticide, it translocates to some extent on the target surface (RhonePoulenc, pers. comm.). Evidently, "pronil WG and SC, owing to their increased dispersibility in water, have relatively greater movement in/on leaf in comparison with EC. Studies on deposit morphology can be utilized to explain in part for the loss of biological activity by weathering, degradation, and evaporation. Pick et al. (1984) observed that EC formulations were retained better on the leaf surface than wettable powders (WP). The EC deposits interact with the wax layer of the leaf to increase rainfastness whereas WP deposits remain particulate and
Fig. 3. SEM photographs of "pronil formulation deposits (400 lm) on cotton leaves after a 15 min rain-washing (130 mm/h): (A) EC formulation; (B) SC formulation; (C) WG formulation.
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are loosely retained on the leaf resulting in lowered insecticidal e$cacy. In another study, addition of surfactants to WP did not increase retention on the leaf (Phillips and Gillham, 1971). In contrast, Young et al. (1996) compared three formulations EC, SC, and WP and found that EC had the highest retention on cabbage leaves. However, after the addition of Agral, an adjuvant, the highest increase in retention was observed for SC. Weathering and degradation of insecticides do not always negatively a!ect biological activity. For example, Hainzl and Casida (1996) studied the photochemical reactions of "pronil and observed that the primary UV degradation product, a tri#uoromethyl pyrazole, retains high bio-activity against insects. Our studies conclude that di!erent formulations of "pronil exhibit varied deposit characteristics and information from similar studies on di!erent target surfaces can be useful to optimize physio-chemical properties of formulations to address issues such as coverage and spreading. Movement and locomotory behavior of an insect pest will also in#uence the toxicant transfer e$ciency. In#uence of the pests' feeding pattern is tightly linked to the survival of the insect. Schneider (1989) stated that &almost all alternative insect pest management tactics are more a!ected by insect movement than is the use of speci"c insecticides'. Hall et al. (1995) investigated the e!ect of feeding and walking parameters and concluded that survival of the insect depended for a major part on the speed of walking. Our results strongly suggest that an attempt to characterize formulation binding properties and insect behavior is needed to understand the uptake of insecticides by the pest. A change in the deposit morphology occurs when a transition is made from examining the toxicant on para"lm to leaves. Consequently, we are initiating e!orts to move beyond the original stage of observing the pick-up of AI from para"lm to leaves. Until a thorough understanding of the individual components of bioavailability and toxicant formulation/pest behavior interactions on target surface is acheived, risk reduction mandates to improve the e$cacy of insecticides at reduced application rates cannot be reliably addressed.
Acknowledgements Research support and salaries were provided by state and federal funds appropriated to Ohio Agricultural Research and Development Center (OARDC), The Ohio State University. We are grateful for the additional "nancial assistance provided by Rhone-Poulenc Ag. Co., RTP, NC. We thank Robert E. Whitmoyer (Head Electron Microscopy Lab, OARDC, Wooster, OH 44691) for helping us with SEM, Rebecca Thompson (LPCAT, OARDC, Wooster, OH 44691) for running the droplet generator and Drs. R.A. Downer, P. Grewal,
and C.W. Hoy for reviewing an earlier draft of the manuscript.
References Adams, A.J., Hall, F.R., 1989. In#uence of bifenthrin spray deposit quality on the mortality of Trichoplusia ni (Lepidoptera: Noctuidae) on cabbage. Crop Protection 8, 206}211. Baker, E.A., Hunt, G.M., Stevens, P.J.G., 1983. Studies of plant cuticle and spray droplet interactions: a fresh approach. Pesticide Sci. 14, 645}658. Colliot, F., Kukorowski, K.A., Hawkins, D.W., Roberts, D.A., 1992. Fipronil: a new soil and foliar broad spectrum insecticide. Brighton Crop Protection Conference, pp. 29}32. Crease, G.J., Ford, M.J., Salt, D.W., 1987. The use of high viscosity oils to enhance the insecticidal e$cacy of ULV formulations of cypermethrin. Aspects Appl. Biol. 14, 307}322. Dorr, G.J., Pannell, D.J., 1992. Economics of improved spatial distribution of herbicide for weed control in crops. Crop Protection 11, 385}391. Duck, N., Evola, S., 1997. Use of transgenes to increase host plant resistance to insects: opportunities and challenges. In: Carozzi, N., Koziel, M. (Eds.), Advances in Insect Control. The Role of Transgenic Plants. Taylor & Francis, London, pp. 1}21. Ford, M.F., Salt, D.W., 1987. Behavior of insecticide deposits and their transfer from plant to insect surfaces. In: Cottrell, H. (Ed.), Critical Reports in Applied Chemistry. Pesticides on Plant Surfaces. Wiley, London, pp. 26}81. Graham-Bryce, J.I., 1983. In: Doyle, P., Fujita, T. (Eds.), Pesticide Chemistry: Human Welfare and the Environment. Vol. 1. Synthesis and Structure Activity Relationships. Pergamon Press, Oxford. Hainzl, D., Casida, J.E., 1996. Fipronil insecticide: Novel photochemical desul"nylation with retention of neurotoxicity. Proceedings of National Academy of Sciences USA Vol. 93, 12764}12767. Hall, F.R., 1997. Spray deposits: opportunities for improved e$ciency of utilization via quality, quantity and formulation. Phytoparasitica 25, 39S}52S. Hall, F.R., Chapple, A.C., Taylor, R.A.J., Downer, R.A., 1995. Modeling the dose acquisition of Bacillus thueingegiensis: In#uence of feeding pattern on survival. In: Hall, F.R., Barry, J.W. (Eds.), Biorational Pest Control Agents: Formulations and Discovery. American Chemical Society, Washington, DC, pp. 68}79. Kanga, L.H.B., Plapp Jr., F.W., 1995. Target site insensitivity as the mechanism of resistance to organophosphates, carbamates, and cyclodiene insecticides in tobacco budworm adults. J. Econ. Entomol. 88, 150}157. Matteson, P.C., 1995. The `50% pesticide cutsa in Europe: a glimpse of our future?. Am. Entomol. 11, 210}219. McCa!ery, A.R., Little, E.J., Gladwell, R.T., Holloway, G.J., Walker, C.H., 1989. Detection and mechanisms of resistance in Heliothis virescens, Proceedings, Beltwide Cotton Conference. National Cotton Council, Memphis, TN, pp. 207}211. Mcwhorter, C.G., Ouzts, C., 1994. Leaf surface morphology of Erythroxylum sp. and droplet spread. Weed Sci. 42, 18}26. Oreke, E.C., Dehne, H.W., Schonbeck, F., Weber, A. (Eds.), 1994. Crop Production and Crop Protection: Estimated Crop Losses in Major Food and Cash crops. Elsevier, Amsterdam. Phillips, F.T., Gillham, E.M., 1971. Persistence of rain-washing of DDT wettable powders. Pesticide Sci. 2, 97}100. Pick, F.E., van Dyke, L.P., deBeer, P.R., 1984. The e!ect of simulated rain on deposits of some cotton pesticides. Pesticide Sci. 15, 616}623. Reynolds, K.M., Bugler, M.A., Madden, L.V., Ellis, M.A., 1987a. New methods using simulated rain to study the dispersal of plant pathogens. Phytopathology 77, 921}929.
V.K. Pedibhotla et al. / Crop Protection 18 (1999) 493}499 Reynolds, K.M., Madden, L.V., Reichard, D.L., Ellis, M.A., 1987b. Methods of study of raindrop impact on plant surfaces with application to predict innoclum dispersal by rain. Phytopathology 77, 226}232. SAS Institute., 1985. SAS User's Guide: Statistics, 5th Edition. SAS Institute, Cary, NC. Sato, K., 1992. Partitioning of insecticide between insect surface wax and water as a factor of toxicity. Appl. Entomol. Zool. 27, 287}289. Schneider, J.C., 1989. Role of movement in evaluation of area-wide insect pest management tactics. Environ. Entomol. 18, 869}874. Schonherr, J., Riederer, M., 1989. Foliar penetration and accumulation of organic chemicals in plant cuticles. Rev. Environ. Contam. Toxicol. 108, 1}70.
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Schreiber, L., Riederer, M., Schorn, K., 1996. Mobilities of organic compounds in reconstituted cuticular wax of barley leaves: e!ects of momodisperse alcohol ethoxylates on di!usion of pentachlorophenol and tetracosanoic acid. Pesticide Sci. 48, 117}124. Sparks, T.C., 1981. Development of insecticide resistance in Heliothis virescens. North Am. Bull. Entomol. Soc. Am. 27, 186}192. Welling, W., 1977. Interaction of insecticide with insects. Ann. Rev. Entomol. 22, 53}78. Young, R.D.F., Thacker, J.R.M., Curtis, D.J., 1996. The e!ects of three adjuvants on the retention of insecticide formulations by cabbage leaves. J. Environ. Sci. Health B 31, 165}178.
Deposit characteristics and toxicity of "pronil formulations for tobacco budworm (Lepidoptera: Noctuidae) control on cotton Venkat K. Pedibhotla *, Franklin R. Hall, Je! Holmsen The Scotts Company, 14111 Scottslawn Rd, Marysville, OH 43041, USA Laboratory for Pest Control Application Technology (LPCAT), Ohio State University, 1680 Madison Ave., Wooster, OH 44691, USA Rhone Poulenc Ag. Company, Research Triangle Park, NC 27709, USA Received 22 December 1998; accepted 22 June 1999
Abstract Three water-dispersible "pronil formulations have been tested for toxicity against second instar tobacco budworm, Heliothis virescens (F.). Topically, EC is the most potent of the three assays followed by WG and SC. However, pick-up assays on para"lm did not show signi"cant di!erences in toxicity between formulations. Droplet spread and deposit morphology of the formulations were studied on cotton. Although the initial droplet spread was observed to be highest for EC, the "nal dried deposits of formulations were not signi"cantly di!erent from each other. The deposit patterns of all three formulations on cotton are di!erent. For EC, the formulation/AI is associated with an annulus (outer ring) whereas the WG and SC are particulate in appearance and dispersed across the deposit. From the rain-washing studies on cotton, SEM photographs indicate that SC has greater retention than EC or WG. 1999 Elsevier Science Ltd. All rights reserved. Keywords: Heliothis virescens; Deposit morphology; Droplet spread; Deposit weathering
1. Introduction Among the major pests attacking cotton, heliothines account for about 91% of the yield loss when no control measures are undertaken (Duck and Evola, 1997). In the United States alone, the total production of cotton was worth $ 25.7 billion against an annual loss of 15.4% due to pests (Oreke et al., 1994). Through the proli"c use of insecticides, heliothines have developed resistance to many classes of insecticides including, cyclodienes, organophosphates, carbamates, and pyrethroids (Sparks, 1981; McCa!ery et al., 1989; Kanga and Plapp, 1995). Despite the growing resistance problem worldwide, synthetic organic chemicals continue to dominate the market for the control of pests. Currently, among the new crop of insecticides developed with unique modes of action, "pronil, a phenylpyrazole, has potential for the management of lepidopterous pests of cotton. Fipronil is primarily
* Corresponding author. Tel.: #1-937-645-2669; fax: #1-937-6447104.
a stomach poison with some contact activity that can be e!ectively used against both chewing and piercing}sucking pests (Colliot et al., 1992). Fipronil exerts its action through blocking the c-aminobutyric acid (GABA)-gated chloride channel in insects (Hainzl and Casida, 1996). Recent US EPA policies (IPM, Risk-reduction, etc.) aim for a reduction in pesticide usage (Hall, 1997). Northern European countries have passed a legislation that a 50% reduction in pesticide use should be mandated by the year 2000 (Matteson, 1995). In general, application of pesticides is an ine$cient process leading to signi"cant losses in the environment and only low amounts of agrochemical reaching the intending target (Adams and Hall, 1989; Graham-Bryce, 1983). In order to enhance the application e$ciency of insecticides, various parameters of the dose-transfer process have to be understood. The dose-transfer process can be broadly classi"ed into spray application and bioavailability. Bioavailability is a biological phenomenon generally used to describe the availability of an applied toxicant on a plant canopy or crop foliage and its transfer to a target pest resulting in a biological e!ect. Formulation/AI properties and their
0261-2194/99/$ - see front matter 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 2 6 1 - 2 1 9 4 ( 9 9 ) 0 0 0 5 0 - 2
494
V.K. Pedibhotla et al. / Crop Protection 18 (1999) 493}499
interactions, surface morphology of the leaf, and pest behavior are some of the features that in#uence bioavailability, consequently a!ecting the pick up toxicant from the target surface (Ford and Salt, 1987). The objectives of this study were two-fold. First, to evaluate and compare the toxicity of three "pronil formulations, an emulsi"able concentrate (EC), a suspension concentrate (SC), and a wettable granule (WG), by pick-up and topical assays against tobacco budworm (TBW), Heliothis virescens. Second, to conduct various studies on physical characteristics of the formulations including droplet spread, deposit morphology, and rainwashing for a better understanding of the bioavailability mechanisms on cotton.
2. Materials and methods 2.1. Insects Tobacco budworm eggs were shipped bimonthly to the Laboratory for Pest Control Application Technology (LPCAT) by Rhone Poulenc Ag. Company, Research Triangle Park, NC. TBW were obtained from a continuously maintained culture, with no known resistance to any insecticides. All experiments were performed using II instar larvae. Larvae were reared on arti"cial diet and were contained in a constant-environment incubator (253C, 70% RH, a photoperiod of 14 : 10 [L : D] h). Arti"cial diet for TBW was prepared and shipped to LPCAT by Rhone-Poulenc in powder form. The diet was stored in freezer at 43C and was periodically used for experimentation. 2.2. Chemicals Three water-dispersible "pronil formulations 800WG (wettable granules), 200SC (suspension concentrate), and 300EC (emulsifyable concentrate), provided by Rhone Poulenc Ag. Company, Research Triangle Park, NC, were evaluated in all experiments. Distilled water was used to prepare appropriate concentrations of assay solutions for all formulations. 2.3. Topical application This test estimates the lethal dose of each formulation following direct application to the dorsal surface of the larva. To each larva, 0.25 ll of solution was applied using a micro-applicator (Isco., Inc., Lincoln, NE). Ortho X-77 at 0.1% v/v was added to formulations to aid wetting. After treatment, each larva was placed in a plastic diet cup along with arti"cial diet (Lepidoptera diet provided by Rhone-Poulenc) and stored in an incubator (273C incubator, 70% RH, a photoperiod of 14 : 10 [L : D] h). To each control larva 0.25 ll of acetone was applied.
Mortality was assessed after 24 and 48 h. Five concentrations were used and 20 larvae were tested per treatment which was replicated three times. 2.4. Pick-up assay on paraxlm This experiment is useful to estimate the lethal dose of the formulation from toxin pick-up alone. Wet "lter paper (9 cm diameter, grade 1, Whatman, Hillsboro, OR) was placed under para"lm disks, in the bottom of a Petri dish (9 cm diameter). Droplets were applied to the para"lm using an Isco (model M) micro-applicator. Each droplet was 0.5 ll and a total of 24 droplets were placed in a circular pattern with a 2 cm diameter untreated region in the center of the dish and a 5 cm untreated ring along the outside margin of the dish. Furthermore, droplets were placed such that droplets in one row were approximately centered in the gaps between droplets in the other row. Five concentrations, 7.2, 3.6, 1.8, 0.9, and 0.45 g a.i./l, of each formulation were used. Arti"cial diet prepared in the laboratory was placed in the center and three larvae per Petri dish were released in the outside untreated region after the deposits dried. Mortality was assessed after 24 and 48 h. For each concentration, "ve Petri dishes were used and each treatment was replicated three times. 2.5. Spread behavior Rate of spreading measurements were performed on cotton leaves. Cotton (Gossypium hirsutum L.) was seeded in plastic pots (12 cm diameter, 4 seeds per pot) containing a commercial growing medium. Upon emergence plants were selected for uniformity and thinned to two plants per pot. The temperature in the green house was maintained at 28$33C day during the day and 24$33C at night. All formulations were applied at 5 g/l and all assay solutions included Nigrosin (0.4%, w/v), a standard dye, to aid detection of the deposit boundary. One microliter droplets were placed on cotton leaves and the area of the droplets were measured upon contact with the leaf surface and after 0.5, 1, 5, 10, and 15 min. Spread measurements were conducted using an image analysis system consisting of a 18}108 mm 6x zoom lens (Fujinon Inc. Wayne, NJ) "tted to a Kodak megaplus camera (CKOD839-4900, Eastman Kodak Co., San Diego CA). All leaf samples were backlit (Fostec system, Fostec Inc. Auburn NY 13021) and the data was captured using a Data Raptor-PCI capture board (Bit#ow Inc. Woburn, MA) installed in a pentium computer. 2.6. Deposit morphology Fipronil formulations at 5 g a.i./l were applied to cotton to examine the droplet structure. Various droplet sizes (200 & 500 lm) were applied to leaves using a
V.K. Pedibhotla et al. / Crop Protection 18 (1999) 493}499
droplet generator. The droplet generator produces uniformly sized droplets with a pulse generator which is activated by a piezo-electric crystal (Reynolds et al., 1987b). The size of droplets was con"rmed by capturing them in corning #uid and measuring under a microscope "tted with an eyepiece graticule. The dried deposit was marked and the leaf area excised for imaging. Scanning electron microscopy (SEM) was used to examine the resulting deposit after drying. 2.7. Deposit weathering A rain simulator was used to determine the in#uence of rain washing on retention of "pronil on cotton (Reynolds et al., 1987a). An upward-pointing Tee-Jet 8002 nozzle (Tee-Jet Spraying Systems, Wheaton, IL) "tted to a rotating bar was used to produce a spray pattern of uniform rain. Distance of the drop fall was 4 m with a water pressure of 25 psi at the nozzle ori"ce. The rainfall amount over a period of time was measured with a rain gauge. The mono-size droplet generator was used to apply droplets with a diameter of 400 lm on the abaxial surfaces of cotton leaves (potted plants). After 24 h, rain washing experiments were carried out on treated plants which were placed under the rain simulator for 15 min in a 130 mm/h rain. Upon drying, leaf portions were excised and viewed under SEM. Each experiment was replicated three times and had control plants which were treated but not rain washed. 2.8. Statistical analysis Probit analysis were performed using PROC PROBIT (SAS Institute, 1985) to estimate the lethal dose (LD ) for topical and lethal concentration (LC ) for pick-up assays. LD and LC s were expressed as micrograms a.i. per larva. LD s and LC s were considered signi" cantly di!erent if their 95% FL did not overlap, which is p(0.01.
3. Results and discussion By comparing the LD s and LC s from topical and pick-up assays, we observed variations in toxicity to the three formulations, WG, EC, and SC (Table 1). In the topical assay, EC (0.07 lg AI) was the most toxic followed by WG (0.54 lg AI) and SC (1.75 lg AI). From pick-up assays, we noted that the LC of all three formulations, WG (1.7 lg AI), EC (1.4 lg AI), and SC (1.2 lg AI) were within the same range. Interestingly, the high toxicity of EC in the topical assay was not re#ected in pick-up assay. This could be attributed to the solventAI interactions with the insect cuticle in#uencing the penetration and binding properties of the EC formulation. For pick-up assays, para"lm has been used as it is
495
Table 1 Topical and pick-up toxicity of "pronil formulations to II instar H. virescens after 48 h Assay
Formulation
LD (lg a.i.)
95% FL
Slope$SE
Topical
WG SC EC
0.54 1.75 0.07
0.51}0.56 1.33}1.90 0.06}0.09
1.69$0.3 1.91$0.35 1.24$0.27
Pick-up
WG SC EC
2.0 1.4 1.7
1.6}2.2 1.2}1.7 1.4}1.9
2.4 $0.6 1.25$0.28 1.1 $0.24
an conventional substitute for leaves. Additionally, this assay was designed speci"cally to determine the pick-up toxicity only by contact and not through feeding behavior. This is accomplished as larvae move through toxin treated deposits for reaching the diet placed in the center of the petri dish. Work on adhesion and uptake of contact insecticides to the insect cuticle has been conducted by Welling (1977). It was observed that the toxicity of such chemicals depend largely on the partition coe$cients between the surface waxes of insect cuticle and water, for uptake and penetration of toxicant. Sato (1992) showed that parathion is 20 times more soluble in the cuticular wax of Spodoptera litura and Mamestra brassicae than carbaryl. Although "pronil is primarily a stomach poison it also exhibits contact activity. Unlike certain lepidopterous larvae, such as the cabagge looper which consumes large amounts of foliage, TBW wander on the leaf surface to reach and feed on the bolls. For this reason, the pick-up assay is crucial to determine the amount of toxicant that is transferred to the TBW larvae during its activity on a surface. Studies on the spread and deposit patterns were conducted on a representative cotton leaf surface because, deposit physiology is often a!ected by the morphological characteristics of the leaf surface (Hall et al., 1995). Baker et al. (1983) observed that spread of oil droplets were restricted on relatively wax-free leaves as compared with waxy leaf surfaces. However, the reverse was found true for non-oil droplets. For oil formulations, deposits will spread over time on plant surface and may penetrate into the underlying cuticle (Crease et al., 1987). For certain pests, this penetrated insecticide may be unavailable resulting in loss of transfer e$ciency via pick-up mechanisms. Although the mechanism of action is unclear, chemical agents in a formulation are known to enhance cuticular permeability through increased spreading consequently providing better crop protection (Mcwhorter and Ouzts, 1994; Schreiber et al., 1996). Foliar uptake of toxicants are dependent on the intrinsic and extrinsic properties of the plant cuticle (Schonherr and Riederer, 1989).
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Fig. 1 shows the spread of 1 ll droplets of the three formulations. The initial deposit sizes of WG and SC were smaller than their "nal deposits. However, for EC formulation the initial deposit size was greater than its "nal deposit size. Correlation between toxicant presentation on the target surface and toxicant pick-up by an insect can be made by spread behavior studies. For example, Crease et al. (1987) from their spread behavior
Fig. 1. Spread of 1 ll droplet of WG, EC, and SC formulations on cotton.
studies demonstrated that the addition of high viscosity carrier oils increased the pick-up of cypermethrin ULV formulations. This is especially true for lepidopterous larvae which have greater contact area with the target compared with other insects (Crease et al., 1987). Our droplet spread studies indicate that the areas of the "nal deposits of all three water-dispersible formulations were not di!erent from each other. This result can be correlated to pick-up assays which showed that all three formulations exhibited similar toxicity to TBW larvae. The deposit morphology of WG, EC, and SC as observed from SEM studies are shown in Figs. 2(A), (B) and (C), respectively. From SEM photographs of the EC deposit (Fig. 2(A)), it appears that the formulation is associated with an annulus (ring-like boundary). However, with SC and WG there were similarities between deposits and it appears that the formulation within the deposit is spread out as particulates on the leaf. For WG, presence of the annulus imparts a &crater-like' appearance to the deposit. It was di$cult to detect WG deposit at smaller droplet sizes (200 lm) on the leaf surface. However, at 500 lm the deposit was visible (Fig. 2(C)). This
Fig. 2. SEM photographs of "pronil formulation deposits on cotton leaves: (A) EC formulation, 200 lm; (B) SC formulation, 200 lm; (C) WG formulation, 500 lm.
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study has shown that SEM methodology can be used to explore and understand the deposit patterns of formulations on plant surfaces. The optimum droplet size for an insecticide is based on the nature of crop foliage, type of the insect pest, and formulation being applied. As early as 1969, Himel determined that (50 lm was the optimum droplet size for insect control. But small-sized droplets ((100 lm) are prone to cause drift problems and if they are too large ('300 lm) then it might result in poor coverage of the target (Dorr and Pannell, 1992). Therefore, the spray droplet sizes for various formulations have to be optimized because a uniformly similar droplet size for all foliar applications cannot be translated into optimal biological e$ciency. Deposit weathering studies, through visualization of the deposit, make an attempt to address the retention properties of formulations to cotton after simulated rain-washing. Although the match between natural rains and the simulated rains may not be exact, there is su$cient data to treat the simulated rains as representative of natural ones (Reynolds et al., 1987a). Figs. 3(A), (B) and
497
(C), show deposit images of EC, SC, and WG formulations, respectively, after the rain tests and illustrate the physical nature of the deposit on the target surface. These SEM images suggest that the rainfall impacted the EC and WG deposits more than SC deposits. It is important to note that rain-washing tests were conducted 24 h after the application of the insecticide on cotton and these tests do not take into account the amount of AI that has been adsorbed and/or translocated into the leaf. Although "pronil is not a systemic insecticide, it translocates to some extent on the target surface (RhonePoulenc, pers. comm.). Evidently, "pronil WG and SC, owing to their increased dispersibility in water, have relatively greater movement in/on leaf in comparison with EC. Studies on deposit morphology can be utilized to explain in part for the loss of biological activity by weathering, degradation, and evaporation. Pick et al. (1984) observed that EC formulations were retained better on the leaf surface than wettable powders (WP). The EC deposits interact with the wax layer of the leaf to increase rainfastness whereas WP deposits remain particulate and
Fig. 3. SEM photographs of "pronil formulation deposits (400 lm) on cotton leaves after a 15 min rain-washing (130 mm/h): (A) EC formulation; (B) SC formulation; (C) WG formulation.
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are loosely retained on the leaf resulting in lowered insecticidal e$cacy. In another study, addition of surfactants to WP did not increase retention on the leaf (Phillips and Gillham, 1971). In contrast, Young et al. (1996) compared three formulations EC, SC, and WP and found that EC had the highest retention on cabbage leaves. However, after the addition of Agral, an adjuvant, the highest increase in retention was observed for SC. Weathering and degradation of insecticides do not always negatively a!ect biological activity. For example, Hainzl and Casida (1996) studied the photochemical reactions of "pronil and observed that the primary UV degradation product, a tri#uoromethyl pyrazole, retains high bio-activity against insects. Our studies conclude that di!erent formulations of "pronil exhibit varied deposit characteristics and information from similar studies on di!erent target surfaces can be useful to optimize physio-chemical properties of formulations to address issues such as coverage and spreading. Movement and locomotory behavior of an insect pest will also in#uence the toxicant transfer e$ciency. In#uence of the pests' feeding pattern is tightly linked to the survival of the insect. Schneider (1989) stated that &almost all alternative insect pest management tactics are more a!ected by insect movement than is the use of speci"c insecticides'. Hall et al. (1995) investigated the e!ect of feeding and walking parameters and concluded that survival of the insect depended for a major part on the speed of walking. Our results strongly suggest that an attempt to characterize formulation binding properties and insect behavior is needed to understand the uptake of insecticides by the pest. A change in the deposit morphology occurs when a transition is made from examining the toxicant on para"lm to leaves. Consequently, we are initiating e!orts to move beyond the original stage of observing the pick-up of AI from para"lm to leaves. Until a thorough understanding of the individual components of bioavailability and toxicant formulation/pest behavior interactions on target surface is acheived, risk reduction mandates to improve the e$cacy of insecticides at reduced application rates cannot be reliably addressed.
Acknowledgements Research support and salaries were provided by state and federal funds appropriated to Ohio Agricultural Research and Development Center (OARDC), The Ohio State University. We are grateful for the additional "nancial assistance provided by Rhone-Poulenc Ag. Co., RTP, NC. We thank Robert E. Whitmoyer (Head Electron Microscopy Lab, OARDC, Wooster, OH 44691) for helping us with SEM, Rebecca Thompson (LPCAT, OARDC, Wooster, OH 44691) for running the droplet generator and Drs. R.A. Downer, P. Grewal,
and C.W. Hoy for reviewing an earlier draft of the manuscript.
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