1547 Performance of Termidor® HE High-Efficiency Termiticide Co-pack with Termidor® HE Technology Against Eastern Subterranean Termites Reticulitermes flavipes and Formosan Subterranean Termite Coptotermes formosanus in Laboratory Trials and Field Applications by T. Chris Keefer1,2, Robert T. Puckett1, & Roger E. Gold1 ABSTRACT Two laboratory and one field study were performed utilizing fipronil (C 12 H 4 Cl l2 F 6 N 4 OS), in the form of Termidor® HE Co-pack with Termidor® HE Technolog y. Laboratory studies included glass tube bioassays and col- lateral transfer effect trials. Glass tube bioassay results indicated that this formulation of Termidor® HE Co-pack was efficacious against Reticulitermes flavipes and Coptotermes formosanus caused 100% mortality of both species by 72 h post-exposure. Collateral transfer effect trials also showed efficacy of Termidor® HE Co-Pack with Termidor® HE Technolog y against both R. flavipes and C. formosanus by causing 100% mortality by 96 h. Termidor® HE Co-pack with Termidor® HE Technolog y was also applied to structures as a post-construction treatment for remediation of subterranean termite infestations. In field trials, 12 termite structures were treated with Termidor® HE Co-pack with Termidor® HE Technolog y (hereafter referred to as Ter- midor® HE) and no subterranean termites were detected through 36 months post-treatment. Key Words: Termidor® HE Co-pack, Reticulitermes flavipes, Coptotermes formosanus INTRODUCTION Fipronil (C 12 H 4 Cl 2 F 6 N 4 OS) is a phenylpyrazole (Tingle et al. 2003) insec- 1Department of Entomolog y, Texas A&M University, 2143 TAMU, College Station, TX 77843- 2143. 1*Corresponding Author E-mail: tckeef@tamu.edu 1548 Sociobiolog y Vol. 59, No. 4, 2012 ticide first discovered in 1987 by Rhone-Poluenc Agro (Tomlin 2000). It was first introduced and registered in the United States in 1996 (Bobe et al. 1998, Ware 2000) for use against piercing, sucking chewing insect pests (Bostian et al.1996). The mode of action for fipronil is interference with the passage of chloride ions through the gamma-aminobutyric acid-regulated chloride channel which disrupts the arthropods central nervous system (Gant et al. 1998). Fipronil has a wide range of uses on many different pests worldwide (Yanese and Andoh 1998). This compound is considered a broad-spectrum insecticide and has been demonstrated to be effacacious against a variety of insects including termites in urban environments (Tomlin 2000). Fipronil, as a liquid termiticide is sold asTermidor® by BASF Corpora- tion (Research Triangle Park, NC). It is a non-repellent termiticide that can be applied as a soil barrier to protect structures from subterranean termite invasion. Subterranean termites cause damage to structures throughout the United States, and cost estimates to control and repair damage caused by these insects are as high as $11 billion annually (Su and Scheffrahn 1998, Su 2002). Compounds that are non-repellent, have a delayed reaction on subterranean termites as compared to repellent products. It has been reported that fipronil can be transferred through the colony from individual to individual by tro- phallaxis, grooming, and contact with contaminated soil (Kard 2003). METHODS AND MATERIALS Laboratory Study I The objective of this work was to evaluate the efficacy of fipronil, in the form of Termidor® HE 0.06% AI and Termidor® HE 0.12% AI in glass tube bioassays against R. flavipes and C. formosanus subterranean termites. Are- nas consisted of glass tubes measuring 15 X 1.5 cm which were capped on both ends with aluminum foil (Fig. 1) (Gold et al. 1994). The following treatments were utilized in this study; Termidor® HE (0.06%) and Termidor® HE(0.12%), and untreated controls (water only). Treatments to soil were made according to the manufacturer’s label, and untreated controls were identically ‘treated’, but with water only. Equal volumes of Termidor® HE and the HE technolog y additive were thoroughly mixed and diluted at a rate equivalent to 5.75 mL (0.06%) solution and 11.75 mL (0.12%) solution in 1549 Keefer, C. et al. — Performance of Termidor® HE High-Efficiency Termiticide Fig. 1. Schematic showing components of glass-tube arenas used in FST feeding cessation experiment (.Waare (2000). 946 mL of water. Soil was treated with the equivalent rate of 4 L per 3 m per 0.3 m of depth. Twenty worker termites and two soldiers of either R. flavipes or C. formosanus were added to each arena after assembly. Soldier termites were added to stimulate colony dynamics and stimulate termite acticity. Post- treatment obser vations were made daily for 7 d. Four replications of each treatment and untreated controls were constructed for this trial. Data collected included distance tunneled and mortal- ity, and these data were analyzed via Analysis of Variance (ANOVA) at P < 0.05 and means separated using Tukey’s HSD (Honest Significant Difference) test at P < 0.05 (SPSS for Windows, V. 18.0). Laboratory Study II The objective of this work was to determine collateral transfer effects, if any, of Termidor® HE 0.125% AI treated soil utilizing varying ratios (donor:recipient) of R. flavipes and C. formosanus subterranean termite species through time. Recently collected termites were utilized in these tri- als and they were allowed to adjust to the laboratory conditions for at least 48 hrs before beginning study. Worker termites were placed in labeled Petri dishes that corresponded to a final untreated arena to allow observation of collateral effects. These worker termites of both species were marked using methods similar to Forschler 1994 with Rust-O-Leum® (Vernon Hills, IL) orange fluorescent paint. Marked termites were allowed to adjust to laboratory environment for 48 h. Treatments to the soil were made with the following products; Termidor® SC (BAS 350 95 I) Lot No. 1219502FI and Termidor® HE technolog y additive (BAS 270 00 S) Lot No. 502012. These products were used to create the Termidor HE Co-pack and were mixed just prior to study initiation. Treatments to soil were made according to the manufacturer’s 1550 Sociobiolog y Vol. 59, No. 4, 2012 label, and were identical to treatments in the Laboratory Study I (described above). Untreated controls were ‘treated’ with water only. Both the treated and the untreated control soils were allowed to dry for 24 h prior to exposing of termites to the soils. Treatment arenas consisted of Petri dishes measuring 9.0 x 2.5 cm. Approximately 34 g of treated soil was placed in each Petri dish. Worker termites of either R. flavipes or C. formosanus were added to each arena after assembly, and were allowed to contact treated soil or untreated control sand for 30 min. After the contact period elapsed, these termites (donors), were placed in an untreated, clean Petri dish (9.0 x 2.5 cm) with moistened #4 Whatman® (Maidstone, England) filter paper, and unexposed termites (recipients). Four replications of each of five donor:recipient ratios and untreated controls were constructed for this trial. The following ratios of donor:recipient termite cohorts were utilized in these trials 20:0, 15:5, 10:10, 5:15, 1:19, 0:20 (untreated marked control), and 0:20 (untreated unmarked control). Two soldier termites of corresponding species were placed in all arenas to simulate colony dynamics and stimulate worker termite activity. Observations of worker termite mortality (donors and recipients) were made at 1, 4 and 24 hours after donor:recipient mixing, then daily until 100% mortality was reached in each treatment. Data were analyzed statisti- cally via Analysis of Variance (ANOVA) at P < 0.05 and means separated using Tukey’s HSD (Honest Significant Difference) test at P < 0.05 (SPSS for Windows, V. 18.0). Field Study The objective of this field study was to determine effectiveness, if any, of Termidor® HE against two species of subterranean termites when applied as a post-construction treatment to infested structures. Eleven structures infested with R. flavipes and one infested with C. formosanus were utilized in this field study. All structures were located in the Houston/Galveston, TX area. Soldier termites were collected from all 12 structures, identified with termite identification keys (Scheffrahn and Hope 1996), and stored in 100% ethanol as voucher specimens. Structure owners were interviewed to verify that none of the 12 structures had received a subterranean termite treatment within the past 12 months prior to the Termidor® HE treatments. Four of the structures were pier and beam construction, which included one 1551 Keefer, C. et al. — Performance of Termidor® HE High-Efficiency Termiticide structure infested with C. formosanus, the other eight structures consisted of monolithic slab construction. All 12 structures had at least one active sub- terranean termite mud tube leading from the soil into the structure that was associated with an exterior wall. A diagram was made of each structure prior to treatment to include all known points of subterranean termite infestation. All subterranean termite mud tubes were documented and marked relative to a permanent benchmark (ie. corner of the foundation). Under the supervision of personnel from the Center for Urban and Struc- tural Entomolog y and BASF, all Termidor® HE applications were made by a licensed pest management professional. All termiticide applications were made following the Termidor® SC Exterior Perimeter/Localized Interior directions for use according to the label with the following exceptions: all exterior drilling was done on 46 cm centers and all trenches were 5 X 10 cm. Volume and concentration of finished dilution applied varied according to the treatment specifications described below. At each of the structures, one half of the desired volume of water was added to the tank and then the appropri- ate volume of Termidor® HE was introduced to the tank, and the remaining volume of water was then added to ensure thorough mixing of the dilution. In setting up for the study, the linear length for each structure was measured prior to the treatment to ensure the proper volume of finished dilution was applied. Six structures each received one of the following treatments; 1. Termidor® HE 0.06% AI applied at 7.5 L of finished dilution/3 linear m /0.30 m of depth. 2. Termidor® HE 0.12% AI applied at 7.5 L of finished dilution/3 linear m /0.30 m of depth. There were no interior applications made at any of the 12 structures. A Great Plains Industry Inc. 01N Series (Wichita, KS) digital flow meter was utilized during this study to measure volumes of termiticide applied at each structure. All structures were treated between August 10 and August 13, 2009. Post-treatment inspections were made on or about 1, 3, 6, 12, 24, and 36 months. Post-treatment inspections included visual assessment as well as the use of mechanical tools such as a Termatrac®, borescope, and/or infrared camera. 1552 Sociobiolog y Vol. 59, No. 4, 2012 RESULTS Laboratory Study I Termidor® HE 0.06% and Termidor HE® 0.12% both caused 100% mortal- ity against both species of termites by 72 h post-treatment (Table 1). Mean mortality associated with the untreated controls was significantly less than (P <0.01) from all three treatments at the end of the trials (Figs. 2 and 3), and was < 10% in both species. In the Termidor® HE 0.06% treatment C. formosanus tunneled a mean distance of 10.0±7.6 mm and R. flavipes tunneled 7.8±2.2 mm (Table 1 and Figs. 4 and 5). This tunneling distances in the Termidor® HE 0.12% AI treat- ment were C. formosanus 16.0±3.9 mm for C. formosanus and 8.3±4.6 mm Fig. 2. Mean percent mortality of Coptotermes formosanus through time when exposed to soils treated with Termidor® HE. 1553 Keefer, C. et al. — Performance of Termidor® HE High-Efficiency Termiticide R. flavipes (Table 1 and Figs. 4 and 5). Both termite species in the untreated control groups tunneled the maximum distance of 50 mm, and there were significant differences (P <0.05) in the distance tunneled by both species of termites in the treatments compared to the untreated controls at the end of the trial. Laboratory Study II Coptotermes formosanus- At the 1 h observation period for C. formosanus, the 10:10 donor:recipient cohort was the only one that demonstrated mortality, but it was not significantly different from the other treatments or untreated controls (Table 2 and Figs. 6 and 7). There were significant differences (P <0.05) in total (donors:recipients) mortality beginning at the 4 h observa- Fig. 3. Mean percent mortality of Reticulitermes flavipes through time when exposed to soils treated with Termidor® HE. 1554 Sociobiolog y Vol. 59, No. 4, 2012 tion period, the 10:10 cohort showed a significant difference (P<0.01) from the rest of the treatments and the untreated controls (Table 2 and Figs. 6 and 7). Starting at the 72 h observation, all of the effects of the treatments were significantly different (P <0.05) from the untreated controls (Table 2 and Figs. 6 and 7). At the 1 and 4 h observation periods, the only donor populations with any mortality were in the 10:10 cohort (Fig. 6). At the 72 h observation, all donor populations in the treatments had 100% mortality and the untreated controls had less than 10% mortality (Fig. 6). At the 1 h observation, there was no mortality in the recipient populations in the treatments (Fig. 7). At the 96 h observation, all recipient populations in the treatments had 100% mortality and were significantly different (P <0.05) from the untreated controls, which had less than 10% mortality throughout the study (Fig. 7). Reticulitermes flavipes At the 1h observation period, there were no significant differences in mortality between the treatments and the untreated controls (Table 3). At the 4 h observation period the 20:0 cohort showed the greatest mortality followed by the 15:5 and then the 10:10 cohorts, respectively (Table 3 and Figs. 8 and 9). These three cohorts were significantly different (P <0.01) Table 1. Mean percent mortality and distance tunneled by Coptotermes formosanus and Reticulitermes flavipes in soils treated with Termidor® HE Co-pack (two concentrations). Treatment % Mortality Distance Tunneled (mm)*** C . f o r m o - sanus* R. flavipes** C. formosanus R. flavipes Termidor HE 0.06% 100.0 a 100.0 a 10.0 b 7.8 b Termidor HE 0.12% 100.0 a 100.0 a 16.0 b 8.3 b Untreated Control 0.0 b 5.0 b 50.0 a 50.0 a P-values <0.01 <0.01 <0.01 <0.01 F-stats 1521.00 1574.33 101.79 197.30 df 15 15 15 15 *All treatments caused 100% mortality in the C. formosanus by 120 h post-treatment. **All treatments caused 100% mortality in the R. flavipes by 96 h post-treatment. ***Maximum distance tunneled could not exceed 50 mm. Means followed by the same letter in the same column were not significantly different (P=0.05) as per Tukey’s Honest Significant Difference (HSD). 1555 Keefer, C. et al. — Performance of Termidor® HE High-Efficiency Termiticide from all of the other cohorts, and the untreated controls. By the 48 h ob- servation, all of the treatments were significantly different (P <0.05) from the untreated controls (Table 3 and Figs. 8 and 9). At the 1 h observation period, there was no mortality in the donor populations in any of the treat- ments (Fig.8). At the 48 h observation all of the donor populations in all the treatments showed 100% mortality, and were significantly different (P <0.01) from the untreated controls (Fig. 8). At the 1 h observation period there was no mortality in the recipient population in any of the treatments (Fig. 9). At the 48 h observation period the recipient populations in the 15:5 cohort was 100%, and at the 72 h observation period mean mortality of all recipient populations in the treatments was 100%. At the 72 h observation, all of the recipient populations were significantly different (P <0.01) from Fig. 4. Mean distance tunneled (mm) by Coptotermes formosanus in a glass tube bioassay through time in soils treated with Termidor® HE (maximum distance tunneled could not exceed 50 mm). 1556 Sociobiolog y Vol. 59, No. 4, 2012 the untreated controls, which had less than 10% mortality throughout the study (Fig. 9). Field Study The mean perimeter length of structures in this test was 57.8±18.6 m. The mean number of pre-trial termite mud tubes per structure was 2.3±8. The mean volume of finished solution applied to each structure was 162.7±52.3 L. There was no detection of subterranean termite activity on any of the 12 structures at any time post-treatment through 36 months. DISCUSSION Exposure of R. flavipes and C. formosanus to Termidor® HE treated soils Fig. 5. Mean distance tunneled (mm) by Reticulitermes flavipes in a glass tube bioassay through time in soils treated with Termidor® HE (maximum distance tunneled could not exceed 50 mm). 1557 Keefer, C. et al. — Performance of Termidor® HE High-Efficiency Termiticide resulted in significantly greater mortality than untreated soils. The distance tunneled by both termite species was also statistically less in treated soils than in untreated control soils. The results show that Termidor® HE was efficacious against R. flavipes and C. formosanus. Termidor® HE 0.06% and 0.12% caused 100% mortality to both R. flavi- pes and in C. formosanus at 72 hrs post-treatment, respectively. Coptotermes formosanus had a slower response to the treatments than R. flavipes at 24 and 48 hrs. The treatments caused 100% mortality, while the untreated controls caused less than 10% mortality for the duration of the study. There was a positive correlation between increased donor numbers and recipient mortality, which provided evidence that there was transfer of ac- tive ingredient among nestmates. This was evident based on the fact that the treatments caused 100% mortality of donor and recipient termites, while the untreated controls caused less than 10% mortality (P <0.05) for the duration of the study. Whether the transfer in this study was by grooming, trophallaxis, movement of contaminated sand, or simply contact, could not be determined Table 2. Total mean percent mortality (both donors and recipients combined) of different cohorts of donor:recipient Coptotermes formosanus when donors were exposed to soil treated with Termidor® HE at 0.12% AI through time. Hours Post-treatment Cohort 1 4 24 48 72 96 0:20* 0.00 a 0.00 b 0.00 b 0.00 c 1.25 b 2.50 b 1:19 0.00 a 0.00 b 1.25 b 5.00 c 87.50 a 100.00 a 5:15 0.00 a 0.00 b 1.25 b 45.00 b 90.00 a 100.00 a 10:10 2.50 a 5.00 a 13.75 b 92.50 a 100.00 a 100.00 a 15:5 0.00 a 0.00 b 75.00 a 96.25 a 100.00 a 100.00 20:0 0.00 a 0.00 b 80.00 a 100.00 a 100.00 a 100.00 a 0:20** 0.00 a 0.00 b 0.00 b 2.50 c 2.50 b 5.00 b P-values 0.28 <0.01 <0.01 <0.01 <0.01 <0.01 F-stats 3.00 6.00 53.47 201.76 246.94 1482.60 df 27 27 27 27 27 27 *untreated unmarked control **untreated marked control Means followed by the same letter in the same column are not significantly different P=0.05. 1558 Sociobiolog y Vol. 59, No. 4, 2012 Fig. 6. Mean % mortality of donor populations in different cohorts of donor:recipient Coptotermes formosanus termites through time when exposed for 30 minutes to soil treated with Termidor® HE at 0.12% AI. Table 3. Total mean percent mortality of different cohorts of donor:recipient Reticulitermes flavipes when donors were exposed to soil treated with Termidor® HE at 0.12% AI through time. Hours Post-treatment Cohort 1 4 24 48 72 0:20* 0.00 a 0.00 a 1.25 a 1.25 a 6.25 a 1:19 0.00 a 0.00 a 20.00 a 78.75 b 100.00 b 5:15 0.00 a 0.00 a 53.75 b 97.50 c 100.00 b 10:10 0.00 a 30.00 b 95.00 c 98.75 c 100.00 b 15:5 0.00 a 43.75 bc 100.00 c 100.00 c 100.00 b 20:0 0.00 a 57.50 c 100.00 c 100.00 c 100.00 b 0:20** 0.00 a 0.00 a 0.00 a 1.25 a 3.75 a P-values <0.01 <0.01 <0.01 4814.50 F-stats 19.37 42.65 150.38 <0.01 df 27 27 27 27 27 *untreated unmarked control **untreated marked control Means followed by the same letter in the same column are not significantly different P=0.05. 1559 Keefer, C. et al. — Performance of Termidor® HE High-Efficiency Termiticide and was outside of the scope of this work. All of the treatments, excluding the untreated controls, in this study caused 100% mortality in all the differ- ent ratios of donor:recipient termites in both species by 96 h post-exposure. Coptotermes formosanus had a slower response to the treatments than did R. flavipes. The higher donor:recipient ratios (20:0 and 15:5) in both species of termite were the fastest to reach 100% mortality. In the field study, the application methods were performed by a licensed pest management professional (PMP) and were done so by the normal practices of a PMP. The application of fipronil in and around the structures was done so that applications were made on, or as close as possible to, subterranean termite entry points. This was critical when applying termiticide for control of the subterranean termites. There has been no detection of subterranean termite Fig. 7. Mean % mortality of recipient populations in different cohorts of donor:recipient Coptotermes formosanus termites through time when exposed for 30 minutes to soil treated with Termidor® HE at 0.12% AI. 1560 Sociobiolog y Vol. 59, No. 4, 2012 activity on any of the 12 structures through 36 months post-treatment. The results of these trials demonstrate that at either concentration, fipronil in the form of Termidor® HE is effective at causing significant mortality of C. formosanus and R. flavipes upon contact. Additionally, collateral transfer of Termidor® HE was demonstrated among both subterranean termite species investigated in these studies. Thus, Termidor® HE is not only transferred among termite nestmates, contact with the compound results in death of the insects. Given these laboratory findings, it is not surprising that when applied to termite infested structures, Termidor® HE successfully remediated all infestations and prevented re-infestation for a period of at least three years when applied at 7.5 L per 3 m per 0.3 m depth. Fig. 8. Mean % mortality of donor populations in varying cohorts of donor:recipient Reticulitermes flavipes termites through time when donors were exposed for 30 minutes to soil treated with Termidor® HE at 0.12% AI. 1561 Keefer, C. et al. — Performance of Termidor® HE High-Efficiency Termiticide REFERENCES Bobe, A., J. Cooper, C.M. Coste, and M.A. Muller. 1998. Behavior of fipronil in soil under sahelian plain field conditions. Pestic. Sci. 52, 275-281. Bostian, A. L., S. Swasdichai, and L. Darus. 1996. Activity of fipronil on diamondback moth. Proceedings of the Third International Workshop: The Management of the Diamondback Moth and Other Crucifer Pests, Kuala Lumpur, Maylasia, 195-198. Forschler, B.T. 1994. 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