**3. Colony elimination in the field cases**

Colony elimination performance of bistrifluron has been examined in the field. In this section the results of the Japanese and Malaysian cases with *Coptotermes* are described. Aside from these studies, Colony elimination performance of bistrifluron was demonstrated against Australian *C. acinaciformis* (Evans 2010) and Malaysian *Globitermes sulphreus* (Neoh et al. 2011).

#### **3.1 Field trial against** *Coptotermes formosanus* **in Japan**

A bait system using bistrifluron as an active ingredient was applied to a *C. formosanus*  colony which naturally occurred in Okayama city, Okayama prefecture in Japan (Aki, 2005). The bait system worked in the following procedure: bait stations containing wood blocks

Colony Elimination of Subterranean Termites by Bait Application

perform these tests.

**4.1 Analytical method** 

for the study is as follows.

Using Benzoylphenylurea Compounds, with Special Reference to Bistrifluron 353

be achieved by application of bistrifluron bait based on the above described procedure (unpublished data). These data indicate that application of bistrifluron bait will successfully eliminate colonies of *Coptotermes* termites within 1–2 months. Although numbers of foraging termites varies from ten thousands to over a million, it will take weeks that single colony fall into the collapse after large part of foraging termites of single colony take more than a critical amount of bistrifluron and become unable to take part in maintaining colony's health. The laboratory study described above also showed that individual workers will become incompetent and die within a couple of weeks. Combining such laboratory results with the fact that colonies having hundreds of thousands to a million of foragers were eliminated in 1–2 months, it is suggested that large part of foragers took a critical amount of bait toxicant in very short time and it is an interesting fact for discussion of termite's feeding behaviour. A manner

**4. Feeding behaviour of termites and kinetics of bistrifluron in the termite body**  To discuss how termites will take up a critical amount of bistrifluron through their feeding acitivities, some laboratory studies were conducted to determine its lethal dose and kinetics in their bodies (Kubota et al., 2008). Firstly analytical method had to be established to

Method to chemically analyze bistrifluron amount in termite body was examined using high performance liquid chromatography (HPLC). The outline of analytical method established

The termites were analyzed by HPLC immediately after they were collected. The termites were kept chilled in a vial on ice until they were crushed and homogenized in a mortar with a pestle after being rinsed with solvent. A preliminary chemical assay demonstrated that there was no peak of bistrifluron in HPLC analysis with termites that had been exposed to blank bait. When a given amount of bistrifluron was mixed well with homogenized termites in solvent, 100% bistrifluron was recovered by the above-described procedure. The amount of bistrifluron recovered from solvent that was used to clean termites that had been exposed to 0.5% bistrifluron bait for 1 or 2 weeks was much smaller than 5% of that recovered from whole bodies. The amount of bistrifluron that remained on the external surface of termites was considered to be negligibly small. Homogenized termites were washed into a flask with acetonitrile and then subjected to ultrasonication (42 kHz ± 6%) for more than 1 hour in an ultrasonic device to extract bistrifluron from termites. The acetonitrile suspension was filtered with a filter, PTFE of 0.45 μm. HPLC analysis was performed by a Shimadzu LC-10Avp (Shimadzu Corporation, Kyoto, Japan), with a column of SUMIPAX ODS A-217 (4.6 mm in internal diameter, length 150 mm, Sumika Chemical Analysis Service, Ltd., Tokyo, Japan). Flow rate, injection volume and wave length were 0.5 ml/min, 20 μl and 254 nm, respectively. Mobile phase was acetonitrile/water = 80/20. Analytical-grade 2, 4, 6–anilinetrichloride was

in which termites will take up bait toxicant will be discussed in the next section.

used as an internal standard. HPLC-grade acetonitrile was used as a solvent.

**4.2 Lethal dose of bistrifluron to workers of** *Coptotermes formosanu***s** 

How much amount of bait should be fed by individual foragers was investigated to examine how much amount of bistrifluron should be necessary to be taken to give a critical effect on them. The similar no-choice test with *C. formosanus* workers as described in the 2nd section was designed to examine how long termites should be exposed to 0.5% (w/w) bistrifluron

were installed in the ground. Every station was investigated as to whether wood blocks were infested with termites at given timings. If any station was infested with termites, infested blocks were replaced with paper baits impregnated with 0.5% (w/w) bistrifluron. The location of their nest was identified in this case: their nest had been established within a tree trunk in the vicinity of the premise being attacked. Therefore a couple of bait stations were installed in the ground around the trunk.

The progress and results were as follows: bait stations containing wood blocks were installed on June 29, 2002. Toxic baits were applied on July 18, 2002 as some wood blocks were infested. After some inspections of bait stations there was no live termite and were a lot of dead soldiers on September 3, 2002 at any place where they had been seen before. Inspection within the nest with a microfiber scope revealed that there was also no living termite in their nest. Therefore it can be concluded that the colony had been eliminated by the 1.5-month bait application.

#### **3.2 Field trial against** *Coptotermes gestroi* **in Malaysia**

Efficacy of bistrifluron as a bait toxicant was evaluated against *C. gestroi* which is the most dominant species in Malaysia (Lee, 2007). The study was performed using premises in Penang Island, which had been suffered from their heavy infestation. Blank paper baits stored in plastic cases (the side to which an infested site was attached was open) were applied onto several infested sites of each premise. Since blank baits had been infested at all the premises, 30-50% of them were replaced with tablet bait made of powder cellulose incorporated with 0.5 or 1.0% (w/w) bistrifluron for each premise. All the bait stations were monitored on a weekly basis. Baits with less than 20% remaining matrix were replaced with new ones. The replaced bait matrix were dried and weighed to determine consumed amount. At the time point when there was no termite activity in all the stations applied to each premise it was concluded that the colony was eliminated.

In the four cases applied with 0.5% bistrifluron baits every colony has been eliminated 6-8 weeks after application of toxic baits. Consumed amounts of toxic baits were 211.4-645.2 g per colony, which were equivalent to 1.06-3.23 g of the active ingredient. In the other four cases applied with 1.0% bistrifluron baits every colony has been eliminated 4 or 5 weeks after application of toxic baits. Consumed mounts of toxic baits were 172.9-833.9 g, which were equivalent to 1.73–8.34 g of the active ingredient. Although how much amount of and how intensively baits are consumed would depend on some factors like colony population and activity, faster elimination was obtained with 1.0% bistrifluron baits than 0.5% baits in this study.

#### **3.3 Discussion on the field studies**

As in most cases location of each nest will be out of reach and unidentified, judgement of colony elimination by absence of termite in their nest is difficult and unrealistic. Whether colony elimination has been achieved should be determined by monitoring all the bait stations for a certain period after no termite activity was observed in all the stations by baiting, like the Malaysian case described above. If status of no termite activity continues for the given period (EPA guideline mentions that it should be more than 12 months) then it should be concluded that the colony applied with bait has been eliminated (US EPA, 2004). On the other hand, in the Japanese case colony elimination was confirmed by direct inspection of inside of the nest. Separately from this case, there were many Japanese cases that colony elimination seemed to be achieved by application of bistrifluron bait based on the above described procedure (unpublished data). These data indicate that application of bistrifluron bait will successfully eliminate colonies of *Coptotermes* termites within 1–2 months. Although numbers of foraging termites varies from ten thousands to over a million, it will take weeks that single colony fall into the collapse after large part of foraging termites of single colony take more than a critical amount of bistrifluron and become unable to take part in maintaining colony's health. The laboratory study described above also showed that individual workers will become incompetent and die within a couple of weeks. Combining such laboratory results with the fact that colonies having hundreds of thousands to a million of foragers were eliminated in 1–2 months, it is suggested that large part of foragers took a critical amount of bait toxicant in very short time and it is an interesting fact for discussion of termite's feeding behaviour. A manner in which termites will take up bait toxicant will be discussed in the next section.
