The responsibility for the interpretation and use of the material lies with the reader

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WHO/CDS/NTD/WHOPES/2006.2

REPORT OF THE NINTH

WHOPES WORKING GROUP MEETING

WHO/HQ, GENEVA 5–9 DECEMBER 2005

Review of:

DIMILIN^£ GR AND DT VECTOBAC^£ DT AQUA K-OTHRINE^£ AQUA RESLIN SUPER^£

CONTROL OF NEGLECTED TROPICAL DISEASES WHO PESTICIDE EVALUATION SCHEME

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© World Health Organization 2006

The designations employed and the presentation of the material in this publication do not imply the expression of any opinion whatsoever on the part of the World Health Organization concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries.

The mention of specific companies or of certain manufacturers’

products does not imply that they are endorsed or recommended by the World Health Organization in preference to others of a similar nature that are not mentioned. Errors and omissions excepted, the names of proprietary products are distinguished by initial capital letters.

All reasonable precautions have been taken by the World Health Organization to verify the information contained in this publication.

However, the published material is being distributed without warranty of any kind, either express or implied. The responsibility for the interpretation and use of the material lies with the reader. In

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CONTENTS

Page

1. INTRODUCTION ... 1

2. REVIEW OF DIMILIN 2% GR AND 2% DT... 4

2.1 Safety assessment ... 4

2.2 Efficacy – background/supporting documents.... 6

2.2.1 Laboratory studies ... 6

2.2.2 Field trials ... 8

2.3 Efficacy – WHOPES supervised trials ... 16

2.4 Conclusions and recommendations ... 26

3. REVIEW OF VECTOBAC DT... 37

3.2 Efficacy – background/supporting documents.. 40

4. REVIEW OF AQUA K-OTHRINE... 48

4.3.1 Laboratory studies ... 52

4.3.2 Field trials ... 57

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5. REVIEW OF AQUA RESLIN SUPER... 65

ANNEX 1 : LIST OF PARTICIPANTS... 89

ANNEX 2 : REFERENCES... 91

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1. INTRODUCTION

The ninth meeting of the WHOPES Working Group, an advisory group to the WHO Pesticide Evaluation Scheme (WHOPES), was convened at WHO headquarters in Geneva, Switzerland, on 5–9 December 2005. The objective of the meeting was to review the reports of testing and evaluation of Dimilin^£ (diflubenzuron) 2%

GR and 2% DT (Crompton, Netherlands) and VectoBac^£ (Bacillus thuringiensis israelensis) DT, 3000 international toxic units (ITU)/mg (Valent BioSciences, USA) for mosquito larviciding; and Aqua K-Othrin^£ (deltamethrin) 2% w/w EW and Aqua Reslin Super EW (= Aqua Resigen; containing permethrin 25/75 cis/trans (10.35% w/w), s-bioallethrin (0.14% w/w) and piperonyl butoxide (9.85% w/w) (Bayer Environmental Science, France)) for space spraying for mosquito and fly control.

The meeting was opened by Dr Lorenzo Savioli, Director, Department of Control of Neglected Tropical Diseases. In his opening remarks he referred to the new name and departmental structure. Dr Savioli stated that the neglected tropical diseases, which include sleeping sickness, schistosomiasis, river blindness, hookworm, elephantiasis, dengue and blinding trachoma, affect several hundred million people and kill at least half a million annually, yet they receive little attention from donors, policy- makers and public health officials. He added that for costs that are relatively modest compared with those needed to control “the big three” – HIV/AIDS, tuberculosis and malaria – an integrated package for control of neglected tropical diseases could have a proportionately greater impact on the health of more poor people and be more equitable for the majority of the poorest and marginalized communities. Dr Savioli noted the importance of vector control as an essential cross-cutting activity for the control of vector-borne diseases.

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Dr Michael Nathan, Team Leader, Vector Ecology and Management, reminded the meeting of the WHO Global strategic framework for integrated vector management (IVM)¹, which provides the guiding principles for assisting Member States in controlling vector-borne diseases. He informed the meeting of the WHO Bulletin (2005) policy and practice article Exploiting the potential of vector control for disease prevention² and commented that the development of guidelines for planning, implementation and monitoring of IVM activities and supporting Member States in vector control capacity strengthening would be priority activities of the Vector Ecology and Management team in the coming years.

Dr Morteza Zaim, Scientist in charge of the WHO Pesticide Evaluation Scheme (WHOPES), presented the objectives and an overview of the scheme to the participants. He noted that the recommendations of WHOPES on the use of public health pesticides are to facilitate the registration of such products by Member States. He also indicated that the reports of the WHOPES Working Group meetings are well received by national registration authorities and control programmes as an excellent source of information and consolidation of available information on pesticides evaluated by WHOPES and that every effort will be made to make the reports more useful and widely available.

Dr Zaim also noted that WHOPES is a four-phase programme and that the development of specifications constitutes the last phase of the scheme. He emphasized that WHO recommendations on the use

1Global strategic framework for integrated vector management. Geneva, World Health Organization, 2004 (WHO/CDS/CPE/PVC/2004.10).

2Townson H, Nathan MB, Zaim M, Guillet P, Manga L, Bos R, Kindhauser M.

Exploiting the potential of vector control for disease prevention. Bulletin of the World Health Organization, 2005, 83:942–947.

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of public health pesticides are valid ONLY if linked to WHO specifications for their quality control.

The meeting was attended by 12 scientists (see Annex 1: List of participants). Dr Mir Mulla was appointed as Chairman and Dr Purushothaman Jambulingam as Rapporteur. The meeting was convened in plenary and group sessions, in which the reports of the WHOPES supervised trials and relevant published literature (see Annex 2: References) were reviewed and discussed.

Recommendations on the use of the above-mentioned products were made.

The group also reviewed and revised the draft guidelines for testing mosquito adulticides for indoor residual spraying and for treatment of nets, to be published as a separate document by WHOPES.

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2. REVIEW OF DIMILIN 2% GR AND 2% DT 2.1 Safety assessment

Dimilin^£ (diflubenzuron) is an insect growth regulator (IGR) of the benzoyl urea family [1-(4-chlorophenyl)-3-(2,6-difluorobenzoyl) urea] that acts by disrupting chitin synthesis and deposition. The toxicity of diflubenzuron was evaluated by the FAO/WHO Joint Meeting on Pesticide Residues (JMPR) in 1981, 1984, 1985 and 2001 (JMPR, 2001).

WHO has classified diflubenzuron as “unlikely to present an acute hazard in normal use”. Diflubenzuron (purity 99.6%) has very low acute toxicity when given by various routes (oral, dermal and inhalation). Diflubenzuron had little toxicity in rats exposed dermally, with an LD50 of >10 000 mg/kg bw, or by inhalation, with an LC50 >2.8 mg/litre of air. Only marginal increases (less than doubling) in methaemoglobin concentrations were seen in mice and rats given 10 000 mg/kg bw of a formulation of diflubenzuron, equivalent to 2500 mg/kg bw, which is above the limit doses used in toxicological tests. Diflubenzuron did not significantly irritate the skin or eyes of rabbits and did not irritate the skin of guinea-pigs exposed in a Magnusson and Kligman “maximization” study.

Diflubenzuron was not a skin sensitizer.

Studies in rats showed that excretion was relatively rapid, >90% of the doses of 5 and 100 mg/kg bw being excreted within 24 hours.

There was no evidence of increased absorption with longer exposure. Diflubenzuron is poorly absorbed through rabbit skin.

There was no evidence of neurotoxicity in routine studies of toxicity.

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Diflubenzuron is unlikely to be genotoxic and is unlikely to pose a carcinogenic risk to humans. In a two-generation study of reproductive toxicity in rats, no adverse effects were found on sperm quality or quantity, reproductive performance, litter size or attainment of developmental landmarks at the highest dietary concentration. There was no evidence of overt maternal toxicity, fetotoxicity or teratogenicity in rabbits and rats at the highest dose (1000 mg/kg bw per day). Diflubenzuron is neither fetotoxic nor teratogenic.

The previously established acceptable daily intake (ADI) of 0–0.02 mg/kg bw, based on the no-observed adverse effect level for haematological effects of 2 mg/kg bw per day in the 2-year studies in rats and the 52-week study in dogs, has been re-confirmed (safety factor, 100).

No reports of adverse effects or poisoning incidents associated with diflubenzuron were found, and no adverse effects have been reported during field use. Although no actual exposures have been described, no disturbances in the health status of the employees in the production and formulation were observed that could be linked to exposure to diflubenzuron. Diflubenzuron has low toxicity to birds, fish, earthworms and aquatic plants but is highly toxic to some crustacea. The following are the extracts from the material safety data sheet of the manufacturer for diflubenzuron technical.

Acute toxicity – oral (rat) LD50 >4640 mg/kg Acute toxicity – dermal (rat) LD50 >2000 mg/kg Acute toxicity – inhalation (rat, 4 hours) LC50 >2.88mg/litre Skin irritation (rabbit) Non-irritating Eye irritation (rabbit) Non-irritating Skin sensitization (guinea-pig) Negative

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2.2 Efficacy – background/supporting documents 2.2.1 Laboratory studies

Florida, USA

The efficacy of diflubenzuron was studied in the laboratory along with 64 compounds against the insecticide-susceptible larvae of Anopheles quadrimaculatus (Dame et al., 1976). Assays were also carried out against DDT-resistant and -susceptible strains of An.

quadrimaculatus and malathion-resistant and -susceptible strains of Aedes taeniorhynchus. The laboratory assays were conducted in 18x28 cm enamel pans containing 1 litre of distilled water. Known concentrations of the IGR were prepared by pipetting 0.1–10 ml of stock solutions of 1 mg/ml of active ingredient in acetone. Then 50 early fourth-stage larvae obtained from a laboratory colony of an insecticide-susceptible strain of An. quadrimaculatus were placed in each pan, and larval food was added daily. The untreated control was maintained for each day’s assay. The pans were held at 27–

29^oC and 65–75% relative humidity. Each day, pupae were removed, rinsed with distilled water and transferred to a container of distilled water in a 25 x25x16 cm cage for observation of eclosion. The dead larvae were counted, removed and discarded.

The number of adults that successfully emerged was calculated.

After initial testing with 3–5 concentrations to determine the general level of activity, a minimum of 3 concentrations giving between 0 to 100% mortality were replicated to obtain the data necessary to establish a dosage–response relationship. The data were adjusted by Abbott’s formula, and IE50 and IE90 were calculated.

The study showed that diflubenzuron was among the most promising compounds, producing extensive larval mortality at all levels and reducing eclosion in the laboratory with an IE90 value of

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0.004 ppm. The results of the tests conducted against insecticide- resistant strains of An. quadrimaculatus (IE90 = 0.003–0.004) and Ae. taeniorhynchus (IE90= 0.002–0.003) showed no substantial differences in the susceptibility of the resistant and susceptible strains to the compound (Table 1).

Florida, USA

Diflubenzuron, methoprene and pyriproxyfen (technical materials) were evaluated against Ae. albopictus, a Florida laboratory population (Ali et al., 1995). In the evaluation, 20 late-third and early-fourth-instar larvae were placed in 120 ml disposable cups containing 100 ml tap water; 5–9 different concentrations were tested on at least three different occasions. Each concentration was replicated three times, and three untreated controls receiving only 1 ml of acetone were maintained; 1 ml of 1% beef liver and yeast (1:3) was added to each cup at 2-day intervals. Treated and untreated cups were examined daily for any larval, pupal or adult mortality, and cumulative mortality was recorded at the termination of the test when adult emergence was completed in control cups and no living larva or pupa remained. A 14-hour photoperiod and 28 + 3 ^oC were maintained. Mortality was corrected for any control mortality and the data subjected to a log-probit regression analysis to estimate larval dosage response. The IE50 values for diflubenzuron, methoprene and pyriproxyfen were 0.00045, 0.0022 and 0.00011 mg/litre active ingredient, respectively.

Following the same procedure, Ali et al. (1999) tested the efficacy of diflubenzuron, methoprene and pyriproxyfen (technical materials) against field-collected Culex quinquefasciatus from urban Dhaka, Bangladesh. The IE50 values for diflubenzuron, methoprene and pyriproxyfen were 0.0014, 0.017 and 0.00029 mg/litre active ingredient, respectively.

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2.2.2 Field trials Florida, USA

Simulated field tests against Ae. taeniorhynchus were conducted in 0.1 m² circular metal pans 20 cm deep lined with 6 ml polyethylene film using “non-commercial” granular formulations of diflubenzuron and methoprene (Rathburn and Boike, 1975). The objective of the study was to determine the initial impact of the insecticide rather than its residual activity. About 2 cm of sandy soil was placed in the bottom of each pan and 4 litres of 25% seawater (7–9 ppt salt) were added. The pans were held in a water bath at a temperature of 29 ^oC + 2 ^oC. The surface of the water in each bath was covered with pieces of Styrofoam to limit heat dissipation from the water surface. Immediately before treatment, 200 third-instar Ae. taeniorhynchus larvae were placed in each pan; each test consisted of two replications of each treatment and the check.

Diflubenzuron, 0.2% GR formulation (sand and vermiculite formulations made from 25% WP), was applied at a dosage rate of 11.2 g/ha active ingredient. Larvae were fed daily. After pupation began, pupae were removed daily, transferred to clean water and held at the same temperature in plastic cups and observation made on adult emergence. With both the sand and the vermiculite formulations of diflubenzuron, at the low dosage rate of 11.2 g/ha active ingredient, the larval mortality was 100% and there was no emergence.

Two small-plot field tests were also conducted in eight specially constructed plots, each approximately 6 m long, 3 m wide and 30–

45 cm deep, arranged in a single line. Each plot was flooded with water from a nearly natural saltwater lagoon, and water level was maintained. The salinity of the water averaged 20.7 and 26.4 ppt salt respectively. Ae. taeniorhynchus larvae from the laboratory colony were reared in saltwater taken from the canal, and

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approximately 2000–3000 larvae (second or third instar) were placed in each plot about 4 hours before treatment. All treatments and the checks were replicated twice. Two formulations were compared in this test: the liquid formulation of diflubenzuron 25% WP diluted in water was applied manually by use of a clothes sprinkler and the granular formulations using a glass jar with a mesh screen top. The granular formulations used in the check plots were the same as those used in the treated plots but without the IGR. All treatments were applied at a rate of 22.4 g/ha active ingredient.

Ten dips per plot were taken pre and post-treatments, five from each side of the plot and, when possible, only where larvae were observed. A minimum of 50 pupae or fewer (when not available) were collected from each plot daily and observed for adult emergence until pupae were no longer present. None or very few pupae appeared in the plots treated with diflubenzuron at 22.4 g/ha active ingredient up to day 5 post-treatment. No pupal skins or emerging adults were noticed in any of the treated plots at 72–96 hours. Collected pupae failed to emerge as adults.

Similar results were obtained with methoprene, except that higher mortalities occurred at the pupal stage.

Although no attempt was made to assess the effect of the treatments on non-target organisms in these tests, several live dytiscid larvae and many corixids (up to several hundred per dip) were noted in both treated and check plots.

California, USA

Granular (0.5%) and wettable powder (25%) formulations of diflubenzuron were evaluated for initial and residual activity at the application rates of 28.0 (0.08 ppm) and 56.0 (0.16 ppm) g/ha active

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ingredient in ponds filled with water from a reservoir against Cx.

tarsalis, chironomid midges and some commonly associated non- target organisms (Mulla et al., 1975). The WP formulation was mixed with water and applied using a squeeze bottle; the granular formulation was applied evenly by hand. Two ponds were used for each dosage and check. The mosquito larvae and some non-target organisms (midge larvae, mayfly naiads, cladocerans, ostracods and copepods) were sampled using a dipper, concentrated in a cup provided with mesh, preserved in 95% ethyl alcohol in plastic vials and examined under stereoscopic microscope. Planktons were sampled using a tow-net mounted on a metal sled, preserved in formaldehyde 3% and examined microscopically. Benthic chironomid midge larvae were sampled by taking 6 x 6-inch bottom mud samples by a scraper in each pond, washing through a mesh screen sieve, transferring to a cup and floating the larvae by adding saturated solution of Mg SO4. The larvae were then grouped and counted under a magnifying lamp. Emergence of chironomid midges was assessed by a placed emergence cylinder in each pond.

Inhibition of emergence of mosquitoes was assessed by placing 20 fourth-instar larvae in two of the isolating units per pond and following them until emergence. Decline in the population of third- and fourth-instar mosquito larvae was apparent from 2–8 days after treatment but was not observed 11 days post-treatment; no appreciable decline was noticed in first-instar larvae, as these resulted from continuous oviposition. Adult emergence from treated larvae isolated in floating units was almost completely inhibited up to at least 11 days post-treatment in all treatments. There was little or no difference in the efficacy of the two dosages.

A marked decline in the population of nektonic chironomid larvae was not detected in the treatments during the 15-day duration of the experiment. Emergence of chironomids, however, was depressed up to 8–15 days after treatment.

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Among the non-target organisms studied, mayfly, Baetis spp., naiads were depressed slightly for 2–4 days post-treatment but recovered to normal levels soon after. Cladocera (Daphnia spp.) were moderately affected by the WP formulation (but not affected by the GR formulation), with density reaching almost zero for 11 days post-treatment. The copepods Cyclops spp. and Diaptomus spp. were affected for 2–8 days. All affected non-target organisms recovered, as did the target organism 11–15 days after treatment.

The ostracods (Cypricercus spp. andCyprinotus spp.) were little or not affected by the treatments, nor were diving beetle larvae and adults and odonate naiads during the duration of these experiments.

California, USA

Three formulations of diflubenzuron (wettable powder (25%WP), flowable concentrate (2% SC) and granular formulation (0.5% GR)) were evaluated for initial efficacy against Psorophora confinnis under field conditions in irrigated alfalfa hay fields and in freshwater ponds (Mulla and Darwazeh, 1976).

In two locations in the Palo Verde Valley along the Colorado River, 1/16-acre plots were selected in alfalfa hay fields, where mature larvae and pupae of Ps. confinnis were abundant 3–4 days after irrigation and the larvae were in the fourth stage at treatment time.

Water depth in the plots was 3–6 inches and vegetation 6–8 inches high. In the first trial, the granular formulation was compared with the standard WP formulation, while in the second trial the SC was compared with WP. The WP and SC formulations were diluted in water and sprayed using a pressurized hand sprayer. The granular formulation was dispersed by hand over the plots. The plots were treated with 28.0 and 56.0 g/ha active ingredient; two plots were treated with each rate and two left untreated as a check in each test.

Immature mosquitoes were sampled by 10 dips per plot before, and 1, 2, 3 or 1 and 2 days after treatment. Reduction in the immature

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population was calculated for the last sampling time, using post- treatment and pretreatment counts. Additionally, 40 surviving larvae were isolated 24 hours after treatment or surviving pupae 48 hours after treatment from each plot and transferred to tap water in paper cups, 20 larvae/pupae in each and followed up for adult emergence.

In irrigated alfalfa hay fields, all three formulations applied at 28.0 and 56.0 g/ha active ingredient produced complete control, with 95–

100% mortality in the larval populations of Ps. confinnis, within 2–

3 days after treatment. Although the rate of pupation in the granular treatment was higher than that of the WP formulation, mortality in subsequent stages was high even with the granular formulation. At 28.0 and 56.0 g/ha active ingredient, both the SC and WP formulations yielded high levels of larval mortality (90–98%).

Trials in freshwater ponds were conducted to determine residual activity against the immatures of Cx. tarsalis and An. francisanus.

The ponds (35 m²) were located near the Salton Sea in the Coachella Valley of southern California, fed by artesian piped water, with a water depth of 30 cm by float valves. The SC, WP and GR formulations were applied at 28.0 g/ha active ingredient. Three ponds were treated with each formulation and three ponds used as checks. For assessment, five dips per pond were taken before and at intervals after treatment. Only third-to-fourth instars were counted for each species, and pupae for both species. The test was terminated when the number of third-to-fourth instars and pupae recovered substantially. Additionally, inhibition of adult emergence was assessed by isolating 20 fourth-instars of each species in each of two isolation units (with fine mesh) placed 2 and 5 days after treatment in each pond. Larval food was provided. Dead larvae or pupae were removed, and inhibition of adult emergence was determined for the treated and untreated units.

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The mortality of surviving larvae in isolation units 24 hours after treatment was 65–75% in the GR treatments and 93–100% in the WP treatments. The IE was 100% in larval/pupal isolates from WP treatments but was 88–95% for the granular treatments, indicating that the WP formulation is more effective than the granules. In treatments with SC and WP, larval mortality was >80% in all isolates, except from the 28.0 g/ha active ingredient rate of WP; 90–

100% pupal mortality was observed at both rates of both formulations. At the higher rate, both formulations produced 100%

inhibition of emergence from larval isolates.

All three formulations applied at 28.0 g/ha active ingredient produced a marked reduction in the immature population of both An. franciscanus and Cx. tarsalis during the post-treatment period of 5–21 days. There was a marked absence of pupa from day 5 onwards. Inhibition of emergence caused by the three treatments was almost 100% from day 2 to the end of the experiment (27 days).

North Carolina, USA

Replicated field trials were conducted with diflubenzuron 25% WP along with three other compounds, including methoprene, to control Ae. taeniorhynchus larvae breeding in temporary pools of water in depressions within diked dredge spoil disposal areas in coastal North Carolina (Axtell et al., 1979). The objective of the study was to determine the initial impact of the insecticides rather than their residual action. Since these pools are dependent upon rainfall, their size depends upon the amount and sequence of rainfall. The IGR was applied with a hand-pumped compressed air sprayer. Each isolated pool was a replicate. The treated pools had surface areas of ca. 70–2900 ft², with most ca. 300–900 ft². The depth of the water was 1–6 inches. In four tests, diflubenzuron was applied at 5.6, 11.2, 22.4 and 44. 8 g/ha active ingredient. Two further tests were

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conducted to compare a granular formulation 1% with WP 25% at the same application rates (28.0 and 56.0 g/ha active ingredient).

The numbers of mosquito larvae and pupae were determined immediately before treatment in each pool from 10 dips taken about equidistant around the pool. Post-treatment samples were taken in the same manner until cast pupal skins were found in any of the pools, which indicated that adult emergence was occurring. The percentage reduction at various post-treatment intervals was calculated for each pool by using the number of larvae and pupae pretreatment and post-treatment counts and the mean of the replicates calculated.

Concurrently, at 1-day post-treatment or at greater intervals, 100–

250 (depending upon availability) fourth-instar larvae were collected from each treated and untreated (control) pools and held equally divided in water from their source in four mesh-topped cups in the laboratory to determine the presence of adults successfully emerging.

In the four tests, the mean percentage reduction of larvae and pupae was 87% at 44.8 g/ha active ingredient on day 1 post-treatment, 64% at 22.0 g/ha active ingredient, 61–89% at 11.2 g/ha active ingredient during day 4 post-treatment and 45–88% at 5.6 g/ha active ingredient for 5 days post-treatment. From the larvae retrieved at days 1 and 3 post-treatment and held in laboratory, the IE obtained ranged from 85–100% at the dosages tested, and rates were dosage dependent.

Two further tests were conducted to compare a 1% granular formulation with WP 25% at the application rates of 28.0 and 56.0 g/ha active ingredient. In the comparative evaluation, both the formulations were found to give effective control (95–100%) at the two application rates. Live larvae and pupae were nearly absent in

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the treated pools for 3–4 days post-treatment. No adult emergence occurred in the laboratory from larvae collected at days 1 and 3 post-treatment. There was no difference between the formulations in their efficacy.

Observations were made in the field on organisms other than mosquitoes in the pools. Although an occasional dead-water boatman and water beetle were found, large numbers were alive in the pools. Live fiddler crabs (Uca spp.) and minnows were observed in the treated pools; no evidence of toxic effects of the chemicals on these non-target organisms was observed.

Methoprene at 44.8 g/ha active ingredient provided complete mosquito control in this habitat.

North Carolina, USA

Diflubenzuron 25% WP and 1% GR was evaluated along with four other insecticides and one IGR, in anaerobic swine waste lagoons, for the control of the mosquito Cx. quinquefasciatus, in North Carolina, USA (Axtell et al., 1980). The sizes of lagoons varied between 0.02 and 0.07 ha. The chemicals were applied using a hand-operated compressed air sprayer to lagoons in which substantial numbers of larvae were present. The spray was applied to the margin of each lagoon in about a 3 m band that could be easily reached from the shoreline. After spraying, pupae and/or fourth-instar larvae (150) were collected from each lagoon and held in the laboratory in fresh water in screen-topped cups (25/cup) to determine the percentage-yielding adults that were mobile and not deformed. Adult emergence from pupae and larvae collected from the untreated lagoon and held in the laboratory was usually 96–

100%. In addition, dipper sampling was made at 10 approximately equal intervals around the margin of each lagoon on each sample date. Only the third and fourth larval instars and the pupae from the samples were counted and used to calculate the average number per

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dip. The two formulations were also compared at the same rate at 112 g/ha active ingredient in simulated lagoons into which a metal drum was sunk into the ground with water and poultry manure added. In each test, there were three replicates.

In the lagoons, diflubenzuron gave no mosquito control at the rate of 33.6 g/ha active ingredient and at 67.2 g/ha active ingredient partially inhibited adult emergence. At 89.6 g/ha active ingredient, mosquito control was improved, with 90–99% of inhibition of adult emergence at 7 days post-treatment in two tests. Some initial reduction in the numbers of larvae and pupae was also evident at this rate. At 112 g/ha active ingredient, diflubenzuron was consistently effective for at least 7 days in drastically reducing the numbers of larvae and pupae in the lagoons, to the extent that the dip counts were at or near zero. No adults emerged from the specimens collected from the treated habitat for 14 days. At days 14, 21 and 28 post-treatment, there was some reduction in adult emergence but this was variable. Both the formulations gave complete mosquito control, with little or no adult emergence, for 14 days post-treatment when applied to simulated lagoons with poultry manure added at three rates.

2.3 Efficacy – WHOPES supervised trials Sistan and Baluchistan, Islamic Republic of Iran

To determine the activity of diflubenzuron in the laboratory, a susceptibility test was carried out on the laboratory strain of larvae of An. stephensi and Cx. quinquefaciatus, following standard method (WHO, 2004; Laddoni, 2005). A 1% stock solution (w/v) of the diflubenzuron (90.1%) was prepared in actone. The stock solution was then serially diluted in acetone to get the desired lower concentration. One ml stock solution of test concentration was

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added to 99 ml chlorine-free water in the containers to obtain the desired target dosage. Batches of 20–25 early third-instar larvae were transferred to containers and one drop of larval food was added at 2-day intervals until a mortality count was made. Four replicates were set up for each concentration (six concentrations) and control. The control received 1 ml acetone only. The test containers were held at room temperature (25 + 2 ^oC) and a photoperiod of 12:12 light/dark. Observation was made every 24 hours until adult emergence. The percentage inhibition of adult emergence (%IE) was calculated. The IE50 and IE90 values for An.

stephensi and Cx. quinquefasciatus were obtained by probit analysis.

Diflubenzuron showed a high level of activity, with the IE50 and IE90 values of 0.003 + 0.007 and 0.01 + 0.028 mg/litre for An.

Stephensi and 0.036 + 0.005 and 0.134 + 0.031 mg/litre for Cx.

quinquefasciatus, respectively. Diflubenzuron at the higher rate of concentration caused 95–100% of mortality in the larval stage, while at the lower concentration larval mortality was not complete and the surviving individuals suffered mortality in the pupal and adult stages. The larvae of An. stephensi were 10.2-fold more susceptible to diflubenzuron than that of Cx. quinquefasciatus (Table 1).

GR 2% formulation of diflubenzuron was also tested in comparison with 25% WP against Anopheles and Culex mosquitoes breeding in artificial ponds and in rice fields of Ghassreghand, south-east Islamic Republic of Iran, to determine the initial efficacy, residual activity and optimum dosage (Laddoni, 2005).

Initially, the formulations were evaluated in artificial ponds (100 x

100 x 50 cm), dug at a distance of about 1 m apart, against Anopheles (An. culicifacies, An. stephensi, An. dthali, An.

superpictus and An. sergentii) and Culex (Cx. tritaeniorhynchus, stephensi

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Cx. pipiens, Cx. pseudovishnui and Cx. quinquefasciatus) species.

The ponds were fully lined with plastic sheeting and filled with soil or mud from the breeding sites of the target vector species (10 cm depth) and subsequently filled with water from the habitat to a depth of 30–40 cm. The water level was maintained throughout the trial period. The ponds were left for 2 weeks until oviposition occurred, and treatment was undertaken when adequate numbers of third- and fourth-instar larvae and pupae were found in the ponds.

To avoid adult emergence, mature pupae were removed daily from the artificial ponds.

The formulations were tested at four application rates (25, 50, 100 and 150 g/ha active ingredient), and the ponds were randomly selected for each dosage and for control with four replicates in each.

Diflubenzuron WP was diluted with water and sprayed using a hand-compression sprayer with a flat fan nozzle; the GR formulation was distributed uniformly by hand after mixing with fine sand. Larval and pupal densities were monitored before treatment using dipper sampling, daily on post-treatment days, until the recovery of 90% immature stage of population at pretreatment stage. The density of early- and late-instar larvae and pupae was calculated based on density/10 dips per pond per day.

The percentage reduction of larval and pupal density, as well as the

%IE on successive days of post-treatment, was calculated in relation to the pretreatment density and %IE in the treated and untreated sites. Additionally, late-instar larvae and pupae were collected along with water from each treated and untreated (check) pond and kept in 200 ml plastic cups inside the holding cages in the laboratory. The dosage at which >90% larval and pupal mortality or emergence inhibition was observed for a longer duration was selected as the field dosage for each habitat.

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In rice fields, the two formulations were tested at three application rates (25, 50 and 100 g/ha active ingredient). Major breeding sites with moderate-to-high mosquito density were allocated randomly to the dosage of each formulation and to the control, with at least four replicates for each dosage. The surface area of the breeding sites ranged between 500 and 800 m², with an average depth of 25 cm and a water temperature of 27 + 3 ^oC. Application of formulations and assessment of efficacy were done as described above. The species composition of the larvae in breeding places was determined by collecting and identifying fourth-instar larvae from untreated breeding places (3–4 times). The habitat was re-treated when the inhibition of adult emergence or reduction of pupal density reached below 50%. The efficacy of different dosages of the formulations against larval density and %IE of Anopheles and Culex was analysed using paired t-test or chi-square test.

At the higher dosage (150 g/ha active ingredient), the formulations showed a similar effect on the larvae of Anopheles and Culex. On day 1 pretreatment, the %IE was in the range 15.6–22.4 for Anopheles and Culex. One day after application, diflubenzuron caused 49.4–88.2% of emergence inhibition. The IE increased thereafter and was 84.4–100% up to day 30 post-treatment at 150 g/ha active ingredient, 14–15 days at 100 g/ha active ingredient, 7–

9 days at 5 g/ha active ingredient and 3–5 days at the lowest dosage (2.5 g/ha active ingredient).

The two formulations suppressed pupal density of Anopheles and Culex by >80% up to days 12–14 post-treatment, at 100 g/ha active ingredient, 8–9 days at 50 g/ha active ingredient, while it was 3–5 days at 25 g/ha active ingredient. The density of late-instar larvae was reduced by >80% at 100 g/ha active ingredient for 7–12 days and at 50 g/ha active ingredient for 8–9 days post-treatment. The percentage reduction in density of late- and early-instar larvae at the

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low dosages was highly variable, ranging from 50–70% for 4–6 days post-treatment.

Overall, the adult emergence and densities of immature stages of Anopheles and Culex did not show any significant differences between the two formulations (P >0.05). At 150 g/ha active ingredient, both formulations had a high adverse effect on a number of aquatic insects belonging to the families of Dytiscidae, Histeridae, Libellulidae and Notonectidae in the ponds. However, recovery usually occurred after 7–10 days post-treatment.

Diflubenzuron at 100 g/ha active ingredient did not show any adverse effect on the non-target aquatic insects in breeding ponds.

In rice fields, application of WP and GR formulations yielded 80–

100% emergence inhibition up to days 8–10 post-treatment and suppressed pupal density of AnophelesandCulex for 5–7 days post- treatment at the two higher dosages. The reduction of density of late-instar larvae caused by IGR was >80% up to days 3–5 post- treatment; the effect on early instars of Anopheles and Culex was highly fluctuating. At 2.5 mg/m² active ingredient, the %IE was

<80% during the first week post-treatment. The IGR did not show any significant effect on immature stages at this application rate.

Nonthaburi, Thailand

In this study, two formulations of diflubenzuron (GR and tablets) were evaluated at five dosages each along with controls to determine the initial, short-term and long-term efficacy of these formulations against Ae. Aegypti in typical and commonly used water-storage jars (capacity: 200 litres) in Thailand (Mulla, 2005).

Two sets of jars were used in the study: one set was kept full for the duration of the experiment without removing water; the other set had half the water removed weekly and was refilled with fresh

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water to simulate water use conditions. The water level in the full jars was maintained by adding water when needed.

In the full jars, 1.0, 0.5, 0.1, 0.05 and 0.02 mg/litre (ppm) of each formulation and two controls were used, each replicated four times, while the dosages in water removal-refilled jars were 1.0, 0.5 and 0.1 mg/litre; each treatment was replicated four times with two sets of control. The jars filled with water were allowed to sit for a week or two before the treatments were applied. The jars were covered with celocrete sheets for 2 months and thereafter with netting to preclude mosquitoes from entering from the outside. The placement of the jars was configured in a block design form to equally distribute positional effects.

To assess the efficacy, 25 third-instar larvae from a laboratory colony were added to each treated jar and control. Before adding the first cohort of larvae, larval food was added to each jar initially and thereafter weekly and every 2 weeks as needed. Larval survival was assessed 2 days post addition of larvae for the first four cohorts, and the emergence of adult mosquitoes was determined by counting the pupal skins 7 days post addition of each larval cohort. The treatments were challenged with new cohorts of larvae (third instar) weekly. The %IE was calculated on the basis of the initial number of larvae in each cohort in each treatment. Evaluation was continued up to 182 days post-treatment. Water temperature during the 28 days of the test was 26–27 ^oC minimum and 32–33 ^oC maximum.

Larval mortality 48 hours post-treatment was quite high, ranging from 56% to 75% for the various dosages and cohorts. However, assessment of pupal skins showed almost complete inhibition of adult emergence in the first four cohorts, indicating additional mortality in lavae or pupae following the 48-hour larval assessment.

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Both formulations of diflubenzuron were equally effective, yielding IE 100% at all dosages (except the 0.02 mg/litre of tablet formulation) in full jars for 161 days. The higher three dosages (0.1, 0.5 and 1 mg/litre) produced about 90% IE on day 168 post- treatment but declined to below 90% IE on days 173 and 182 post- treatment.

In the removal-refill jars, all three dosages (1.0, 0.5 and 0.1 mg/litre) of both formulations produced almost 100% IE for 105 days post-treatment. However, at 112 days post-treatment the low dosage of 0.1 mg/l active ingredient of granules yielded only 81%

IE, declining to 79%, 77% and 22% IE after 119, 126 and 133 days post-treatment, respectively; the same low dosage of tablets yielded almost 100% IE for 126 days. The two higher dosages of both formulations produced close to 100% IE for 147 days post- treatment. The granules at the two higher dosages yielded 80% IE for 168 days post-treatment.

Water removal and refill somewhat reduces the longevity of diflubenzuron in water-storage jars against Ae. Aegypti larvae. In jars without water removal, the practical dosages of 0.05 to 0.1 lasted for about 168 days, while the 0.1 and 0.5 dosage in jars with water removal and refill lasted for 168 days (granules) and 147 days (tablets).

The study shows that both formulations have the same level of efficacy and that the low dosages (0.05 to 0.1 mg/litre) can control larvae for about 5 months. The higher dosages (0.5 and 1 mg/litre) yielded somewhat longer control for 168–173 days post-treatment.

Pondicherry, India

Two formulations of diflubenzuron (GR and DT) were tested in comparison with WP formulation against Cx. quinquefasciatus in

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three types of larval habitats (cesspits, drains and disused wells) at three application rates (25, 50, 75 and 100 g/ha active ingredient) in Cuddalore, an urban area 22 km from Pondicherry, India (Sadanandane et al., 2005). Additionally, the DT formulation was tested at a still higher dosage (1 tablet/m²).

Adult emergence and larval and pupal densities were monitored using emergence traps and dipper sampling, every 2–3 days before treatment for 1–2 weeks, and after treatment until the abundance in the treated habitats reached pretreatment level. The habitats with comparable densities were assigned randomly to control and treatments. The percentage reduction of larval, pupal densities and the %IE on each day of sampling was calculated to compare the effect of the dosages.

The number of days during which the emergence inhibition or the reduction of densities of pupae/larvae exceeded 80% was considered as the duration of effectiveness. The inhibition of emergence and the reduction of the density of larvae and pupae were below 50% in all the three types of habitats selected and at all the dosages of diflubenzuron tested (25, 50, 75 and 100 g/ha active ingredient) on the first day post-treatment. More than 80%

emergence inhibition or reduction of larval/pupal density could be observed from days 2–3 post-treatment onwards at the effective dosages. The duration of effectiveness was relatively longer and more pronounced, as determined by the %IE, because of the cumulative effect, compared with the density of pupae and larvae.

The residual effect of diflubenzuron was generally longer in disused wells than in drains and cesspits at all the application rates.

In cesspits, both 2% GR and 25% WP formulations were equally effective, yielding >80% IE for 7 days at all application rates. The effective control and the residual activity were independent of the dosages. In the DT formulation, the lower three dosages did not

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give effective control, i.e. >80% IE. The higher dosages (100 g/ha active ingredient and 1 tablet/m²)gave >80% IE for 7–10 days; the activity did not differ significantly between these two dosages. All the selected dosages need to be applied at weekly intervals. In drains, the 25% WP formulation yielded >80% IE for 7 days at 50, 75 and 100 g/ha active ingredient, and these dosages were better than 25 g/ha active ingredient. Only 100 g/ha active ingredient of GR produced >80% IE for 7 days. The DT formulation did not give effective control at the four dosages of 25–100 g/ha active ingredient. A still higher dosage was required, and 1 tablet/m² was found effective in giving >80% IE for 14 days post-treatment. In wells, the 25% WP formulation gave >80% IE for 2 weeks at all application rates, with no significant difference between the dosages. The 2% WG formulation was not effective at 25 g/ha active ingredient. At higher dosages (50, 75 and 100 g/ha active ingredient), this formulation produced >80% reduction for a period

>2 weeks; the %IE did not vary between the three dosages. In this habitat, the DT formulation was effective only at 1 tablet/m², giving

>80% IE for 7 days.

In conclusion, 25 g/ha active ingredient of 25% WP formulation could be the field application dosage for cesspits and wells, and 50 g/ha active ingredient for street drains, to be applied at weekly intervals. The dosages 25, 50 and 100 g/ha active ingredient of 2%

GR could be the optimum field dosages for application in cesspits at weekly intervals, in disused wells every 2 weeks and in drains at weekly intervals, respectively. The 2% DT formulation has to be applied at 100 g/ha active ingredient in cesspits weekly and at 1 tablet/m²every 2 weeks in drains and weekly in disused wells.

Penang, Malaysia

The efficacy and residual effects of IGR diflubenzuron (DT and GR) and VectoBac (Bti) (DT and WG, both 3000 ITU/mg) were

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determined against Ae. aegypti in glazed earthen jars and plastic containers (Zairi Jaal, 2005). The methodology and results of VectoBac evaluation are summarized in section 3.3. The earthen jars used were 40 cm high with a rim diameter of 48 cm at the top and 30 cm at the base. The plastic containers were 45 cm high, with a rim diameter of 44 cm at the top and 27 cm at the base. In both types of containers, 50 litres of water were used. The two formulations of diflubenzuron were applied at two dosages (0.4 and 0.08 mg/litre active ingredient). Each dosage and a control with untreated water were replicated five times. The jars/containers were set around the residential houses outdoors, under shade (in places protected from rain), covered with a fine-mesh net (mesh size less than 0.5 mm) to prevent field mosquitoes from laying eggs. The water was allowed to stand for at least 24 hours before the experiment. All the formulations were first weighed before being manually introduced onto the water surface in the jars/containers. A total of 25 laboratory-cultured mosquito larvae (third instar) of Ae.

aegypti were introduced separately into each container at 24 hours, on day 7, at weeks 2, 3 and 4 and subsequently every 2 weeks up to 12 weeks. Larval mortality was recorded at 24 and 48 hours post- treatment. Any surviving larvae or pupae were transferred into a container and observed in the laboratory for emergence inhibition.

A day before the introduction of the larvae at each time interval, water was topped up to the water-level mark for all treated/untreated jars/containers to compensate for evaporation. The trials for each formulation were terminated when the mortality rate or emergence inhibition fell below 50%.

With diflubenzuron and in the earthen jars, 65% and 75% mortality was recorded 2 days after treatment with GR and DT formulations respectively. One week later, diflubenzuron DT and GR at both 0.08 and 0.4 mg a.i./l were effective against Ae. aegypti up to week 16, with larval mortality exceeding 95%.

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In the plastic containers, 58% mortality was recorded at both 2 days and 1 week after treatment. Diflubenzuron DT and GR at both 0.08 and 0.4 mg a.i./l were effective against Ae. aegypti up to week 16, with larval mortality exceeding 95%.

The adult emergence was almost 100% in both the formulations and at all the dosages throughout the period of study. For the untreated control, both earthen jars and plastic containers showed lower than 5% larval mortality after 24 hours and more than 86% adult emergence after 7 days throughout the whole experimental period.

Diflubenzuron DT and GR formulations were both equally effective as larvicides against Ae. aegypti for a minimum duration of 16 weeks.

2.4 Conclusions and recommendations

Dimilin^£ (diflubenzuron) is an IGR of the benzoyl urea family that inhibits the synthesis of chitin and hence interferes with moulting.

Mosquito larvae treated with diflubenzuron die during ecdysis because the ecdysing larvae fail to completely shed the old cuticle;

those that survive die in the pupal stage or during eclosion of adults.

Diflubenzuron has low-acute and chronic toxicity to mammals, with no indication of carcinogenicity, mutagenicity or teratogenicity.

Diflubenzuron is classified by WHO as unlikely to present acute hazard in normal use.

Diflubenzuron is of low toxicity to birds, fish and aquatic plants. It is highly toxic to some crustacea and macro-invertebrates. In most studies related to impact on non-target biota, it has been shown that

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resurgence of affected target and non-target organisms occurs rather quickly.

Laboratory studies have indicated that diflubenzuron is highly effective against mosquito larvae and pupae at the dosages of 1–10 µg/litre active ingredient. Aedes species were found to be more susceptible to diflubenzuron than Anopheles and Culex species (Table 1).

Previously published studies on the activity of diflubenzuron granules and wettable powder formulations against the natural populations of Culex and Anopheles species have shown that they were effective for 11–27 days at the application rates of 28.0–

112.0 g/ha active ingredient. These trials were conducted in a variety of mosquito breeding habitats, such as rice fields, ponds, lagoons and irrigated pastures (Table 2).

In the WHOPES supervised trials (Table 3), the 2% DT, 2% GR and 25% WP of diflubenzuron have been evaluated against Anopheles and Culex species in rice fields, wells, cesspits, ponds and street drains. The GR and WP formulations were effective for 7–14 days at the application rates ranging from 25–100 g/ha active ingredient. Both formulations were equally effective. The DT formulation was effective at 100 g/ha active ingredient in cesspits and street drains. In polluted habitats and deeper bodies of water with high organic load, higher dosages were needed.

Against the container-breeding mosquito, Ae. aegypti, the efficacy of GR and DT formulations lasted for 4–5 months at the dosages of 0.02–1.0 mg/litre active ingredient. Both formulations exhibited comparable efficacy (Table 3).

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Noting the above, the Meeting recommended:

x The use of diflubenzuron 2% GR and 25% WP for mosquito control in open bodies of water at the dosage of 25–100 g/ha active ingredient. The higher dosages are required in polluted and vegetated habitats. Lower dosages may be sufficient for the control of flood-water mosquitoes.

x The use of diflubenzuron 2% DT and 2% GR for the control of container-breeding mosquitoes, such as Ae. aegypti, at the dosage of 0.02–0.25 mg/l active ingredient, with an expected residual activity of 2–4 months. The higher rates of application are recommended for containers with exposure to sunlight or with a high organic content.

x Industry to consider placing grooves on tablets to facilitate their breaking and the proper dosing for application to smaller containers.

x WHO should carry out a safety assessment of diflubenzuron for application to drinking-water.

Note: WHO recommendations on the use of pesticides in public health are valid ONLY if linked to WHO specifications for their quality control.³

3 WHO specifications for public health pesticides are available on the WHO home page on the Internet at http://www.who.int/whopes/quality/en/.

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Activity of diflubenzuron against Anopheles, Culex and Aedes mosquito larvae in the y es Location Formulation Larval instar Lethal concentration (mg/litre active ingredient) IE50IE90 quadrimaculatus DT-susceptible) Florida, USA Technical 4 0.003 0.004 quadrimaculatus ) 4 0.002 0.003 eniorhynchus usceptible) 4 0.001 0.002 eniorhynchus sistant) 4 0.001 0.003 Tehran, Iran (Islamic Republic of)

Technical 3 0.003 0.010 ciatus 3 0.036 0.134 pti (Ghana strain)Montpellier, France Technical 4 0.0005 0.0035 bopictusFlorida, USA Technical4 0.00045 0.00084 ciatus Dhaka, Bangladesh Technical 4 0.0014 0.0034

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Efficacy of diflubenzuron (25% WP) against various mosquito species in field conditions es HabitatLocation Dosage Mortalitya (%)Emergence inhibition (%) Reference 22.4 g/ha 75–100 omaculis An. melanimon Irrigates pastures California, USA 28–56 g/ha 100 Schaefer et al., 1975 28 g/ha 100 92–99.5 Flooded mangrove Florida, USA 56 g/ha 100 100 Dame et al., 1976 nsis Irrigated ditch Gezira, Sudan 15.2 g/ha 80–95 (7) 85–100 El Safi and Haridi, 1986 22.4 g/ha 100 (9) 95–100 Irrigated pastures California, USA 50.4 g/ha 100 (9) 100 Schaefer et al., 1975 onfinnis Irrigated pastures California, USA 5.6–56 g/ha 96–100 (2–3) 100 Mulla and Darwazeh, 1975 ber of days; percentage reduction of larval/pupal density measured by dipper samples.

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inued). Efficacy of diflubenzuron (25% WP) against various mosquito species in field s es Habitat Location Dosage Mortalitya (%)Emergence inhibition (%) Reference ciatus Earthen drains, cement tanks, water cress field

Jakarta, Indonesia 1 mg/litre 96 (8) 99 (6) Self et al., 1978 0.1 mg/litre 0–14 0.2 mg/litre 80 (3) 0.5 mg/litre 97–99 (5–6)

ciatus Drains Delhi, India 1.0 mg/litre 98–100 (4) No observation Sharma et al., 1979

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inued). Efficacy of diflubenzuron (25% WP) against various mosquito species in field s es Habitat Location Dosage Mortalitya (%)Emergence inhibition (%) Reference 1 mg/litre 90–100 (28) 2 mg/litre 95–100 (28)

Septic tank Parak, Malaysia 4 mg/litre 95–100 (56)

No observation Lam, 1990 10 20andDrains Assam, India 40

93–96 (3) No observation Baruah and Das, 1996 ber of days; percentage reduction of larval/pupal density measured by dipper samples.

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Efficacy and persistence of diflubenzuron formulations against immatures of Culex, elesand Aedesmosquitoes as determined in WHOPES supervised small- and/or studies Test sites Mosquito species Formulation Dosage (active ingredient) Efficacy days (>90% IE) ge jars (200-litre) Ae. aegypti 2% DT 0.02 mg/litre 152 0.05 mg/litre 161 0.1–1.0 mg/litre 168 Water-storage jars (half-refilled) Ae. aegypti 2% DT 0.1 mg/litre 126 0.5–1.0 mg/litre 147 Water-storage jars (200-litre) Ae. aegypti 2% GR 0.02–1.0 mg/litre 161 Water-storage jars (half-refilled) Ae. aegypti 2% GR 0.1 mg/litre 105 0.5 mg/litre 152 1.0 mg/litre 168 0.05–1.0 mg/litre 147 n jars Ae. aegypti 2% DT 0.08–0.4 mg/litre 112 2% GR 0.08–0.4 mg/litre 112 Plastic containers Ae. aegypti 2% DT 0.08–0.4 mg/litre 112 2% GR 0.08–0.4 mg/litre 112

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continued. Efficacy and persistence of diflubenzuron formulations against immatures of heles and Aedes mosquitoes as determined in WHOPES supervised small- and/or ale field studies Test sites Mosquito species Formulationa Dosage (active ingredient) Efficacy days (>90% IE) (1–15 m2 )Cx,quinquefasciatus25%WP 25–75 g/ha7 100 g/ha 10 2% GR 25–100 g/ha 7 2% DT 100 g/ha 7 1 tablet/m2 10 Drains (4–6 m2 )Cx,quinquefasciatus25% WP25 g/ha4 50–100 g/ha 7 2% GR 25–75 g/ha4 100 g/ha 7 2% DT 1 tablet/m2 15 Wells (1.3–4 m2 )Cx,quinquefasciatus25% WP25–100 g/ha 7–14 2% GR 25g/ha 4 50–100 g/ha 14–17 2% DT 1 tablet/m2 7 = tablet for direct application; GR = granule; WP = wettable powder.

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continued. Efficacy and persistence of diflubenzuron formulations against immatures of heles and Aedes mosquitoes as determined in WHOPES supervised small- and/or studies Test sites Mosquito species Formulation Dosage (active ingredient) Efficacy days (>90% IE) n (Islamic blic of) Simulated artificial ponds Anopheles 25% WP 15 mg/m2 30 10 mg/m2 13 5 mg/m2 9 2.5 mg/m2 5 2% GR 15 mg/m2 26 10 mg/m2 13 5 mg/m2 9 2.5 mg/m2 4 Culex25% WP 15 mg/m2 30 10 mg/m2 9 5 mg/m2 7 2.5 mg/m2 3 2% GR 15 mg/m2 30 10 mg/m2 14 5 mg/m2 7 2.5 mg/m2 3