The control of anopheline mosquitoes, vectors of. malaria has long been by the use of residual adulticides. applied to the interiors of dwellings.

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1 ABSTRACT The control of anopheline mosquitoes, vectors of malaria has long been by the use of residual adulticides applied to the interiors of dwellings. The most commonly used types of equipment in these spraying operations are compression sprayers with flat fan nozzles applying wettable powder formulations as a high volume spray. Ultra low volume (ULV) spraying is a technique now widely established in many pest control operations, especially in the tropics where access to large volumes of water may be a problem. ULV spraying has not however been widely used for malaria vector control because of a lack of suitable equipment. This study describes the use of a hand-held ULV electrostatic sprayer, the ICI Electrodyn, in this type of control operation. The Electrodyn sprayer in its commercially available agricultural form, the Bozzle sprayer, and its oil-based formulations were evaluated in the laboratory to assess wall coverage, floor contamination and formulation persistence on representative surfaces. The sprayer and its formulations were also tested in the field in a comparative trial with compression sprayers and wettable powders. The performance of the sprayer was considered good enough to warrant further investigation, and possible redesigning is suggested to produce a machine more suited to the specific requirements of the malaria control operation. The advantages of the machine in terms of ease of use, time savings and increased safety i

2 factors is discussed, and the role of the sprayer in some alternative community regimes is approaches to malaria control, such as participation and revised dose and cycle considered. ii

3 CONTENTS PAGE ABSTRACT...i CONTENTS... ii LIST OF FIGURES... viii CHAPTER ONE - INTRODUCTION HISTORY OF MALARIA CONTROL DDT PIRIMIPHOS METHYL PYRETHOIDS SPRAYING TECHNIQUES - HIGH VOLUME AND ULV SPRAYING DOSE AND CYCLE OF INSECTICIDE SPRAYING OPERATIONS AIMS OF STUDY...30 CHAPTER TWO - ELECTROSTATIC SPRAYING INTRODUCTION ELECTRODYNAMIC DROPLET GENERATION THE DESIGN OF THE ELECTRODYN SPRAYER THE APPLICATION OF ELECTROSTATICS IN MOSQUITO CONTROL CHAPTER THREE - DROPLET AND DEPOSITION STUDIES INTRODUCTION DROPLET SAMPLING AND ANALYSIS SWATH DIMENSION AND DISTANCE FROM WALL Method Results Discussion iii

4 3.4 THE USE OF DEFLECTRODES Introduction Method Results Discussion DROPLET DEPOSITION IN CORNERS Introduction Method Results Discussion THE EFFECT OF NOZZLE POTENTIAL Introduction Method Results Discussion THE DESIGN OF THE DEFLECTRODES Introduction Method Results Discussion DROPLET DISTRIBUTION NEAR THE FLOOR Introduction Method Results Discussion DEPOSITION WITH HIGH FLOW RATES Introduction Method iv

5 3.9.3 Results Discussion THE EFFECT OF FORMULATION ON DROPLET CHARACTERISTICS Introduction Method Results Discussion FIELD STUDIES ON DEPOSITION Introduction Method Results Discussion CHAPTER FOUR - LABORATORY BIOASSAYS INTRODUCTION METHODS Mosquito Culture Treatment of Test Surfaces Bioassay Procedure RESULTS DISCUSSION Insecticide Effectiveness CHAPTER FIVE - FIELD TRIALS EXPERIMENTAL HUT TECHNIQUES EXPERIMENTAL HUT SITES Location and Site Description Experimental Huts Mosquito Species in Huts v

6 5.2.4 Collecting Methods Treatment of Results Mortality Repellency Deterrency Feeder Survivor Index FIELD TRIAL Spraying Methods Mosquito Mortalities FIELD TRIAL Spraying Methods Susceptibility Testing Mosquito Mortalities DISCUSSION OF CONTROL SUCCESS Anopheline Mortalities Culicine Mortalities THE EFFECTS OF INSECTICIDES ON BEHAVIOUR Repellency Deterrency Feeding Success CHAPTER SIX - DISCUSSION AND CONCLUSIONS SPRAYER PERFORMANCE FORMULATION EFFECTIVENESS POTENTIAL ADVANTAGES OF THE ELECTRODYN SYSTEM CONSIDERATIONS REGARDING THE USE OF THE ELECTRODYN SPRAYER IN THE FIELD FURTHER DEVELOPMENTS CONCLUSIONS vi

7 REFERENCES SUMMARY ACKNOWLEDGEMENTS APPENDIX 1.1 DISTRIBUTION OF DROPLETS IN CORNERS... i APPENDIX 1.2 EFFECT OF NOZZLE POTENTIAL ON DROPLETS.iii APPENDIX 1.3 APPENDIX 1.4 EFFECT OF DEFLECTRODE SHAPE ON DEPOSITION... V DEPOSITION CHARACTERISTICS NEAR FLOOR..vii APPENDIX 1.5 DEPOSITION WITH HIGH FLOW RATES... xiii APPENDIX 1.6 EFFECT OF FORMULATION ON DROPLET CHARACTERISTICS... xvi i i APPENDIX 1.7 APPENDIX 2.1 APPENDIX 3.1 APPENDIX 3.2 FIELD DEPOSITION STUDIES...xxiii BIOASSAY DATA... xxiv FIELD SUSCEPTIBILITY TESTS... xxvi i DATA FROM TRAP HUT TRIALS xxix APPENDIX 3.3 DATA FROM TRAP HUT TRIALS xxxvii vii

8 LIST OF FIGURES PAGE FIGURE 2.1 Schematic Diagram of Bozzle Unit FIGURE 3.1 FIGURE 3.2 FIGURE 3.3 FIGURE 3.4 FIGURE 3.5 Diagram of Spraying Methods for Corners...52 Deposition in Corners...54 Effect of Nozzle Potential on Deposition..59 Types of Deflectrode...63 Effect of Deflectrode Shape on Deposition FIGURE 3.6 Effect of Wall Position on Deposition FIGURE 3.7 Effect of High Flow Rates on Deposition FIGURE 3.8 Effect of Formulation on Droplet Characteristics FIGURE 4.1 DDT Bioassay FIGURE 4.2 Pirimiphos Methyl Bioassay FIGURE 4.3 FIGURE 4.4 FIGURE 4.5 Permethrin Bioassay...99 Cypermethrin Bioassay...99 Bioassay with Blank EF FIGURE 4.6 Effect of Exposure Time on Mortality MAP 1 Magugu Area, Tanzania MAP 2 Plan of Louvre Trap Hut Site MAP 3 Plan of Verandah Trap Hut Site FIGURE 5.1 FIGURE 5.2 FIGURE 5.3 Basic Trap Hut Design Concrete Pillar and Ant Prevention Channel Louvres (open) with Louvre Trap in Position FIGURE 5.4 Verandah Trap Hut FIGURE 5.5 Diagram of Verandah Trap Hut FIGURE 5.6 Diagram of Louvre Trap Hut viii

9 FIGURE 5.7a Weekly Mean Totals of Mosquitoes - Verandah Trap Huts FIGURE 5.7b Weekly Mean Totals of Mosquitoes - Louvre Trap Huts FIGURE 5.8a Weekly Mean Totals of Mosquitoes - Verandah Trap Huts FIGURE 5.8b Weekly Mean Totals of Mosquitoes - Louvre Trap Huts FIGURE 5.9 Mortality/FSI of A.arabiensis - Cypermethrin EF 0.4 g/m2 (Grass Roof Verandah Trap Hut FIGURE 5.10 Mortality/FSI of A.arabiensis - Cypermethrin WP 0.4 g/m2 (Grass Roof Verandah Trap Hut FIGURE 5.11 Mortality/FSI of A.arabiensis - DDT WP 2 g/m2 (iron Roof Verandah Trap Hut FIGURE 5.12 Mortality/FSI of A.arabiensis - DDT EF 2 g/rr\2 (Mud Roof Verandah Trap Hut) FIGURE 5.13 Mortality/FSI of A.arabiensis - Pirimiphos Methyl EF 2 g/m2 (Grass Roof Louvre Trap Hut) FIGURE 5.14 Mortality/FSI of A.arabiensis - Pirimiphos Methyl EF 2 g/m2 (Iron Roof Louvre Trap Hut) FIGURE 5.15 Mortality/FSI of A.arabiensis - Pirimiphos Methyl WP 2 g/m2 (iron Roof Louvre Trap Hut) FIGURE 5.16 Mortality/FSI of A.arabiensis - Pirimiphos Methyl WP 2 g/m2 (Mud Roof Louvre Trap Hut) FIGURE 5.17 Mortality/FSI of A.arabiensis - Cypermethrin EF 80 mg/m2 (Grass Roof Louvre Trap Hut) FIGURE 5.18 Mortality/FSI of A.arabiensis - Cypermethrin EF 80 mg/m2 (Iron Roof Louvre Trap Hut) FIGURE 5.19 Mortality/FSI of A.arabiensis - Cypermethrin WP 80 mg/m2 (Mud Roof Louvre Trap Hut) ix

10 Mortality/FSI of A.arabiensis - Cypermethrin WP 80 mg/m^ (Grass Roof Louvre Trap Hut)... Mortality/FSI of A.funestus - Cypermethrin EF 80 mg/m^ ("iron Roof Louvre Trap Hut)... Mortality/FSI of A.funestus - Cypermethrin EF 80 mg/m2 ("Grass Roof Louvre Trap Hut)... Mortality/FSI of A.funestus - Cypermethrin WP 80 mg/m^(mud Roof Louvre Trap Hut)... Mortality/FSI of A.funestus - Cypermethrin WP 80 mg/m2 (Grass Roof Louvre Trap Hut)... Mortality/FSI of A.arabiensis - Cypermethrin EF 40 m g / m 2 (iron Roof Verandah Trap Hut)... Mortality/FSI of A.arabiensis - Cypermethrin EF 40 mg/m2 (Mud Roof Verandah Trap Hut)... Mortality/FSI of A.arabiensis - Cypermethrin EF 40 mg/m2 (Grass Roof Verandah Trap Hut)... Mortality/FSI of A.funestus - Cypermethrin EF 40 mg/m2 Tlron Roof Verandah Trap Hut)... Mortality/FSI of A.funestus - Cypermethrin EF 40 mg/m2^grass Roof Verandah Trap Hut)... Mortality/FSI of A.funestus - Cypermethrin EF 40 mg/m2 (Mnd Roof Verandah Trap Hut)... Mortality/FSI of C.p.quinquefasciatus Cypermethrin EF 80 mg/m2 (iron roof Louvre Trap Hut)... Mortality/FSI of C.p.quinquefasciatus Cypermethrin EF 80 mg/m2 (Grass Roof Louvre Trap Hut)... Mortality/FSI of C.p.quinquefasciatus Cypermethrin WP 80 mg/m2 (Mud roof Louvre Trap Hut) x

11 5.34 Mortality/FSI of C,p,quinquefasciatus Cypermethrin WP 80 mg/m2 (Grass Roof Louvre Trap Hut) Mortality/FSI of C.p.quinquefasciatus Cypermethrin EF 40 mg/m2 (iron roof Verandah Trap Hut) Mortality/FSI of C,p.quinquefasciatus Cypermethrin EF 40 mg/m^ (Grass Roof Verandah Trap Hut) Mortality/FSI of C.p,quinquefasciatus Cypermethrin EF 40 mg/m2 (Mud roof Verandah Trap Hut) Repellency of Cypermethrin 0.4 g/m2 EF and WP against A.arabiensis Repellency of DDT 2 g/m2 e F and WP against A.arabiensis Repellency of Cypermethrin 80 mg/m2 EF (Iron Roof Louvre Trap Hut) Repellency of Cypermethrin 80 mg/m2 EF (Grass Roof Louvre Trap Hut) Repellency of Cypermethrin 80 mg/m2 WP (Grass Roof Louvre Trap Hut) Repellency of Cypermethrin 80 mg/m2 WP (Mud Roof Louvre Trap Hut) Repellency of Cypermethrin 40 mg/m2 EF (Iron Roof Verandah Trap Hut) Repellency of Cypermethrin 40 mg/m2 EF (Mud Roof Verandah Trap Hut) Repellency of Cypermethrin 40 mg/m2 EF (Grass Roof Verandah Trap Hut) Deterrency of Cypermethrin 0.4 g/m2 EF and WP (Verandah Trap Huts) Deterrency of DDT 2 g/m2 e F and WP (Verandah Trap Huts) Deterrency of Cypermethrin 80 mg/m2 EF (Iron Roof Louvre Trap Hut) Deterrency of Cypermethrin 80 mg/m2 EF (Grass Roof Louvre Trap Hut) Deterrency of Cypermethrin 80 mg/m2 WP (Grass Roof Louvre Trap Hut) xi

12 FIGURE 5.52 FIGURE 5.53 FIGURE 5.54 FIGURE 5.55 FIGURE 5.56 FIGURE 5.57 FIGURE 5.58 FIGURE 5.59 FIGURE 5.60 FIGURE 5.61 FIGURE 5.62 FIGURE 5.63 FIGURE 5.64 FIGURE 5.65 FIGURE 5.66 FIGURE 5.67 Deterrency of Cypermethrin 80 mg/m2 WP (Mud Roof Louvre Trap H u t ) Deterrency of Cypermethrin 40 mg/m2 EF (Iron Roof Verandah Trap Hut) Deterrency of Cypermethrin 40 mg/m2 EF (Mud Roof Verandah Trap Hut) Deterrency of Cypermethrin 40 mg/m2 EF (Grass Roof Verandah Trap Hut) Feeding Success of A.arabiensis in Hut Treated with DDT WP (2 g/m2) Feeding Success of A.arabiensis in Hut Treated with DDT EF (2 g/m2) Feeding Success of A.arabiensis in Hut Treated with Cypermethrin (0.4 g/n\2).200 Feeding Success of A.arabiensis in Iron Roof Verandah Trap Hut Treated with Cypermethrin EF (40 mg/m2 ) Feeding Success of A.arabiensis in Mud Roof Verandah Trap Hut Treated with Cypermethrin EF (40 mg/m2) Feeding Success of A.arabiensis in Grass Roof Verandah Trap Hut Treated with Cypermethrin EF (40 mg/m2) Feeding Success of A.arabiensis in Iron Roof Louvre Trap Hut Treated with Cypermethrin EF (80 mg/m2) Feeding Success of A.arabiensis in Mud Roof Louvre Trap Hut Treated with Cypermethrin WP (80 mg/m2 ) Feeding Success of A.arabiensis in Grass Roof Louvre Trap Hut Treated with Cypermethrin (80 mg/m2) Feeding Success of A.funestus in Iron Roof Verandah Trap Hut Treated with Cypermethrin EF (40 mg/m2 ) Feeding Success of A.funestus in Mud Roof Verandah Trap Hut Treated with Cypermethrin EF (40 mg/m^) Feeding Success of A.funestus in Grass Roof Verandah Trap Hut Treated with Cypermethrin EF (40 mg/m2 ) xii

13 FIGURE 5.68 FIGURE 5.69 FIGURE 5.70 FIGURE 5.71 FIGURE 5.72 FIGURE 5.73 FIGURE 5.74 FIGURE 5.75 FIGURE 5.76 Feeding Success of A.funestus in Iron Roof Louvre Trap Hut Treated with Cypermethrin EF (80 mg/m2) Feeding Success of A.funestus in Mud Roof Louvre Trap Hut Treated with Cypermethrin WP (80 mg/m2 ) Feeding Success of A.funestus in Grass Roof Louvre Trap Hut Treated with Cypermethrin (80 mg/m2 ) Feeding Success of C.p.quinquefasciatus in Iron Roof Verandah Trap Hut Treated with Cypermethrin EF (40 mg/m2 ) Feeding Success of C.p.quinquefasciatus in Mud Roof Verandah Trap Hut Treated with Cypermethrin EF (40 mg/m2) Feeding Success of C.p.quinquefasciatus in Grass Roof Verandah Trap Hut Treated with Cypermethrin EF (40 mg/m2) Feeding Success of C.p.quinquefasciatus in Iron Roof Louvre Trap Hut Treated with Cypermethrin EF (80 mg/m2) Feeding Success of C.p.quinquefasciatus in Mud Roof Louvre Trap Hut Treated with Cypermethrin WP (80 mg/m2 ) Feeding Success of C.p.quinquefasciatus in Grass Roof Louvre Trap Hut Treated with Cypermethrin (80 mg/m2) xiii

14 CHAPTER ONE INTRODUCTION 1.1 HISTORY OF MALARIA CONTROL Malaria is one of the most important diseases in the history of man. Much progress has been made over the last forty years in reducing the incidence of malaria, but the disease stills exists as a threat to more than two thousand million people worldwide (Busvine 1978). Great advances have undeniably been made in reducing the areas at risk from malaria, notably in the Mediterranean countries; but the major successes have really been made only at the periphery of the distribution of the disease, and in areas of the highest endemicity, Africa, the problem remains very acute. such as Central In areas such as South East Asia and Central America, after ten years or so of improvement, malaria is now coming back. Overall the total number of people worldwide freed from malaria rose from 317 million in 1961 to 728 million in 1971, but by 1976 had fallen to 436 million (Busvine 1978). Before World War II, the control of anopheline mosquitoes had been achieved primarily through attacking the larval stage by drainage, by introduction of the larval predator Gambusia affinis and by applications of either Paris Green, a crystal compound of copper acetoarsenite applied at 1.1 kg/ha, or larvicidal oil at 235 1/ha (Russell 1955). Spraying with a pyrethrum emulsion was also carried out with some success, for example in 1

15 India and South Africa (Ross 1935). In 1943, DDT became widely available for the first time, and was initially used against anopheline larvae, however the value of DDT as a residual treatment was quickly recognised. The current insecticidal approach to malaria control was developed following studies made by the Rockefeller Foundation in Sardinia between 1946 and Initially the aim was to eradicate the local vector, Anopheles labranchiae by larviciding with DDT, but with a switch to the use of DDT as a residual treatment in dwellings came a realization that malaria eradication was possible by interrupting the transmission of the Plasmodium parasite instead of total elimination of the vector. In this way, malaria was totally eradicated from Sardinia, although the vector continued to be present, albeit in lower numbers than before. The success of the residual treatment approach to malaria control is due to the house being the point of vector-host contact for these species. A female anopheline mosquito is infective only if it survives the period of time following the taking of an infected blood meal necessary for the completion of the sexual cycle of the Plasmodium. This critical time varies with different Plasmodium species; for example it is 8-10 days with P.vivax and days with P.falciparum. The mosquito feeding cycle is typically 2-3 days, so it is not until the 4th or 5th feeding cycle after the initial infection that the mosquito itself becomes infective. Therefore 2

16 residual spraying aims to eliminate the possibility that an infected female entering a house to feed survives to an infective age (Macdonald 1957). In the years between 1949 and 1955, joint World Health Organization and national malaria control programmes were set up in many countries, based mainly on DDT residual spraying. Many areas were successfully treated, for example, in India, 30 million people were protected by 1953, and only a year later, the figure was as high as 64 million. This resulted in a reduction of the proportion of malaria cases to total dispensary cases from 11% to 4% by 1958 (Pal 1962). In the Philippines, just one year's extensive spraying reduced the parasite rate from 41.6 to 4.6, and the infant parasite rate to 0 (Dy 1954). Large areas of the Middle East were under treatment by 1955, and all of the six countries in Europe affected by malaria, where the total number of malaria cases fell from 4-5 million prior to 1945 to a few thousand by 1955 (Brown et. ajl 1976). National control operations in Africa were more limited, with organized spraying only being carried out in South Africa, Swaziland, Rhodesia (now Zimbabwe) and the islands of Madagascar and Mauritius, with varying degrees of success (W.H.O 1957a, Dowling 1951, 1953, Pampana 1951). Malaria was eradicated from the U.S.A at this time, and many South American countries successfully brought about some level of control. Over the period of National Control Campaigns, much 3

17 was learnt about the dosage and number of treatments required annually in different areas, and certain drawbacks concerning the use of DDT became apparent. The significance of the excito-repellent effect of DDT on anophelines, first noted in the laboratory by Kennedy (1947), was realised. In Uganda, 38% of A.qambiae entering mud huts treated with 2 g/m^ DDT survived (de Zulueta et al^ 1961). Other residual insecticides were beginning to be used, in particular gamma-hch (lindane) and dieldrin. By 1955 however, despite the successes, there were still approximately 200 million cases of malaria worldwide, with 2 million deaths. In 1955, the Eighth World Health Assembly proposed a world malaria eradication campaign, co-ordinated by W.H.O. This campaign, designed as a number of internationally co-ordinated national programmes supported by multilateral or bilateral funds, aimed at total coverage of all dwellings in malarious areas using a residual insecticide. The plan was to synchronize operations so that neighbouring national campaigns would protect each other against re-introduction of the disease. Each national campaign (in the absence of previous operations) was estimated to last for eight years following a proposed set of phases (W.H.O 1957b): 1. Preparatory Phase. Initial survey of each country to establish limits of malarious areas, to decide on priority of treatment, staff recruitment and pilot operation. This would last up to one year. 2. Attack Phase. Operations were aimed to give total 4

18 insecticide coverage of all dwellings in the proposed areas. After one year, evaluation of success was carried out by house to house investigation of malaria cases. The attack phase was to be maintained until transmission was interrupted and the parasite eradicated. This was expected to take four years but in practice was often longer. 3. Consolidation Phase. Elimination of residual pockets of transmission by drugs or re-spraying. This was to be continued until no new indigenous cases had been discovered over a three year period. 4. Maintenance Phase. The malaria eradication organization in the area was to be disbanded or redeployed, with introduced malaria cases being dealt with by the area public health department. If the absence of detected indigenous cases could be shown to have been based on an adequate surveillance system, and if alleged imported cases were so proven, the country or major area could be registered with the World Health Organization as malaria free. The campaigns were based on residual treatments, mainly DDT, but the following insecticides were also used: dieldrin, particularly in South America, and gamma- HCH (lindane), mainly in Southern Africa. The programme, although very ambitious, did have major successes. The attack phase reached its peak in 1961, and by 1968 nearly 80% of the total population in malarious areas were covered by some stage of the campaign, and malaria 5

19 eradication was successfully effected in 36 countries (the majority of these being outside the tropics) (Brown et a_l 19 76). Full accounts of the progress of these campaigns can be found in the W.H.O Chronicle (W.H.O ). By 1968 however, despite the obvious successes of the programme, it was clear that the majority of the continental countries in tropical regions were still at the attack phase over large areas, and insecticide resistance was fast becoming a major problem. Physiological resistance in Anopheles to DDT probably first appeared in Greece and the U.S.A in In 1955 dieldrin resistance was shown in A.gambiae in Africa. In 1980 the W.H.O Expert Committee on Vector Biology and Control (Fifth Report) noted that 51 species of anopheline mosquitoes were resistant to one or more insecticides: 34 were resistant to DDT, 47 to dieldrin o and 30 to both DDT and dieldrin. Organophjsphate resistance has been recorded in ten species and carbamate resistance in four. Pyrethroid resistance has been demonstrated in laboratory strains and also by A.sacharovi in Turkey. The widespread use of insecticides in agriculture has resulted in the rapid development of resistance in many species of anopheline mosquitoes. In consequence, an increasingly greater range of insecticides has been used, and the relatively high cost of these has created major problems for many Third World countries. Malathion, despite costing five times as much, was substituted for DDT in treating malaria foci, reappearing 6

20 in consolidation areas in India and Turkey, and in the attack phase in Iran, Afghanistan and Iraq. In El Salvador, malathion resistance was shown by A.albimanus, and therefore propoxur was used in many areas of Central America at 20 times the cost of DDT. However by 1971, propoxur resistance was shown by A.albimanus in El Salvador and Nicaragua (Georghiou 1972). The insecticide resistance problems were compounded by the development of chloroquine resistance in the Plasmodium parasite, which has since become widespread. The occurrence of aspects of behaviour in anophelines which contribute to control failures have been termed "behaviouristic avoidance" (Muirhead Thomson 1960, W.H.O 1960). This term covers all cases where a mosquito shows an ability to avoid lethal contact with the treated surface on which, in the absence of treatment, it would normally rest; and includes instances where the irritability and avoidance are natural ("protective avoidance") or developed as a result of insecticide pressure ("behaviouristic resistance"). There are also other aspects of behaviour which may be altered by contact with insecticides, notably reduction in feeding or feeding success. There has been much work to try and establish clearly the existence of ethological or behaviouristic resistance. It has been well demonstrated that the general phenomenon of behaviouristic avoidance is shown by several species in varying degrees, but to show that this has developed as a 7

21 result of insecticide pressure is rather more difficult. Elliott and de Zulueta (1975) reviewed a number of papers on the subject, and concluded that the daytime resting patterns of the great majority of malaria vectors included facultative daytime exophily even before the habit became advantageous in terms of survival. They showed that several DDT spraying operations had resulted in apparent changes in the behaviour of the population between the pre-ddt period and the earliest period of its use, and possibly also after some years of use. The authors did conclude that there were numerous examples in the literature of changes in the behaviour of various species towards reduced contact, by day or by night, with sprayed surfaces, that may possibly be more than the effect of the sum of reactions of individuals to the irritant, excitant or locomotor-stimulant properties of DDT. This has proved difficult to confirm by selection of strains in the laboratory, and although not disproved, only a few studies suggest a basis for the selection of behaviour patterns which confer protection to the treated areas (Gerold and Laarman 1964). There have been several reports of species returning to endophily after spraying had ceased, whereas there is no recorded case in which exophily acquired possibly as a response to insecticidal treatment continued after cessation of spraying. This is totally different to acquired physiological resistance, which does not necessarily disappear after the insecticide pressure is removed. There are other differences between physiological and ethological 8

22 resistance; the former has appeared in a number of taxonomic groups which are protected against a wide range of chemicals, while the latter, if it really does exist, is confined to a single genus protecting against one chemical by systems where operation and inheritance are unknown and has proved impossible to measure accurately or reproduceably. The initial optimism regarding total malaria eradication has thus waned in the light of the many problems that have developed in the course of vector control operations. While newer control measures, such as the use of repellents, biological control and genetic control are undoubtedly important, the use of residual insecticides remains the most effective method of attack; and the need is for more effective, cheaper insecticides. Vector control programmes are being jeopardised in many areas because of the high cost of replacing DDT with newer insecticides and even due to the rising cost of DDT itself (W.H.O 1975). Coupled with this is the need for more effective treatment measures, including more accurate targetting of the insecticide to reduce wastage and thus cut down costs, and the necessity to look closely at approaches to spraying programmes in terms of dosages, number and timing of treatments, and personnel requirements. These will be considered in more detail in sections 1.5 and 1.6 of this chapter. Residual spraying must also be considered as part of an overall strategy of integrated control, including other non-chemical methods of control such as biological control agents, genetic 9

23 manipulation etc. in conjunction with insecticides. 1.2 DDT DDT is without doubt the most important insecticide in the history of malaria control. The introduction of DDT in 1943 made it possible to consider the control of anopheline mosquitoes in terms of malaria eradication on a global basis (Brown et ajl 1976). The wealth of literature on the use of DDT is testament to its importance in the field of malaria control. It would be impossible to attempt a comprehensive review of this literature here, and what follows is therefore a general background to the history and importance of DDT and the problems associated with it, to justify its inclusion as an insecticide for study in this investigation. DDT was initially used in larviciding programmes, but following detailed studies in Sardinia from , where 90% of the inhabitants were infected with malaria, emphasis switched to residual treatment of dwellings with DDT in order to interrupt malaria transmission rather than attempt to eradicate the vector (Logan 1953). DDT residual spraying was the primary weapon in the national malaria campaigns until 1956, also of of the world malaria eradication campaign until 1968 and the revised strategy of control or eradication up to the present time. Brown ej: a_l estimated in that the global incidence of malaria could not exceed 120 million compared with the 300 million cases estimated to have 10

24 occurred annually before the introduction of DDT (Bruce- Chwatt and Haworth 1965), when the total population at risk was approximately half what it was in More recently there has been some decline in the use of DDT. The persistence of DDT in the environment and its accumulation in food chains, resulting in longterm damage to certain wildlife populations? and also the build-up of DDT in human body fat has lead to a ban on its use in many of the more developed countries. While this is feasible in those countries which are able to afford the more expensive alternative insecticides, it is not possible for many of the Third World countries to consider alternatives while DDT remains a relatively cheap answer to the problem. Despite the accumulative properties of DDT in the human body, there is no evidence that it is harmful to humans in the levels present, and during its intensive history of use, the only toxic effects observed have been the result of accidental ingestion. The environmental contamination associated with DDT used in malaria control is also very low. In residual spraying operations, the amount of outdoor contamination resulting from spraying a village is approximately one thousandth of that arising from treating a cotton field of the same size (Brown 1973). In fact no deleterious effects on wildlife have ever been observed during an attack phase in any country, although no studies have been made of subsurface effects, since the widespread use of organochlorines has tended to mask 11

25 any slight effects which may be present due to antimalaria operations. Another major problem which has forced the use of alternative insecticides, has been the development of physiological resistance, particularly in areas where DDT has been used extensively in control operations against pests other than mosquitoes (Chapin and Wasserstrom 1981), and also the irritancy of DDT to mosquitoes resulting in behaviouristic avoidance, as discussed in the previous section. A survey was carried out in 1980 by Smith and Lossev (1981) for the World Health Organization, to obtain information on pesticides and equipment requirements for national vector control programmes in developing countries. This covered insecticide usage from 1978 to 1979 and estimated projected requirements for 1980 to The survey showed continued heavy usage of DDT in the years 1978 and 1979, particularly in the African and American regions, but also considerable amounts in South East Asia and the Eastern Mediterranean regions. Although it is still used in the European region (especially Turkey), DDT has mostly been superceded by malathion. In the African region, DDT accounted for 92% (291.9 Metric Tonnes a.i.) and 93% (452.3 M.T. a.i.) of all insecticides used for mosquito control in 1978 and 1979 respectively, and it was estimated that the demand for DDT would rise to over 2000 M.T. a.i. per annum in In South America, DDT 12

26 accounted for 94% (4704 M.T. a.i.) and 96% (4781 M.T. a.i.) of all insecticides used in malaria control in 1978 and 1979, and estimated requirements for 1982 were over 4000 M.T. a.i. per annum representing 93% of all insecticides used. Clearly DDT is still a very widely used insecticide, and is likely to remain so for some considerable time to come. Smith (1982) concluded that with regard to residual spraying against malaria vectors "where there is no, or only limited resistance to it, DDT is still the insecticide of choice, because it is comparatively inexpensive, has a long residual effect, and an unparalleled record of safety to man". by Smith and Lossev (1981) showed an estimated The survey five-fold increase in the demand for DDT between 1979 and 1984 in the African region. The significance of DDT irritancy, first shown by Kennedy (1947), who demonstrated excitation in mosquitoes exposed to sub-lethal contacts with DDT deposits, is now known to have substantial impact on some control programmes and will be considered in greater detail in a later section. 1.3 PIRIMIPHOS METHYL Pirimiphos methyl (OMS-1424; 0,0-dimethyl 0-(3- methyl-4-nitrophenyl) phosphorothionate) is one of the newer organophosphorous insecticides. Certain organophosphates, in particular malathion and fenitrothion, are already in widespread use as residual 13

27 insecticides to control malaria vectors, especially in areas where the anophelines are DDT and dieldrin resistant (W.H.O 1975). However high costs and increasingly widespread resistance to organophosphorous compounds have intensified the search for newer effective insecticides. Organophosphate resistance has been reported in ten species of Anopheles (W.H.O 1980), mostly to malathion and fenitrothion, but also to fenthion, chlorpyrifos, chlorphoxim, phoxim, parathion, jodfenphos, temephos and methyl parathion. Pirimiphos methyl has been tested extensively in the laboratory against mosquitoes and has given promising results (Wilson, Labrecque and Thomas 1975; Wilson, Labrecque and Weidhaas 1971, 1973). It has also been tested up to Stage V in the W.H.O Programme for Evaluating and Testing New Insecticides (this scheme has recently been revised and is now the W.H.O Pesticides Evaluation Scheme - WHOPES, and consists of four phases (W.H.O 1982)). Stage V trials in the old scheme refer to village scale trials. Stage IV trials (experimental hut trials) have been carried out at Bobo-Dioulasso, Upper Volta, in specially constructed huts of both local types (mud walls with flat wood and mud roof, and mud walls with conical thatch roof), and also at Semerang, Indonesia. At Bobo-Dioulasso in 1972, pirimiphos methyl was sprayed at 2 g/m^ as two treatments each three months apart. The results were inconclusive because of low mosquito numbers and problems with ants, but it was concluded that the treatment showed sufficiently 14

28 promising activity to warrant re-evaluation (Mouchet and Vervent 1973). A 25% WP formulation was tested at Bobo- Dioulasso in 1973, again at 2 g/m^. The target vectors, as in the previous year, were A.gambiae and A.funestus. The insecticide again appeared promising but the trial was impaired by the same faults as in Bioassays with Aedes aegypti gave high though relatively shortlived mortalities (Chauvet et al 1973). A trial at Bobo- Dioulasso in 1975 with pirimiphos methyl impregnated cotton mosquito nets at 0.2 g/m2 gave good results against naturally entering A.gambiae and A,funestus (70% mortality for four weeks and 75% for eight weeks respectively), but poor results in bioassays with Ae.aegypti, the mortalities dropping to less than 50% after 15 days (Brun and Sales 1976). The trial at Semerang in 1978, where a 50% EC formulation of pirimiphos methyl was sprayed at 2 g/m2f gave mixed results; only 21.4% mortality of naturally entering A.aconitus was obtained, while bioassays with the same species gave 100% mortality for 8 weeks on wood and 91% for 8 weeks on bamboo (W.H.O VBCRU-II rept. 1978). Another house-scale trial at Semerang with the same formulation and dosage gave 36% mortality of naturally entering A.aconitus, and bioassays with this species showed over 90% mortality for 18 weeks on wood and over 85% mortality for 8 weeks on bamboo (Pradhan et. ajl 1979). There have been several village-scale trials (Stage V) with pirimiphos methyl. Rishikesh et a^l (1977) 15

29 carried out a trial with a 25% WP formulation in 1976 near Kaduna, Nigeria. A selected group of villages were sprayed at 2 g/m2 in three rounds of spraying at 3 month intervals, and resulted in a great reduction in A.gambiae and A.funestus hut resting densities, compared to those levels found in an untreated control village, for three months after spraying. Overall mean reduction for all three rounds was 9 6% for A.qambiae and 99% for A.funestus. Exit trap densities were reduced by 99% for both species. Man biting densities were less satisfactory, especially for A.qambiae, but still fairly good. Overall mean reductions were 79% indoors and 77% outdoors (A.gambiae) and 96% indoors and 84% outdoors (A.funestus). This was assumed to be due to the existence of exophilic and exophagic/zoophilic subpopulations in the ecological niches between the villages. Three more trials were carried out in Semerang, Indonesia; in 1978 a 25% WP was sprayed at 2 g/m2 in a group of hamlets against the DDT-resistant vector A.aconitus. Effective control was obtained for weeks (Shaw et. aul ). A further trial was carried out with a 50% EC formulation at 1 g/m2 in Semerang, and although the overall result was hampered by low vector densities, this dosage appeared to be effective for about 5-6 weeks (Supalin et crl 1979 ). Other successful village-scale trials have included the control of A.balabacensis in North East India, one application of 2 g/m^ gave complete control of endophagic anophelines in the Philippines for six months, and in 16

30 Pakistan a treatment of 1 g/m2 interrupted malaria transmission for several months (W.H.O, ed Smith 1982). Pirimiphos methyl thus appears to be a promising insecticide for use in areas where the vector populations are resistant to DDT. It is already becoming widely used in control operations, not only because it has a satisfactory residual effect coupled with an effective vapour toxicity, but also it is less toxic to humans than fenitrothion while having a similar level of activity. 1.4 PYRETHRQIDS The development of the synthetic pyrethroids established a new order of toxicity to insects combined with low levels of human toxicity (Elliott 1971, Elliottet al 1973, 1974). The earlier compounds, such as resmethrin, however were quickly broken down in the presence of ultra-violet light. Later work resulted in the synthesis of still more toxic and persistent compounds, which include permethrin (NRDC 143, OMS-1821), cypermethrin (NRDC 149, OMS-2002) and deltamethrin (formerly decamethrin) (NRDC 161, OMS-1998). All these compounds showed promising results in the laboratory on a variety of surfaces (Barlow and Hadaway 19 75, Barlow et. al 1977) although in bioassays with Anopheles stephensi on plywood, cypermethrin was found to be rather disappointing in view of its high toxicity when applied topically, but it was suggested that this was due to its formulation characteristics, the more viscous 17

31 cypermethrin being less easily picked up than the others. Permethrin tested at Stage IV level in the W.H.O Programme for the Evaluation and Testing of New Insecticides gave somewhat disappointing results. Coosemans and Sales (1977) in Upper Volta, found that only 15% of all A.gambiae entering and leaving experimental huts were killed by a treatment of 500 mg/m2 permethrin WP, and suggested that it was the irritant properties of permethrin at this dosage that caused the mosquitoes to leave the huts before picking up a lethal dose. aecfeti Taylor This was supported by bioassays with caged Aedes which gave high mortalities for around 10 weeks. et al^ (1981) treated a few village huts in Kenya with a lower dosage (125 mg/m2), and obtained at least 85% control until 20 weeks after treatment (control here is expressed as percentage reduction from the mean resting count in untreated huts). mortality or hut exit numbers, No data was given for and thus the concept of "control" here may simply indicate that the mosquitoes left the hut in greater numbers. A Stage V (village-scale) trial was carried out in Nigeria (Rishikesh et al 1978) with permethrin WP applied at 500 mg/m2 as two treatments 12 weeks apart, again showed long persistence and satisfactory bioassay mortalities especially after the second of the two treatments, with reduced hut resting densities. Again a high survival rate among escaping mosquitoes was shown, and the authors concluded that deltamethrin was superior 18

32 in performance (sprayed at 50 mg/m2) both with regard to higher mortalities and longer persistence on mud surfaces. Cypermethrin however, appeared much more promising than permethrin in Stage IV trials. Hervy et a_l (1982) sprayed cypermethrin WP at 500 mg/m^ in experimental huts in Upper Volta and obtained very high mortalities for at least 18 weeks, but also showed a marked decrease in the entry rate of mosquitoes. A further trial carried out in Upper Volta (Hervy and Sales 1982), compared two treatment dosages (500 mg/m^ and 250 mg/m^) and also spraying techniques - full and selective treatment. two The selective treatment consisted of spraying the hut ceiling only, since these are the preferred resting sites of the local endophilic anophelines. Good residual effect was shown on all substrates at both dosages, but only the total treatment of 500 mg/m^ showed high mortalities, and both selective treatments showed the lowest mortalities. The authors concluded that not only does cypermethrin have a deterrent effect on mosquitoes, it also has an irritant action which makes any selective treatment useless, since irritated mosquitoes either leave the huts or fly to rest on the untreated surfaces. Therefore, of the dosages tried, the authors recommended only 500 mg/m^ applied as a total treatment. The initial promise of pyrethroids shown in the laboratory was lessened when tested in the field, but the relationship between dosage and irritancy/mortality has 19

33 been shown to be complex and needs much further exploration to bring out the full potential of these insecticides. In this study, both permethrin and cypermethrin were tested in the laboratory, but cypermethrin was chosen for use in the field because of its superior performance in W.H.O field evaluations. 1.5 SPRAYING TECHNIQUES - HIGH VOLUME AND ULV SPRAYING The most widely used equipment for the application of residual insecticides has long been hand operated compression sprayers; stirrup pump sprayers have also been used, but to a much lesser extent (Symes et ajl 1962, Smith and Lossev 1981). The World Health Organization gives certain specifications for sprayers and is responsible (W.H.O 1974); for extensive evaluation of different makes commonly used compression sprayers include Hudson, Cooper-Pegler, Caleazzi, Gloria, Senior, Osatu, Mesto and Smith. Any of the range of hydraulic nozzles can be used with the compression sprayers, but the most suitable for the treatment of dwellings is the flat fan nozzle. This is because it is easier for a spray operator to assess the evenness of application provided the nozzle tip is maintained at an optimum distance of 46 cm (18 in.) from the surface (Knipe 1955). The standard nozzle for the W.H.O-recommended compression sprayers (Number 8002) discharges 760 ml/min as a flat fan-shaped spray at a spray angle of at the recommended working 20

34 pressures ( kpa or psi). A swath width of cm is applied when the nozzle is held at 45 cm from the surface (Fontaine 1978). Erosion is one of the biggest problems associated with these sprayer nozzles, particularly when used with wettable powders (the most commonly used formulations in this type of vector control). Erosion of nozzle tips can cause increased output rates and an unsatisfactory distribution pattern. Use of a compression sprayer has many disadvantages in addition to nozzle erosion. Preparation of the sprayer before treatment commences is both laborious and can be hazardous as the concentrated insecticide product must be measured and mixed with water in the correct proportions. The sprayer must be calibrated at given pressures; the method also requires access to large volumes of clean water at all spraying sites. The ultra low volume (ULV) method of insecticide application has been developed for use in pest control programmes because it results in considerable savings in money,time and labour when compared with high volume applications of water- and oil-diluted insecticides. ULV can be defined as the miniumum volume per unit area required to achieve economic control (Anon. 1971). The types of ULV application equipment tested for use in mosquito control include vehicle mounted and portable motorized aerosol generators, portable motorized 21

35 mist generators, spinning disc sprayers and aircraft mounted systems (Fontaine 1978, Boize et a J L 1975). While the aircraft mounted systems are only of value in outdoor treatments, the other sprayers have all been tested inside houses. Motta Sanchez et al (1976) compared the use of a Leco ULV Heavy Duty Type Aerosol Generator and three types of light-weight motorized knapsack mistblowers: the Fontan (R-ll), the Hudson (model 4712) and the Capri (11F) CSA, with a conventional intradomiciliary treatment using a Hudson "X-pert" high volume compression sprayer. The target vector was Aedes aegypti in selected villages in Colombia. The main advantage of the ULV treatments in this trial were shown to be speed of spraying and cost of treatment; cost benefit analysis showing that the mistblower applications produced best results for the cost. Fenitrothion and fenthion were applied, and although control was good with all forms of treatment, the high volume Hudson treatment gave the greatest residual effect, and the mistblowers gave higher kills than the aerosol generator. The three mistblowers also gave some mechanical problems, with faulty starting, engine overheating and leakage arising from loose connections. A similar trial in Enugu, Nigeria with ULV aerosol generators spraying malathion, suffered from at least 50% of the droplets bouncing off the walls and ending up as wasted deposits on the floor (Fontaine 1983). Thus it seems there is a need for ULV equipment to 22

36 apply residual treatments directly to walls and ceilings of dwellings in order to combine the speed and efficiency of ULV spraying with long residual effect. The ULV mistblowers and aerosols are better suited to Aedes rather than Anopheles control. An alternative method to provide a residual treatment was an air-assisted spinning cup sprayer (Boize et a_l 19 75), but this was not pursued due to the power requirement for moving air. A range of spinning disc sprayers have been tested, but the majority are considered unsuitable due to the wide range of droplet sizes produced. If droplets are too large (over 70 i diameter) or too small (less than 30 j) they will tend to sediment on the floor, while small droplets are also an inhalation hazard. The Micron spinning cup sprayer however gave better control over droplet size (32-43^ VMD at 10-12V DC), thus reducing floor fall-out. Bioassays indicated good residual effect against DDT resistant 2 mosquitoes with treatments of malathion down to 0.2 g/m and cypermethrin. A major drawback with this method in the field however, was the power source; a rechargeable battery was required and recharging this in remote rural areas proved to be a problem. So it is clear that while the concept of ULV spraying has many advantages over conventional treatments, many of the machines available are better suited to space spraying rather than residual applications, and that spinning disc or cup sprayers, although promising, are not ideal. 23

37 The trend in modern spray application towards ULV spraying necessitates greater care in the field. Some of the problems inherent in ULV spraying have been highlighted by Thompson (1973). Since the concentrations of active ingredient are greater with ULV spraying it follows that any errors in calibration or measurement are correspondingly magnified. The same applies to alterations in emission rates from worn or faulty equipment. Thompson also points out that the rate per unit area is not definitive and that serious consideration must be given to the number of droplets per unit area and the concentration of active ingredient in each droplet, factors which have been detailed by Mount (1970) in studies on space sprays for mosquitoes. Thus the move in application technology towards ULV spraying and the extension of this concept, controlled droplet application or CDA, which aims for closer control of droplet size, are being followed in mosquito control, but these techniques still have some way to go before they can replace high volume treatments for residual applications in malaria control. The World Health Organization Expert Committee on Vector Biology and Control, when reviewing strategies for pesticide application equipment (W.H.O 1977) recommend further research "to develop a range of different types of equipment, from hand carried machines to nozzles fitted to aircraft, by means of which droplet size could be controlled within very narrow limits". They also stated that "consideration must be given to avoiding operator

38 contamination, reducing power requirements and minimizing losses to and contamination of the environment. The capital and recurrent costs of equipment, its robustness and its ease of maintenance also need consideration". 1.6 DOSE AND CYCLE OF INSECTICIDE SPRAYING OPERATIONS The dosage rate is the amount of insecticide to be applied to a unit of surface area and is expressed as grams (or milligrams) of active ingredient per square metre of surface (g/m^). The spray cycle is the interval between successive applications of insecticides. There are recommended target rates for commonly used insecticides - DDT for example is expected to remain effective against susceptible mosquitoes for at least six weeks after one application at 2 g/m^ (Fontaine 1978). In practice however, the rate varies depending on local conditions. The factors which affect dosage and cycle requirements in a particular area include the formulation of the insecticide, the type of surface to which it is to be applied, the influence of the local climatic conditions, the seasonal abundance of the vector, the vector species encountered, the susceptibility of the mosquito populations to the chosen insecticide and the disease transmission period. Consideration must also be given to the cultural and social patterns of the local people in relation to housing practices (whitewashing of walls, smoke deposits, replastering and rethatching etc.). 25

39 In British Guiana, where the houses are typically of sawn wood, a DDT treatment of 1.6 g/m^ was finally applied at intervals of 18 months and effectively controlled the vector A.darlingi (Macdonald and Davidson 1953) whereas in Uganda, DDT applications of 2.2 g/m2 evfry six months failed to control the vector even though it was known to be susceptible to DDT. In India and Sri Lanka, the DDT dosages used were much lower and applied at shorter intervals, for example A.culifacies in Delhi was successfully controlled by 0.55 g/m^ sprayed at 6-8 week intervals. Many insecticides have caused problems besides those of physiological resistance or irritancy, due to rapid loss of toxicity on some surfaces (especially mud). The loss of toxicity of an insecticide is due either to adsorption, absorption or decomposition, and the relative importance of these processes has been the subject of some debate. of DDT on mud, Downs et al (1951) studied the deactivation and concluded that chemical deterioration in the presence of various metallic salts, such as anhydrous ferric oxide, was responsible. Much more evidence however points to sorption being the major cause (Hadaway and Barlow 1951, 1952), since it is possible to recover chemically unchanged DDT below the surface. Adsorption is thought to take place by diffusion (Barlow and Hadaway 1958) rather than from the vapour phase as was previously postulated (Hadaway and Barlow 1952). When all the particles of a non-volatile insecticide such 26

40 as DDT have disappeared from a treated surface no further insecticidal action occurs, whereas with a volatile insecticide such as gamma-hch or pirimiphos methyl, the vapour action will continue to exert a toxic effect after the insecticide has been sorbed into the mud. Bertagna (1959) cites evidence to show a relationship between the percentage of aluminium and iron oxides present and the sorptive capacity of the mud. Other factors affecting sorption include mud particle size, surface area per unit weight of mud, relative humidity (an increase in % RH decreases the rate of initial sorption, Hadaway and Barlow 1952) temperature (increases in temperature increase sorption rates, Barlow and Hadaway 1955), and insecticide particle size (small particles are more effective biologically but are sorbed much more rapidly, Hadaway and Barlow 1952). There has been much debate over the insecticidal efficiency required to control malaria. It has long been known that malaria eradication is possible without total vector eradication, but in practice it is very difficult to establish the relationship between mosquito reduction and interruption of transmission, since so many variables are involved. Garrett Jones and Grab (1964) attempted to assess insecticidal impact on the mosquito's vectorial capacity. Vectorial capacity is a density dependent expression, and is defined as the average number of innoculations with a specified parasite originating from one case of malaria in unit time, that a vector 27

41 population would distribute to man, if all the vectors biting the case became infected. They concluded that a critical level of vectorial capacity can be set, i.e. the level below which downward transmission trends occur, but it is not possible and would be misleading to suggest a similar critical level of insecticidal impact, since this will differ according to the conditions in each endemic area. Macdonald and Davidson (1953) attempted to give tentative suggestions for mortality rates as a guide to insecticidal efficiency in broad categories, and these are shown in Table 1.1. The authors simplified this further by stating that the demands of most natural circumstances would be met by attaining a mortality rate of about 65% per day among all mosquitoes entering a treated shelter, and some common ones by an insecticide with a 45% mortality rate. Thus insecticide choice and determination of dosage and cycle are subject to many influences so it is difficult to apply general rules, and all that can really be concluded is that it is necessary to have a flexible - approach to a spraying programme within the constraints of available funding, labour and time. Revised strategies regarding implementation of control programmes are also being given careful consideration. The concept of vector control at community level has been explored by Mouchet (1982). This involves the members of a village or community 28

42 TABLE 1.1 PROBABLE INSECTICIDAL EFFICIENCY WITHIN TREATED SHELTERS NECESSARY FOR CONTROL OF MALARIA (from Macdonald and Davidson 1953) DEGREE OF ENDOPHILISM OF VECTOR1 ORIGINAL MOSQUITO DENSITY COMPLETE2 MODERATE3 MEDIUM4 MORTALITY RATE (%) V.dense (250 bites/night) Dense (100 bites/night) Moderate (10 bites/night) Mild (1 bite/night) ithe first figure refers to a species with an anthropophilic index of about 10%, and the second to one with an index of 100% p Species entering treated shelter every day 3Species entering treated shelter 3 days out of 4 ^Species entering treated shelter 1 day out of 2 taking over responsibility for insecticidal treatments under the supervision of a local primary health worker or spraying specialist. This would help cut down the cost and time constraints which hinder many large scale operations, but the requirements of such a scheme would be that the equipment used must be easy to calibrate and must be simple to use and maintain. The insecticide formulations must also be easily prepared and measured with minimum hazard. Long term strategic planning is now based on theoretical work involving the construction of 29

43 mathematical models to assess the relationships between host and vector, and the impact of insecticide treatments, environmental and social factors, and insecticide and drug resistance (Ross 1916,1917, Macdonald 1950,1957, Aron and May 1982). Such an approach is necessary not only to give valuable information regarding optimum spray times etc. to assess effectively the likely success of but also integrated control programmes. The need for long persistence by residual treatments is now arguable, since the mathematical models have now shown clearly the relationships between malaria transmission and mosquito abundance (Aron and May 1982) and in areas of seasonal mosquito abundance it should only be necessary for a residual treatment to be effective for a short period over times of mosquito prevalence; this approach to residual spraying should also be advantageous in lengthening the time taken for onset of resistance to occur in a mosquito population. 1.7 AIMS OF STUDY The need for new approaches to malaria control is clear; due to problems such as insecticide resistance, rising costs and failures by chemotherapy. Residual spraying is still the most important and cost effective method available in most areas. This thesis examines the possibility of developing a hand-held ULV sprayer suitable for use in both nationally-organized spraying 30

44 programmes and community-level projects. ULV spraying has several advantages over established high volume techniques, but suitable equipment has been hard to find for this type of spraying. The comparatively recent development of electrostatic spraying techniques appears to offer significant advantages over existing ULV sprayers, and these will be considered in detail in the next chapter. 31

45 CHAPTER TWO ELECTROSTATIC SPRAYING 2.1 Introduction The concept of electrostatics as applied to the field of pest control was first introduced by Pierre Hampe duster. in the 1940's in the form of an electrostatic crop The system proved unreliable in practice however and the machine was found to be too unwieldy. The work was continued in the 1960's by Felici, using a rotary cylinder electrostatic generator, also using dusts, but failing due to the equipment being too large, costly and complicated. The triboelectrogasdynamic (tegd) sprayer invented by Coffee in the late 1960's, although a great improvement, soon became obsolete as farmers were using liquid rather than dust fomulations (Coffee 1978). Emphasis therefore changed to investigating various means of charging liquids. A number of different approaches were followed, mostly involving the use of some established means of droplet production coupled with consecutive or subsequent droplet charging. The twin fluid nozzle developed by Law (1976) was one example, whereby droplets are formed by the effect of air shearing the liquid and subsequently electrified by induction charging. Another approach has been the development of a spinning disc sprayer which charges the droplets on the disc (Arnold and Pye 1980). 32

46 The most radical development in electrostatic spraying however, has been the advent of the electrohydrodynamic, or more simply electrodynamic nozzle (Coffee 1978). This system uses a very novel approach in that, instead of relying on a conventional means of droplet production, an electrical field is used to generate the droplets, charge them and propel the spray. The basis of the system is a "no-moving-parts" device, now known as the Electrodyn sprayer, manufactured and marketed by I.C.I. Plant Protection Division. 2.2 Electrodynamic Droplet Generation The production of droplets electrodynamically is a relatively simple concept. Pesticide, flowing from a reservoir through a simple annulus nozzle at high voltage, is subjected to an intense coulombic field force on emergence. This produces a standing wave at the liquid surface, and from the crest of each wave is a uniform jet of charged liquid, similar to the ligaments produced by a rotary atomizer. Droplets are produced by the break up of these jets. The wave shape is maintained by gentle hydrostatic pressure produced by the gravity pesticide feed. The uniformity of the wave and jet dimensions mean that droplet size control is more accurate than has ever been possible before, with vmd/nmd ratios very close to 1.0. An earthed electrode located near the nozzle 33

47 maintains field strength in spite of the constantly varying distance between the nozzle and the earthed target during spraying. The characteristics of the droplets are governed by the following factors: 1) the nozzle potential, 2) the electrode geometry, 3) the flow rate, 4) the viscoelastic and electrical properties of the liquid (Coffee 1980). In the commercially available machine, all of these factors are pre-set by the manufacturer according to the requirements of the spraying operation. Droplet size control is dependent on the relationship between droplet diameter and electrical charge, which is roughly an inverse square relationship (Coffee 1979) and gives a charge to mass ratio for small droplets approximately corresponding with the Rayleigh Criterion for charged droplet stability: r 36 Y q m2 q2d2 3/2m where: r = radius of droplet 0 = permittivity of free space T = surface tension of liquid m = mass of droplet q = charge on droplet d = density of liquid Thus the charge (q) can easily be varied in the sprayer using a simple potentiometer to adjust the low 34

48 voltage input and thus the high voltage at the nozzle. The constant field force applied to each droplet while moving towards the target has the effect of reducing the other forces acting on the droplet (gravity, air movements etc.). The problem of satellite droplets has always been a feature of many spraying systems. Satellites are very fine droplets formed from a thread connecting the main droplet to the rest of the ligament or liquid on the nozzle (Matthews 1979), and as well as reducing the overall nmd of the droplets, may be small enough to be an inhalation hazard. The satellite droplets produced by the Electrodynamic nozzle differ in their behaviour from the primary droplets. Although the charge-to-mass ratio of these satellites has been shown to be similar to that of the primary droplets, the ratio of electrical to inertial forces is substantially different. The size difference is such that the inertial forces acting on the satellites are about 1000 times smaller. The tiny droplets thus have insufficient inertia to escape the locally intense electrical field near the nozzle and are propelled towards the counter electrode where they are trapped (Coffee 1980). The advantages of such a system, besides that of narrow droplet spectra, include a "wrap around" effect on the target, i.e. with the nozzle held vertically above a target, droplet deposition can be attained even on target 35

49 surfaces facing directly away from the nozzle. The similar polarity of the droplets and their consequent mutual repulsion results in a very uniform droplet distribution on the target; and possibly most important, the attraction of the droplets for the nearest earthed surface results in much more accurate targetting and has the potential to reduce pesticide wastage significantly. 2.3 The Design of the Electrodyn Sprayer The I.C.I Electrodyn Sprayer in its hand-held version has a novel design concept; the pesticide is supplied sealed in a special disposable unit which combines both a bottle and a nozzle made of conducting plastic, known as the Bozzle (see Fig. 2.1). The advantages of the Bozzle system include the fact that no mixing or measuring of pesticide is necessary before use, the pesticide is supplied in a special oil-based formulation at a given concentration. No container cleaning is required after spraying as the unit is disposable; as well as being an important time and labour saving feature, the safety aspects of such a system are obvious, since operator/pesticide contact is reduced. The nozzle is part of the disposable Bozzle unit, thus no nozzle maintenance is required and no error is introduced into the spraying operation by the use of worn nozzles. The Electrodyn sprayer is designed to be a system containing no moving parts. This results in significant 36

50 FIG. 2.1 SCHEMATIC DIAGRAM Or BOZZLE UNIT (AFTER COFFEE 1981) (A) CONNECTION TO EARTH IN ELECTRODYN HANDLE (B) CONNECTION TO VOLTAGE SOURCE IN ELECTRODYN HANDLE (C) HIGH VOLTAGE PLASTIC NOZZLE (D) AIR INLET TO MAINTAIN CONSTANT GRAVITY PRESSURE 37

51 advantages over other sprayers, since it means that there is none of the wear and tear associated with the moving parts in most conventional sprayers, thus reducing maintenance requirements. As none of the components move, the system requires an extremely low energy input. Apart from the small amount of energy expended by pesticide flow in maintaining wave characteristics at the nozzle, droplet formation is due entirely to the conversion of electrical energy. The usual flow rate for an Electrodyn nozzle is about 0.1 to 0.2 ml/sec, and this requires ion neutralisation at a rate of about 2 X 10-6 coulomb/sec. At a typical nozzle potential of 20 kv, this represents a power demand of 40 mw, and assuming a 50% potential conversion efficiency, would require a total input power of less than 0.1 W (Coffee 1980). compared with other spraying systems of a similar When type, this requirement is extremely low. Table 2.1 compares the performance of the Electrodyn with that of another hand-held CDA system. TABLE 2.1 COMPARATIVE POWER REQUIREMENTS (Source: data from I.C.I Plant Protection Division) SPRAYER TOTAL ENERGY NO. AND TYPE TOTAL SPRAYING REQUIREMENT OF BATTERIES TIME Rotary Atomizer 8000 mw 8 HP2 6 hrs Electrodyn Sprayer 100 mw 4 U2 60 hrs 38

52 2.4 The Application of Electrostatics in Mosquito Control The concept of ULV spraying was discussed in the previous chapter, and it was shown that there was a significant gap in the equipment available for malaria vector control since there was a lack of suitable or commercially available equipment capable of applying a residual ULV deposit directly to walls and ceilings without other problems arising. The I.C.I Electrodyn sprayer appears to fit the requirements for such a ULV sprayer in malaria control. It is particularly effective as a ULV sprayer, as the more accurate targetting capabilities of the system have meant that effective rates of application are substantially reduced: down to 1/5 of that previously used in other ULV operations. The possibility of using other electrostatic sprayers was considered; the Rothamsted APE 80 spinning disc sprayer (Arnold and Pye 1980) was tested as an alternative to the Electrodyn but the protoype was unwieldy in confined situations, especially as the rotary disc spread the droplets over a larger area and made accurate targetting difficult without significant wastage. Therefore the I.C.I Electrodyn sprayer was the machine on which all further work was centered. When considering the Electrodyn sprayer for use inside buildings, there were a number of features 39

53 requiring special attention. The first problem was the need for specific oil-based formulations; whereas the most commonly used formulations in residual spraying against mosquitoes are wettable powders, chosen because of their greater persistence and superior performance, particularly on porous surfaces. It was therefore necessary to investigate the performance of the Electrodyn formulations (EFs) with regard to initial toxicity and long-term persistence. A further problem, envisaged in the basic structure of the hand-held Electrodyn sprayer originally designed for spraying agricultural crops such as cotton, was that the spray was directed down onto a crop canopy, whereas spray has to be directed horizontally onto walls in dwellings or upwards to ceilings. Although it is not a problem travel target, for the droplets generated electrodynamically to in a horizontal or vertical direction towards a the droplet characteristics are controlled by a constant gravity induced liquid pressure at the nozzle. Thus when using the sprayer above the operator's head, for example when spraying the upper part of a wall or ceiling, it is possible that problems could occur in obtaining sufficient coverage, or the resulting coverage could be uneven. One safeguard to prevent this problem would have been the installation of a mechanical pump between the bottle and nozzle. This was considered undesirable for two reasons: firstly that it would eliminate one of the most important features of the 40

54 system, the concept of a sprayer without moving parts, and thereby introduce all the problems of wear and maintenance that the system otherwise avoids; and secondly that it would add considerably to the basic cost of the sprayer and would require extensive redesign of the Bozzle. Thus it was decided to test the sprayer in its standard agricultural form, and after assessing the performance, to consider redesigning the sprayer at a later stage should this prove unavoidable. Electrostatic spraying in general, and the I.C.I Electrodyn sprayer in particular, appears to be a very promising system for applying a residual ULV deposit to the walls and ceilings of dwellings. The accuracy of droplet size control and targetting of insecticide offer significant advantages over other types of high volume and ULV equipment. The innovative design of the Electrodyn Bozzle sprayer not only results in considerable savings in energy, time and labour, but also reduces the hazards associated with certain aspects of spraying operations. The Electrodyn sprayer was designed primarily for agricultural use, ±>ut was tested in its existing form to try and avoid costly redesigning. 41

55 CHAPTER THREE DROPLET AND DEPOSITION STUDIES 3.1 INTRODUCTION These experiments studied the coverage and droplet characteristics of deposits when the Electrodyn sprayer was used to treat walls and ceilings under laboratory conditions. Results of some analyses on deposition in huts during spraying in field trials is also included. In the laboratory studies part of a hut was simulated by constructing two adjacent walls, floor and one sloping ceiling panel connected to one wall, from a Dexian frame with plywood panels. Magnesium oxide slides could be placed at various positions over the hut in order to look at droplet coverage in specific areas. Whether the deposition of droplets on glass slides is truly representative of deposition on a wood or mud surface is difficult to judge since charged droplets may be preferentially attracted to some materials; however, for comparative purposes, the slides do show relative deposition patterns. The slides were initially fixed to the wall using double sided carpet tape, but this was time consuming when dealing with large numbers of slides, and later wooden clothes pegs were use,d to hold the slides. These pegs were fixed to the plywood panels with drawing pins and thus could be easily moved to the areas under study. Studies with oil sensitive paper showed no visible alterations in the spray pattern due to the presence of the pegs; it was thought possible that the metal springs in the pegs and the drawing pins might 42

56 preferentially attract droplets, but this was not a problem. Two versions of the Electrodyn sprayer were used in these experiments. The first was a research prototype, consisting of a brass nozzle connected to a removable plastic bottle, between which different sized restrictors could be placed to alter the flow rate. Flow rate could also be altered by changing the annulus size. There was also a voltage regulator connected to the generator which allowed control of nozzle potential over a range between about 18 and 28 kv. The second machine used was a small version of the commercially available Electrodyn Bozzle stick. This had variable flow rate depending on the size of Bozzle used (see Chapter 2, section 2.3), but when fitted with batteries, the nozzle potential was constant (nominally 25.5 kv, but this does change with relative humidity (Harberd pers. com)). It is possible to change the nozzle potential with this machine by taking out the batteries and connecting the input to the generator to a variable voltage control. The primary aim of the studies was to look at specific problems associated with the use of the Electrodyn sprayer in houses, to ascertain whether the standard commercial machine could be used, or whether substantial redesigning of the system was necessary; for cost reasons, the commercial Bozzle Electrodyn with as few modifications as possible was desirable. On 43

57 initially considering the possible problems, the fact that the nozzle was gravity fed was envisaged as probably the most likely cause of poor deposition. Preliminary experiments carried out with the research version of the Electrodyn showed that an acceptable deposit could be obtained on a flat wall surface at nozzle potentials greater than 21 kv with a range of flow rates. The importance of coverage in this type of mosquito control is particularly important, as the mosquito is static when at rest on the surface and therefore if it chances to rest on an unsprayed or partly sprayed area it may escape any toxic effect. The importance of good coverage is increased when an irritant insecticide is used since the deposit may then cause the mosquito to make continued short flights until it reaches an unsprayed site. This was confirmed by field trials with selective treatments of cypermethrin at 0.5 and 0.25 g / m i n which very poor mortalities were recorded when only the ceilings were sprayed (Hervy et al^ 1982, Hervy and Sales 1982). The preferential resting habits of certain mosquito species also mean that adequate coverage over all resting sites must be obtainable, for example Smith (1962) found that Anopheles gambiae in the experimental huts at Magugu, Tanzania, rested almost entirely on the roof. 44

58 3.2 DROPLET SAMPLING AND ANALYSIS The two most commonly used parameters in droplet assessment are number median diameter (nmd) and volume median diameter (vmd). The number median diameter divides the measured droplet sample into two equal parts by number, and consequently emphasizes the small droplets in the sample. The volume median diameter divides the droplets into two equal halves by volume, i.e. the total volume of spray is divided into two, half of which contains droplets larger than the vmd while the other half has droplets droplets smaller than the vmd. The ratio between the vmd and nmd is a useful indication of the range of droplet sizes in the sample, and the closer the ratio is to 1.0, the more uniform the droplet size in the sample (Matthews 1979). There are a number of techniques to determine droplet size. In this study, the most appropriate method was to collect the droplets on a suitable surface, so that this collecting surface could be placed on different areas of the wall surface. The collecting surface used was magnesium oxide, since MgO slides are easy to prepare and can be stored for a limited time before and after sampling, prior to analysis, as well as being easily analysed with the equipment available. The slides were prepared by burning a piece of magnesium ribbon below a glass microscope slide to give a uniform coating of magnesium oxide approximately 2.5 X 3.2 cm in area. The 45

59 thickness of the magnesium oxide coating depended on the expected droplet sizes, the bigger the predicted droplets the thicker the required deposit. A droplet between 20 and 200 p diameter impacting on this surface forms a crater which is 1.15 times larger than the true droplet size (May 1950), and the difference between the measured crater size and the actual droplet size is the spread factor, the reciprocal of which is used to convert crater measurement to true droplet size; for magnesium oxide, this is The slides were analysed using an Optomax Image Analyser (Micro Measurements Ltd, Saffron Walden) which essentially measures areas of detected features in picture elements. A calibration factor is used to convert the picture element count to true measurements for a given magnification. The Image Analyser was interfaced vw-bh initially with a Hewlett Packard 9825A computer, and later with an Apple II computer. The program used counted a minimum of 60 droplets before statistically analysing any change in droplet number or volume. When there was a change, counting continued and re-analysis was carried out after a minimum of 20 droplets had been measured. As soon as there was no change, the program calculated nmd, vmd, vmd/nmd ratio, number of droplets per cm2, dosage applied (1/ha) and number of droplets per litre. 46

60 3.3 SWATH DIMENSION AND DISTANCE FROM WALL Method Large sheets of white card were fixed to a part of the wall away from the influence of the adjacent wall or floor and sprayed with the Bozzle sprayer fitted with a Bozzle with a flow rate of 0.1 ml/sec. Automate red tracer in an Electrodyn blank formulation was sprayed so that the deposit would show on a white card. White card was also laid on the floor to assess floor fall out, and the sprayer was held at increasing distances from the wall. The swath was measured at its widest point and the length of swath down the wall was also measured Results The swath dimensions at various sprayer distances from the wall are shown in Table 3.1. Table 3.1 SWATH DIMENSIONS ON WALL DISTANCE FROM WALL (cm) SWATH WIDTH (cm) SWATH DEPTH (cm) VISIBLE FLOOR FALL OUT YES YES YES YES 47

61 3.3.3 Discussion The dimensions given should be taken as a broad estimate only, since it is difficult to determine objectively where the edge of the swath occurs. Thus a subjective estimate was made of the point at which the droplet density had reduced to less than about 25% of the density at the centre. The swath was generally shield shaped with the majority of droplets in a horizontal band across the swath. The swath could be broadly divided into three areas of droplet density, a high density small circular central area, a lower density wide horizontal band and the lowest density above and below this band, although droplets were recorded outside this area in varying densities. The swath at 20 cm from the wall had a somewhat different shape, losing the bottom "point" of the shield shape and extending further downwards at the peripheries. At this flow rate, the maximum distance that the sprayer can be held away from the wall is 50 cm but at this distance the swath is difficult to control. to maximise swath width and hence reduce spraying Ideally time, the sprayer should be held at about 40 cm from the wall. Considerable floor contamination was apparent at all distances and up to 120 cm away from the nozzle in most directions, and therefore it is possible that operator contamination could become a problem. 48

62 3.4 THE USE OF DEFLECTRODES Introduction In order to maximise the coverage on wall and ceiling, a simple adaptation to the Electrodyn sprayer was fitted. Two extra electrodes ("deflectrodes") were connected to the high voltage generator, and consequently were of the same potential as the nozzle and thus had the effect of deflecting droplets away from them. Short insulated deflectrodes had been developed and tested ICI and used to modify the swath in spraying crops by such as cotton (Coffee 1981b), but the deflectrodes built and fitted for this study were much longer (59 cm) and were made of bendable uninsulated conducting rod. Thus the shape and orientation of the two arms could be changed to find the most efficient design (see Section 3.7 of this chapter). The deflectrodes were fitted projecting downwards from the handle behind the nozzle Method As in the previous experiment, swath width was determined with the sprayer held at various distances from the wall, with white card and automate red + Electrodyn blank. The deflectrodes were straight and angled slightly forward, and at the ends were 33.5 cm apart. 49

63 3.4.3 Results The swath dimensions are shown in Table 3.2. width was taken to be the total horizontal width, Swath before the densities started visibly tailing off. The figure in brackets is the width of the central dense band of droplets. Table 3.2 SWATH DIMENSIONS WITH DEFLECTRODES DISTANCE FROM WALL (cm) SWATH WIDTH (cm) SWATH DEPTH (cm) VISIBLE FLOOR FALL OUT (45) 30 FEW DROPLETS (51) 35 FEW DROPLETS (56) 64 FEW DROPLETS Discussion The same reservations apply here as in the previous section, i.e. that the swath dimension is a subjective figure and should be treated only as an approximation. The effect of deflectrodes is to eliminate the bottom of the shield shape and to widen the whole swath. The swath is therefore band shaped and has a more even droplet distribution; only two main areas of droplet density were visible instead of three as in the previous experiment. The main effect of deflectrodes however was to concentrate the swath on the wall, with only a few scattered droplets visible on the floor, not even droplet 50

64 coverage as was found without deflectrodes. No contamination was observed on objects behind the deflectrodes, as was observed without deflectrodes, and this could be important in terms of reducing or even eliminating operator contamination. 3.5 DROPLET DEPOSITION IN CORNERS Introduction This experiment was examined the deposits of pesticide towards a corner of a hut. The electrostatic forces acting on the droplets cause them to impact on the nearest earthed surface, so one would expect that if the nozzle was pointed directly at the corner, the majority of droplets would be collected at the nearest points on the wall (positions A and B in Fig. 3.1) and the fewest at the point of the corner (position C in Fig. 3.1). Similarly if a different spraying approach was used and the corner sprayed as the edge of two connecting swaths, the first swath on one wall and the second on the adjacent wall (see Fig. 3.1), one might expect that the influence of the adjacent wall as a target for impaction and the tail-off in droplet density towards the edge with no possibility of overlap will cause a reduction in droplet deposition in the corner. Therefore these two spraying methods were tried to see which was the more efficient in giving droplet coverage in the corner. 51

65 FIG. 3.1 DORSAL VIEW OF SPRAYING METHODS FOR CORNERS METHOD 2 Arrows denote direction of spray towards flat target 52

66 3.5.2 Method Two spraying methods were used in this experiment. Method 1 refers to spraying with the nozzle aimed directly at the corner, i.e. with the deflectrode arms equidistant from the corner. Method 2 refers to spraying one side of the corner only, running the left deflectrode arm down the line of the corner. The deflectrode used was elbowed forwards in shape (see Fig. 3.4) Section 3.7 of this chapter). Two rows of magnesium oxide slides were placed at 5 cm intervals (measured from the left side edge of the vertically placed slides) outwards from both sides of the corner. The small Bozzle Electrodyn sprayer was used with a Bozzle of 0.1 ml/sec flow rate and filled with EF blank. Two vertical passes were made over the slides with an approximate spraying speed of 0.2 m/sec. This was repeated twice in order to give four replicates of each slide at a given distance from the corner with each spraying method Results The distribution of deposit volume is shown in Fig. 3.2 I (full data in Appendix 1.1). The distribution of droplet size and droplet density is shown in Fig. 3.2 II and Appendix 1.1. Volume applied is calculated assuming the formulation contains 100% of active ingredient. 53

67 FIG. 3.2 DEPOSITION IN CORNER I. VOLUME APPLIED DROPLET NUMBER / cnr _ VOLUME APPLIED [g/m II DROPLET SIZE AND DENSITY o o VMD Method 1 VMD [ju] 5A

68 3.5.4 Discussion Both spraying methods resulted in significantly reduced pesticide coverage at distances less than 10 cm from the corner (Fig. 3.2 I). Assuming the target dosage when spraying with method 1 is 2.4 g/m^ (taken as the maximum applied in the swath, i.e. the mean of the doses recorded at 15 cm from the corner on the left wall and the 10 cm position on the right wall), the deposit obtained over the 10 cm corner strip by spraying with the nozzle aimed directly at the corner (method 1) was 0.44 g/m2, representing only 18.3% of the full dose. With spraying method 2 (aiming the nozzle at the right hand wall only) if the target dose was 2.2 g/m^ (measured from the peak dose at 15 cm on the right hand wall) the mean dose at less than 10 cm from either side of the corner is 0.65 g/m2r or 29.6% of the full dose. It appears therefore, that spraying method 2 is slightly more efficient than method 1, and when considered in an actual field situation will be substantially better, since the swath which will be made on the other side of the corner will result in a second partial coverage of the left and right hand walls close to the corner, and this should bring the deposit up to about 60% of the target dose. Therefore, when spraying in the field, it is recommended that the corner is treated with two adjacent swaths, one on each side of the corner, running the nearest deflectrode arm as close as possible to the corner without actually touching the wall (since this would 55

69 discharge the sprayer and affect droplet formation and deposition). It is then necessary to repeat one of the swaths in order to achieve at least 90% of the target dose close to the corner. This will inevitably result in some overdosing of one swath per corner, but this appears unavoidable with the present equipment. When the droplet sizes and densities are considered (Fig. 3.2 II), it can be seen that the droplet sizes do not change significantly across the swath with either spraying method. The droplet densities are however, substantially reduced over the same area as reductions in dosage were observed, and thus it appears that the reduced dosage in the corner is simply due to reduced impaction of droplets in this area, as was predicted at the start of the experiment. 3.6 THE EFFECT OF NOZZLE POTENTIAL Introduction The established relationship between the potential of the nozzle and droplet size with the Electrodyn sprayer is that in the absence of other variables, an increase in nozzle potential causes a decrease in droplet size (and hence an increase in droplet density) (Coffee 1980). When considering the effects of this on a particular area within a vertical swath, the situation becomes more complex. Droplets of different sizes can be expected to impact on the wall at different positions. 56

70 Larger droplets are likely to fall further downwards before impaction, and this was demonstrated in Section 3.3 of this chapter. When the droplets are subjected to the extra influence of deflectrodes, their path to impaction becomes less easy to predict (see Section 3.4 of this chapter). The aim of this experiment was to examine an area in mid swath, to see how the droplet spectrum changed with nozzle potential. A similar area on the floor was also assessed. Deflectrodes were also tested to see whether they made a difference to either droplet production or impaction Method Four magnesium oxide coated slides were fixed to the wall in a horizontal line 80 cm above the floor and 10 cm apart. Four slides were also placed on the floor in a similar line parallel to the wall and 45 cm from it. The slides were sprayed with the research Electrodyn sprayer held at 92 cm above the floor (i.e. with the nozzle 12 cm above the height of the wall slides) and 45 cm from the ^wall, directly above the floor slides. Electrodyn blank formulation was sprayed for 5 seconds with a flow rate of 0.12 ml/sec. The nozzle potential was altered over a range between 18.0 and 28.0 kv and measured with a Brandenburg High Voltage Meter. At each potential, two replicates were made giving data based on 57

71 8 wall and 8 floor slides for each potential. The experiment was carried out firstly with no deflectrodes, and then the short insulated deflectrodes were fitted and the experiment was repeated Results The effects of nozzle potential on droplet size, droplet density and dosage applied are shown in Fig. 3.3 Full data showing the means of the measured parameters are given in Appendix 1.2. Regression analysis was carried out for nozzle potential against nmd, vmd, droplet density and volume applied, and analysis of variance about the regression line was used to establish whether there was a significant relationship between nozzle potential and the given parameters (i.e. to see whether the slope of the line b differed significantly from 0 ) Discussion In the area sampled, there is no clear relationship between droplet size and nozzle potential either with or without deflectrodes. When the three highest nozzle potentials only were considered, the expected relationship can be seen (decreasing droplet nmd and vmd with increasing potential), but below 23.0 kv the mean size drops again. This is presumably because there is a maximum size which can be picked up at this point on the wall, and the larger droplets produced by the lower 58

72 FIG. 3.B EFFECT OF NOZZLE POTENTIAL ON DEPOSITION I DROPLET SIZE VOLUME APPLIED [g/m ] DROPLET NUMBER /cm III. VOLUME APPLIED 59

73 potentials impact at points lower down the wall. The vmds of the droplets collected on the floor are significantly larger than those on the wall at a given potential. There is however, a significant relationship between the droplet density and nozzle potential both with and without deflectrodes. This is because the potential not only affects the droplet numbers produced by the nozzle but also the impaction efficiency of the droplets. It should also be remembered that changes in nozzle potential are also changes in deflectrode potential, and thus the efficiency of the deflectrodes can be expected to alter with potential. At potentials of 23.0 and above there were higher droplet densities on the wall and lower densities on the floor when deflectrodes were used, but these differences were not significant except for the decrease in density on the floor at 28 kv. Thus it appears that these deflectrodes only affect floor fall out in the areas measured at 28.0 kv. It is possible that measurements taken over the whole swath may show more differences with the use of deflectrodes. When deflectrodes were used there was a significant relationship between nozzle potential and volume applied to the wall (see Appendix 1.2), although this relationship was not significant without deflectrodes. This appears to reinforce the argument that impaction efficiency is affected by deflectrode potential, although the deposit obtained CIS 60

74 significantly greater on the wall at 25.5 and 28.0 kv. Therefore it appears that nozzle (and hence deflectrode) potentials of at least 25.5 kv are necessary for the deflectrodes to be fully effective. Nozzle potential changes are not easy to predict for a given area of the swath, because of the different routes to impaction of the droplets produced. 3.7 THE DESIGN OF THE DEFLECTRQDES Introduction The small insulated deflectrodes were tested in the previous experiment, and shown to have limited effects at higher voltages. The larger deflectrodes used in previous experiments (see Sections 3.4 and 3.5) were designed to have a stronger deflecting action, and were therefore tested to see if changing the shape of the deflectrode could improve the efficiency with regard to two factors: maximising wall deposition and minimising floor fall out. This was carried out by sampling at several heights in the swath instead of at one height as in the previous experiment, to try and overcome any sampling error due to altered deposition patterns Method Nine magnesium oxide slides were placed on the wall in three rows of three slides. The slides in each row were 20 cm apart, the first row was 12 cm below the 61

75 height of the nozzle and the other rows were at 15 cm intervals below this. The sprayer was held horizontally at 0.9 m above the floor. Similarly nine slides were placed on the floor in three rows, three per row 20 cm apart, the first row was 15 cm from the bottom of the wall and the other two 15 cm apart. Oil sensitive paper was also laid down to extend sampling in each direction, (i.e. two strips towards the wall and three strips parallel to the wall) to check that the slides were collecting the majority of the floor fall out and not missing any major areas. The small Bozzle Electrodyn sprayer was used with a Bozzle flow rate of 0.1 ml/sec to apply blank EF for a duration of 5 seconds. The deflectrodes initially consisted of two pieces of flexible conducting rod fitted to the handle 24 cm behind the point equivalent to the nozzle centre. The two arms were strengthened by a small connecting cross piece fixed 8 cm down from the handle. When straight, the two arms diverged to a distance of 36 cm apart. Three basic shapes of deflectrode were initially tried; these were: 1) elbowed forwards towards the target, 2) straight, perpendicular from the handle, and 3) straight, angled slightly forwards from the cross piece towards the target (see Fig. 3.4, numbers 1,2 and 3 respectively). These three shapes were tested initially, and from the results, a fourth design was constructed from the most promising of the three. This consisted of an extra cross piece fitted 11 cm up from the distal ends 62

76 FIG. 3.4 TYPES OF DEFLECTRODE (not fo scale) Dimensions (in cm )

77 of the deflectrode arms (Fig. 3.4 number 4). The slides were also sprayed using the sprayer without deflectrodes. For each deflectrode type, two replicates were made Results Mean values for the nmd, vmd, droplet density and g/m2, based on the 18 slides on each of the wall and floor are shown in Fig. 3.5 and Appendix 1.3. Volume applied is calculated assuming 100% a.i. in the formulation Discussion The droplet sizes measured on the slides are not easy to explain. The first three types of deflectrode all produced significantly larger droplets on both the wall and the floor (see Fig. 3.5 I). It is difficult to say from this whether the deflectrodes are actually affecting atomization, resulting in an overall larger vmd of the droplets produced, or whether this is simply another effect of altered deposition patterns resulting in the slides not sampling the whole swath efficiently. The observed action of defectrodes is to push the whole swath higher up the wall, and therefore if the larger droplets are simply pushed up to impact on at least the lowest slides, this would raise the mean vmd of the droplets collected. However this assumes that the droplets maintain the same relative positions within the swath and this may not necessarily be true, since the 64

78 FIG. 3.S THE EFFECT OF DEFLECTRODE SHAPE ON DEPOSITION III VOLUME APPLIED 65

79 deflectrodes are likely to act on different sized droplets in different ways. Very little is known about the action of the deflectrodes on the trajectory of individual droplets, and therefore precisely what is happening here is unclear. The picture is further confused by the results with the deflectrode with the extra bar, where the vmd is significantly lower than the others, and not significantly different from the sprayer without deflectrodes. Since it is unlikely that this deflectrode does not have an effect on the atomization if the other types do, the result must be due to altered deposition, and reflects the problems of sampling over the whole swath where certain sized droplets may be concentrated in specific small areas. It is possible that the extra bar pushed the larger droplets horizontally sideways, thus widening the whole swath, but this on does not explain the smaller droplet size collected the floor where oil sensitive paper did not show any visible deposits outside the range of the slides. When droplet density is examined, a clearer picture emerges (Fig. 3.5 II). All deflectrodes showed a significantly higher droplet density on the wall and lower density on the floor when compared with the sprayer without deflectrodes. The droplet numbers on the floor were not significantly different for the the first three deflectrode shapes tried, but were significantly reduced by the addition of the extra bar. The elbowed and straight vertical deflectrodes deposited similar 66

80 densities on the wall, the straight angled deflectrode was significantly better. This result led to the extra bar being fitted to the straight angled shape. With the addition of the extra bar, the droplet density on the wall increases significantly. When the floor deposits (in g/m^) were calculated as a percentage of the wall deposits, the following results were obtained: DEFLECTRODE % NO DEFLECTRODE 70.6 ELBOWED 67.0 STRAIGHT VERTICAL 40.9 STRAIGHT ANGLED 20.2 EXTRA BAR 3.7 It should be noted that these figures are not representative of the percentage fall out likely to be found in a field spraying operation, since when sampled over the whole floor area, the percentages will be much lower than when sampling close to the wall. However the figures do give a useful indication of the relative efficiencies of the different deflectrodes. Clearly, any deflectrode not only improves wall coverage but reduces floor contamination; and of the shapes tried, the straight angled deflectrode with the extra cross bar was the most effective. This however is not necessarily the most effective deflectrode possible, it may be that a solid charged shield would be better, but other 67

81 considerations such as weight and ease of use are important. Floor contamination is an important consideration in mosquito control operations. Rishikesh et al (1977) studied floor fall out in village huts during spraying with high volume compression sprayers (Hudson X-Pert) fitted with 8002 flat fan nozzles, and obtained mean fall out of up to 10.3% in rectangular huts. Gratz and Dawson (1963) sampled different areas of the floor and showed that with a target dose of 1.5 g/m2, mean floor deposits were 1.24 g/m2 in the corners, 1.06 g/m2 at the sides and 0.39 g/m^ away from the walls. This was due to bounce- off from the walls and fall out from the ceilings. It is standard practice in such high volume spraying operations when calculating mean dose rates to allow 10% extra for wastage, and therefore any improvement on this figure will cut down operational costs considerably. Thus the very low figure with the deflectrode with the extra bar appears promising. Toxicological studies following mosquito control operations have also shown that the greatest drop in plasma cholinesterase levels were found in the 0-6 years age group, and because of the high amount of floor contact by young children, this has given rise to some concern. Gratz and Dawson (1963) recommended that if residual insecticides of any greater toxicity than those in use at the time were introduced, consideration should be given to minimising floor contamination. 68

82 It is difficult to assess the total floor contamination likely to be obtained with the Electrodyn, but these results appear promising, although greater contamination may possibly occur when the sprayer is moved up and down the swaths rather than held static as in this experiment. 3.8 DROPLET DISTRIBUTION NEAR THE FLOOR Introduction One of the problems of coverage which became apparent when the sprayer was used in the field trials was that of insufficient deposit being applied on the lower parts of the wall near the floor. Estimations made in the field suggested that little or no coverage was obtained at distances less than about 30 cm from the floor. This was mainly due to the physical presence of the deflectrodes protruding downwards from the handle which prevented the nozzle from being as near to the floor as perhaps was necessary, and care had to be taken not to touch the floor with the deflectrodes since this would discharge the nozzle and result in drips. The electrical field created by the-deflectrodes pushing the swath upwards was also partly to blame, as well as the fact that the wall floor junction was effectively a "corner" and therefore had all the problems of coverage associated with the corners (see Section 3.5). 69

83 3.8.2 Method Magnesium oxide slides were placed on the wall in four horizontal rows (nine slides per row, 8 cm apart). The rows were at 5, 20, 35 and 50 cm from the floor. The rows were initiated with the first slide 8 cm from the corner. A similar set of 36 slides were placed on the floor in four rows of nine slides 8 cm apart at 5, 20, 35 and 50 cm from the wall. The small Electrodyn Bozzle sprayer fitted with the straight angled deflectrodes with extra cross bar was used, since this was found to be the most effective design (see Section 3.7) and also was the deflectrode type used in the field trials. Electrodyn formulation blank was sprayed in a Bozzle having a flow rate of 0.1 ml/sec. The slide layout was designed to be sprayed as three separate adjoining swaths, covering three slides width each, and each swath was sprayed twice, once in an upwards and once in a downwards direction at a spraying speed of approximately 0.1 m/sec. The experiment was then repeated with another two sets of 72 slides (36 wall and 36 floor slides) Results The results are shown in Fig. 3.6 and Appendix 1.4, based on the means of the three slides per position. The Optomax results for the slides at 5 cm from the floor are not given, because insufficient droplet numbers 70

84 FIG. 3.6 EFFECT OF WALL POSITION ON DEPOSITION I. DROPLET SIZE 71

85 impacted at this height to make slide analysis possible. Similarly none of the floor slides in any replicate picked up enough droplets to be able to analyse them. Two-way analysis of variance was carried out on the data (see Appendix 1.4) to look at the two influencing factors, distance from the floor and distance from the corner. Dosage applied was calculated assuming a 50% a.i. formulation, since this was a typical concentration used in the field trials Discussion The droplet size (Fig. 3.6 I) on the slides at 35 and 50 cm from the floor shows a significant trend towards larger droplets closer to the corner (P<0.01 and P<0.05 for 35 and 50 cm respectively, tested using analysis of variance about the regression line). The slides at 20 cm however, showed no such trend. The slides at 20 cm did have significantly larger droplets than those at 35 and 50 cm, except for the slide closest to the corner, but this was due to the slides at the higher levels having larger droplets on the slide nearest the corner, than the slide at 20 cm having smaller droplets. further The larger droplets would be expected to fall than the smaller ones when the sprayer stops at floor level, so this was not an unexpected result. When droplet density is considered, there are clearly problems at heights below 35 cm from the floor 72

86 (Fig. 3.6 II). There was no significant difference between the droplet densities on the slides at 35 and 50 cm from the floor, but the slides at 20 cm had significantly fewer droplets. Again the influence of the corner brought droplet densities on the higher slides down to values similar to that on the slide at 20 cm, was expected from the results shown in Section 3.5. as When considered in terms of dosage applied, the deposits at 20 cm above the floor are significantly less than those at 35 cm and above. The mean dosages applied at 35 cm and 50 cm are 1.29 and 1.31 g/m2 respectively, while at 20 cm it is only 0.79 g/m^, i.e. 60.8% of that applied higher up. Clearly then, there is a point somewhere between 35 and 20 cm where the chemical deposits start reducing due to reduced droplet impaction, and by 5 cm from the floor, droplet impaction is so low that the chemical deposit is not measureable by these methods. While this reduced or absent coverage may not be a problem in many control situations, there are some areas where the vector species rest predominantly on the lower parts of the wall, for example 60% of Anopheles farauti, the main vector in the D 'Entrecastleaux Islands were found to rest on hut walls below 3 ft (Spencer 1965), and for the Electrodyn to be of use in all control conditions, this problem must be given consideration. It is not enough simply to recommend repeat horizontal swaths around the bottom of / the wall, since, although this will improve the deposit at 20 cm from the floor, the areas lower than this are unlikely to be significantly improved. 73

87 There was no significant difference in the volume applied between the slides nearest the corner and those further away, although a slide set closer than 5 cm from the corner would almost certainly have shown a reduced deposit (see Section 3.5). One encouraging result of this experiment was the extremely low floor deposits. Only scattered droplets impacted on the floor slides in numbers too few to count, and therefore floor contamination was extremely low. This is an excellent result considering the sprayer was in motion while spraying, not static as in the previous experiment, and also meant that the deflectrodes effectively prevented the floor from becoming an alternative target when the nozzle was close to the floor. Without deflectrodes, droplet impaction on the floor can be expected as soon as the nozzle becomes near enough to the floor for this to be a rival target, and since depth of swath has been shown to be around cm, this is likely to be the height at which significant floor impaction will occur. 3.9 DEPOSITION WITH HIGH FLOW RATES Introduction The first field trial indicated the necessity for speeding up the rate at which pesticide could be applied with the Electrodyn to hut walls (see Section 5.3). The majority of studies were carried out with 0.1 ml/sec 74

88 Bozzles, which were also used in the first field trial. When the second trial was carried out, 0.15 ml/sec Bozzles were available. Following this field trial, the 0.15 ml/sec Bozzles were studied in the laboratory in order to see whether the deposition characteristics were significantly altered by the higher flow rate, with particular reference to deposition over the lowest parts of the wall. As discussed in Chapter 2, droplet size is a function of nozzle potential, flow rate and formulation resistivity. In this experiment, flow rate only was altered Method The same slide layout and spraying method were used as in the previous experiment (see Section 3.8.2) in that 36 slides were fixed to the wall at 8 cm intervals from the corner in four rows of nine slides at 50, 35, 20 and 5 cm from the floor. The 36 floor slides were also used, again laid in' four rows of nine slides (8 cm apart) at 50, 35, 20 and 5 cm from the wall. Oil sensitive paper was again used to check for contamination in areas outside that covered by the slides. Blank EF was sprayed through a Bozzle with a flow rate of 0.15 ml/sec, using the small Bozzle Electrodyn sprayer. The spraying speed was approximately 0.2 m/sec instead of 0.1 m/sec as in the previous experiment. This 75

89 was to prevent too many droplets impacting on the slides which could have made accurate analysis difficult. The slides were again sprayed twice as three vertical swaths, once in an upwards and once in a downwards direction. The experiment was repeated once to give two sets of slides Results The droplet sizes, droplet densities and volume applied are shown in Fig. 3.7 and Appendix 1.5. Volume applied is again calculated assuming a 50% chemical concentration in the formulation. As in the previous experiment, the slides at 5 cm contained so few droplets that analysis was impossible and therefore no results are included for these. Similarly, only two out of the total of 72 floor slides contained sufficient droplets to make analysis possible, and even these had such low densities that the calculated distribution was not statistically significant and therefore the figures may not be entirely accurate. The two slides had the following droplet characteristics: VMD (M) Droplet number/cm2 Volume applied (g/m2 ) Thus the dosage on the slides was not only relatively low, but when considered as a fraction of the total area sampled, the contamination was extremely minimal. 76

90 FIG. 3 7 EFFECT OF HIGH FLOW RATES ON DEPOSITION I. DROPLET SIZE III. VOLUME APPLIED 77

91 3.9.4 Discussion The greater spraying speed meant that direct comparisons with the 0.1 ml/sec flow rate Bozzles used in the previous experiment were not possible, although relative patterns of droplet distribution are comparable. The higher flow rate Bozzles did not alter deposition at 5 cm from the floor; as in the previous experiment, deposition here was so low that it was not possible to measure it by these methods. The slides at 35 and 50 cm showed similar density patterns to those obtained with 0.1 ml/sec Bozzles in the previous experiment, and as before, the slides at 20 cm showed significantly reduced droplet densities (see Fig 3.19 II). With the 0.1 ml/sec Bozzles, the VMD of the droplets on the slides at 20 cm was significantly greater than that of the slides at 35 or 50 cm; however with the 0.15 ml/sec Bozzles although there was an overall difference (see Appendix 1.5), the only clear difference was between the two slides closest to the corner at 20 cm and those in a similar position higher up (see Fig. 3.7 X); however the nmds were generally larger at 20 cm. It is possible that if the higher flow rate produced larger droplets, these may have fallen further and impacted at a point somewhere between 5 and 20 cm from the floor. However, since the droplet density at 20 cm had already dropped to about 25% of that found at 35 and 50 cm, the 78

92 density of the larger droplets is likely to be so low that they would have little overall impact on deposition at these levels. In fact, flow rate changes need to be relatively large to have a significant effect on droplet size (Endacott pers. comm.) and the 50% change in flow rate used here is unlikely to have much effect on the droplet spectrum. The influence of the corner was again apparent (see Section 3.5) with significantly lower densities on the slides closest to the corner. The extremely low floor contamination even with these higher flow rates was, however, very encouraging, as it was initially thought that increasing the flow rate could result in more droplet fall out THE EFFECT OF FORMULATION ON DROPLET CHARACTERISTICS Introduction All the laboratory studies on deposition characteristics have been carried out on Electrodyn formulation blank, which is a cyclohexanone/white oil mixture. It has been noted (see Chapter 2) that the resistivity of the formulation affects droplet size. The viscosity of the formulation also indirectly affects droplet size by altering flow rate, but a large change in viscosity is required to change the flow rate enough to cause significant changes in droplet spectra. A variety of different formulations were used in the field trials 79

93 (see Chapter 5 section and 5.4.1), and in order to see how much effect the formulation has on droplet characteristics, a 45% formulation of DDT EF was compared with an EF blank, to give some idea of the relative differences between the' blank and a representative formulation containing active ingredient Method This experiment was not concerned with spraying a wall, so instead of fixing targets to a vertical surface with its attendant problems of sampling the complete swath, the spraying was carried out on a horizontal surface. The research version of the Electrodyn sprayer was mounted with the nozzle held 60 cm above a conveyer belt. Plywood boards (30X30 cm) were carried on the conveyer belt at a speed of m/sec below the nozzle. Magnesium oxide slides were laid on the boards in a row of five slides, 3 cm apart, the row was laid at 90 to the direction of the conveyer. Two boards were sprayed at each setting so that each result is derived from the mean of 10 slides. With each of the formulations, a range of nozzle potentials were used, from kv, and these were measured at the nozzle using a Brandenburg High Voltage Meter Results The droplet sizes and densities for the two 80

94 FIG. 3.8 EFFECT OF FORMULATION ON DROPLET CHARACTERISTICS I. DROPLET SIZE III. VOLUME APPLIED ' 81

95 formulations are shown in Fig. 3.8 I and II respectively, and Appendix 1.6. The volume applied was calculated assuming the blank EF had the same concentration of active ingredient as the DDT formulation, i.e. 45%. Regression analysis was carried out and a regression line fitted to the data for VMD, droplet density and volume applied. Analysis of variance about the regression line was calculated to establish any significant relationship between these parameters and nozzle potential (see Appendix 1.6) and two-way analysis of variance was carried out with formulation type and nozzle potential as the two factors (see Appendix 1.6) Discussion The established inverse relationship between nozzle potential and droplet size was apparent in this experiment, in that an increase in nozzle potential resulted in a significant decrease in VMD (see Fig. 3.8 I). The blank EF produced significantly larger droplets than the DDT EF at all except the lowest potential. The droplet density also showed a significant relationship, with an increase in density with increased potential, as this is obviously related to droplet size. The DDT EF produced significantly higher droplet densities at all except the two lowest potentials (18 and 20.5 kv). When the volume applied was considered, it appears that the droplet size has the biggest impact on dosage, since the blank EF produced significantly higher dosages at nozzle 82

96 potentials greater than 20.5 kv. The ultimate difference in dosage between the two formulations is due to the viscosity of the DDT formulation being greater than that of the blank EF (the viscosities were measured as 11 cp and <2 cp respectively), which resulted in a lower flow rate than with the blank. Therefore formulation clearly has a significant effect on the depostion characteristics of the sprayer, and this must be considered when relating laboratory studies to field trials. The effect of viscosity on the flow rates, which in turn affects droplet size is less important in determining the ultimate droplet spectrum than the resistivity of the formulation, since in this experiment, the more viscous DDT formulation produced smaller droplets than the blank FIELD STUDIES ON DEPOSITION Introduction The laboratory studies provided essential background to the deposition characteristics of the sprayer, but it is the use of the sprayer in the field which ultimately shows the important aspects with regard to coverage. However there are a number of problems associated with deposition studies in the field, and these will be considered in more detail later. When the field trials were carried out with the Electrodyn sprayer, it was decided to attempt to assess 83

97 the coverage by chemical analysis of the deposits on the walls and ceilings. This had a major drawback in that the main aim of the trial was to assess mosquito mortality in the huts, and therefore the number of filter papers which could be laid on the walls was limited if they were not to interfere with the overall coverage and hence performance of the insecticide. Ideally a separate hut should have been sprayed with the aim of only assessing wall deposits by chemical means, but a shortage of suitable huts made this impossible Method Circular filter papers (7.0 cm diameter) were taped to the hut walls and ceilings in a line extending from the roof apex down to the floor. Up to five papers were taped to the ceiling, and up to five on the wall. The relative positioning depended on the number used; during the second field trial, filter papers were limited in number and it was impossible to obtain fresh supplies in time, so fewer were used per hut, and not all the huts were sampled. The spraying was then carried out as described in Chapter 5 (section 5.3.1), and the papers were allowed at least 30 minutes to dry out before removing them from the walls and sealing them in envelopes. The envelopes were stored in as cool conditions as were possible, and where a refrigerator was available this was used. On return to England the papers were submitted to GLC analysis to measure the chemical 84

98 deposit on them Results The dose rates on the filter papers are given in Table 3.3 and are calculated from the mg/paper data derived from the analysis which were corrected for an 85% recovery rate. Results are given for cypermethrin only. The DDT sprayed in the first field trial was also sampled, but the results were marred by incomplete extractions by the chemistry section in Tanzania which undertook the analysis; following this all papers were brought back to England for extraction and analysis by ICI. There are also no results for the huts sprayed with cypermethrin WP in the second field trial; this was due to the shortage of filter papers as already mentioned. In the table the figures 1-5 simply refer to relative positions on the wall or ceiling, 1 being the highest paper. As the number of papers varied from 3-5 per wall or ceiling, the distances between papers are not necessarily the same, i.e paper 3 does not necessarily indicate the same postion on the wall in all huts, it will be lower in the huts with only 3 papers on the wall. 85

99 TABLE 3.3 FILTER PAPER ANALYSES OF CYPERMETHRIN DEPOSITS ON HUT WALLS AND CEILINGS FORMULATION TARGET DOSE ( mg/m2) 1 DE POSITS 2 ON FIL1 3 'ER PAPE 4 RS (mg/ir 2>_ 5 X 1) CEILING DEPOSITS WP EF EF EF* EF EF* EF ) WALL DEPOSITS WP EF EF EF* EF EF* EF * denotes hut with iron roof 86

100 Discussion With the huts sprayed at a target dose of 400 mg/m2, there was consistent underdosing of both the wettable powder and the Electrodyn formulation treated huts. However, the papers were kept for several weeks at the field site, where no refrigerator was available, and therefore may have suffered some chemical degradation. The relative deposits are comparable, and the lowest dose was found on the ceiling of the EF treated hut which averaged only about 15% of the maximum dose. The WP treatment showed significantly higher deposits on the ceiling than the EF; there was however, lower dosages on the wall with the WP compared to the EF, but the difference was not significant. (see Appendix 1.7). The low dose on the ceiling was possibly due to the long spraying time necessary to apply cypermethrin EF at 400 mg/m^ (see Chapter 5 section 5.4), which led to arm fatigue and possibly to shorter spraying times than were necessary to achieve full dose. The following year, when lower doses of cypermethrin were sprayed (80 and 40 mg/m^), the measured deposits were greater than the target dose. " The papers were stored in a refrigerator almost immediately in this case, as they were taken from the field site more rapidly, and this should have prevented any chemical breakdown. The overdosing on the papers is not easily explained since checking the formulation quantities 87

101 before and after spraying did not show this problem, but the number of papers sampled was relatively small and the results should therefore be treated with caution. The use of filter papers to measure spray deposits was described by Hudson (1971) for these huts, where the chemical (in this case methoxy-ddt) was mixed with kiton red dye and the dye deposits measured with an Absorptiometer. Hudson (1971) also found up to about 50% overdosage in the huts, and attributed this to the spray operators paying special attention to the filter papers. Smith and Webley (1969) also used the technique when assessing deposits of DDT in these huts and in huts at Taveta, Kenya, and noted considerable variation in the samples taken at different positions in the hut, and suggested that larger samples than 20 papers per hut were necessary to assess total hut deposits accurately. It therefore appears that further studies on hut deposition are necessary to be able to assess the total hut deposition accurately. It is encouraging that any underdosing of cypermethrin which may have occurred in the first field trial did not appear to be a problem in the second field trial. When the huts are assessed together there were however, significantly higher deposits on the walls than the ceilings in the huts sprayed at 80 and 40 mg/m2f but of the 15 papers on the various ceilings, only one in the 80 mg/m^ hut and one in the 40 mg/m^ hut were recorded as having less than the target dosage. 88

102 Thus coverage on the ceilings was less than those on the walls, but how much less is not predictable without more samples. Gratz and Dawson (1963) used filter papers to assess fenthion deposits applied with Galeazzi "OM" compression sprayers, and also found reduced deposits on the ceilings; they attributed this to three factors: drip-off from ceilings, fall-out of smaller drops before impaction, and the difficulty in controlling overhead. the speed of spraying when holding the lance While the first two factors are not likely to be the cause here, the problem of sprayer control overhead could contribute to the problem with the Electrodyn, and also possibly tilting of the Bozzles may result in uneven flow rates especially if the Bozzles are less than half full. 89

103 CHAPTER FOUR LABORATORY BIOASSAYS 4.1 INTRODUCTION The most commonly used formulations of insecticides in the treatment of dwellings against malaria vectors are wettable powders. This is because of their superior persistence on porous surfaces such as mud, due to lower absorption resulting in more insecticide remaining on the surface and accessible to the mosquitoes (Bertagna 1959, Fontaine 1983). Emulsifiable concentrates have been used with some insecticides, notably malathion, fenitrothion chlorpyrifos, dichlorvos and propoxur (Smith and Lossev 1981). Studies on formulation effectiveness have compared wettable powders with emulsifiable concentrates and with oil and kerosene formulations. Malathion as a 50% wettable powder, 84% emulsifiable concentrate and as a technical ULV formulation was evaluated on plaster and dried soil against Anopheles stephensi at 4 g/m^. The wettable powder was the most effective on both surfaces, and the EC and technical malathion were somewhat less effective and showed similar persistences (Barlow et aj^ 1982). Hervy and Sales (1981) however compared malathion wettable powder and emulsifiable concentrate in experimental huts in Upper Volta, and showed similar persistencies for the two formulations. DDT has been applied in solutions of kerosene with 90

104 some success; at approximately 3 g/m2, DDT in pure kerosene was effective for 20 weeks, at a lower dosage of 0.77 g/m2 a kerosene emulsion in water was effective for 20 weeks against A^_ minimus and A.vagus in India on mud, bamboo and thatch surfaces (Ribbands 1947). Oil based formulations have been tested in the field; Davidson (1953) sprayed a DDT oil-bound suspension at 309 mg/ft2 (equivalent to 3.33 g/m2) and found anopheline mortalities of 55-65% for two months in experimental huts; however the problems associated with this trial are discussed in a later section (see Section 5.51), and the mortalities found may not be truly representative of the treatment. Evidence suggests that the persistence of oil-based formulations on porous surfaces may be relatively short compared with wettable powders. The aim of this set of experiments was to investigate the persistence of the oil based Electrodyn formulations on plywood stored under tropical conditions. The ultimate effectiveness of an insecticide is its biological impact, and this was studied by means of bioassays with a suitable test insect, rather than chemical analysis/ which would indicate the amount of insecticide present not the amount available for pick-up or the extent of any vapour action. The test insecticide selected for bioassay was Aedes aegypti (L.). In the field the target mosquito species would be various Anopheles; for laboratory 91

105 purposes however, Aedes aegypti is a much easier species to keep in culture for long periods of time, and is similar in susceptibility to common malaria vectors such as Anopheles quadrimaculatus Say, A.maculipennis Meigen and A.gambiae (Giles) (Gahan et a_l 1945, Wharton 1955). 4.2 METHODS Mosquito Culture A culture of Aedes aeqypti (L.) was established from stocks held at Imperial College, Silwood Park. The culture was maintained at 25+2 C, 60-65% RH, on a light regime of LD 12:12. The larvae were reared in large bowls and fed on dried wholemeal breadcrumbs. The pupae were removed from the larval containers daily and transferred to emergence cages. The adults were given access to 10% glucose solution at all times, and the females were blood fed on 1-4 week old chicks two or three times per week. The chicks were anaesthetised with 0.05 ml of a veterinary anaesthetic (Vetalar ) and sedative (Ronpun ) mixture for approximately 20 minutes to facilitate the mosquito feeding. Filter paper was folded into cones and placed in petri dishes of water in the stock cages, to provide a damp laying surface for the females. Filter papers plus eggs were then either transferred immediately to larval rearing bowls, or were dried and stored for rehydration up to two months later, depending on egg abundance and experimental requirements. 92

106 4.2.2 Treatment of Test Surfaces The test surface consisted of 30X30 cm pieces of plywood (3-ply). Plywood was selected as a representative relatively non-porous, chemically inert building material, which shows similar residual properties to grass and straw thatch, palm leaf and bamboo (Hadaway and Barlow 1965). These were moved on a conveyer belt at a speed of m/s below the Electrodyn research sprayer, fixed so that the nozzle was held at a height of 0.53 m over the boards. A number of preliminary studies were carried out with magnesium oxide slides to investigate different combinations of annulus size, restrictor, nozzle potential and formulation to give suitable target doses. In practise it was necessary to increase flow rate by removing the restrictor from the system altogether in order to achieve the relatively high dosages required, and in some case two passes under the sprayer were needed. The following insecticides were sprayed, all as Electrodyn formulations: DDT - 50% pirimiphos methyl - 50% permethrin - 8% cypermethrin - 3% The plywood boards were stored vertically in an

107 open sided cardboard box, with spacers between them to allow air to circulate around the treated surface, and kept at 25+2 C, 60-65% R H. In order to ensure that the deposits on the board were even, as the metal construction of the conveyer belt may have influenced electrostatic deposition, three boards were covered with lines of magnesium oxide slides and sprayed with EF blank using a nozzle potential of 28 kv. The board was then subdivided into sectors and slides from the sectors were analysed with the Optomax Image Analyser (see Section 3.2 for details of slide analysis). Statistical analysis showed no significant difference between the droplet sizes and densities in the different parts of the board and therefore coverage was assumed to be equal over the board surface Bioassay Procedure The mosquitoes used in the bioassays were all females of 5-7 days old, all of which had received one blood meal the day before the bioassay. Thus the insects were standardised as far as possible with regard to sex, age and nutritional state, all factors which have been shown to influence susceptibility to insecticides (Hadaway and Barlow 1956). All bioassays were carried out in mid afternoon, which was the period of onset of the mosquitoes' darkness, and therefore was the time of greatest activity. The same time was used for each test, 94

108 because circadian rhythms of sensitivity have been shown in some flies (Shipp and Otton 1976). The treated plywood boards were tested at one or two week intervals following treatment. Mosquitoes were exposed to the boards in inverted glass funnels, 10 cm in diameter at the widest point, the stem of which had been removed and the opening stopped with cotton wool. The boards were held vertically in a dexian frame, and the funnels held in place with rubber bands; thus if the mosquitoes were knocked down, they would not necessarily remain in contact with the treated surface, as would happen in the field. The exposure time used was one hour. Fifteen females were introduced into each funnel, with three funnels per board. An untreated control board, also with three funnels containing fifteen females in each, was tested every time a treated board was assessed. Mosquitoes were transferred to the funnels from emergence cages using aspirators. At the end of the exposure period, a piece of card was inserted between the funnel and board. When the mosquitoes were removed from the funnels, the board was covered by a netting cage open at the bottom with an entry sleeve on one side, in order to capture any mosquitoes which had not been knocked down that may have flown from the board when the funnel was taken off. The mosquitoes were then transferred to holding cages, again using aspirators. Separate aspirators were used for treated and untreated boards to prevent contamination of control mosquitoes. The holding cages consisted of paper cups with the tops covered with 95

109 mosquito netting and a small entry hole cut in the side which was stoppered with cotton wool. The mosquitoes were held in the cages for 24 hours with fresh water available, after which time a mortality count was made. Mortality was considered as when the mosquitoes showed no visible signs of life. After each bioassay the funnels and holding cages were well washed with detergent and water to prevent any insecticide contamination in the following bioassay. A further set of bioassays was carried out to assess the effects of altering exposure time. Two boards were treated, one with pirimiphos methyl EF and the other with DDT EF, both at the relatively low dosage of 0.5 g/m^ t and the following day caging female Aedes on the boards for intervals ranging from 1 minute to 1 hour. Apart from exposure time, experimental procedure was the same as that used in the other bioassays, with three funnels containing 15 females each per board, and again assessing mortality after 24 hours. 4.3 RESULTS When control mortality exceeded 20% the results for the treated boards were discarded and the bioassay repeated. When control mortality was between 5 and 20%, Abbott's correction was used (Abbott 1925): % TEST MORTALITY - % CONTROL MORTALITY CONTROL MORTALITY X100 96

110 The corrected percentage mortalities for the four insecticides are shown for periods of up to 24 weeks in Figs Fig. 4.5 shows mortalities associated with a blank Electrodyn formulation applied at the same volume per m2 as the pirimiphos methyl EF, compared with control mortalities on an untreated board. Fig. 4.6 shows the effect of altering exposure time with two insecticides, pirimiphos methyl and DDT. 4.4 DISCUSSION It is difficult to relate laboratory bioassays directly to results which can be expected in the field, because of the many factors which have an effect on wild populations of mosquitoes, but the bioassays were an important preliminary to field trials to indicate whether the persistency obtained under laboratory conditions was long enough to justify field trials with the Electrodyn formulations. The bioasays were perhaps a more rigorous test of the insecticide efficacy than under field conditions, since they were carried out at a time of peak mosquito activity; the mosquitoes had fed the day before and therefore had fully digested the blood meal. In the field it is usual for the mosquito to feed and then immediately rest on the wall or roof, and in this condition are much less active than mosquitoes which have not fed for 24 hours. Another factor is that the density of mosquitoes in the bioassay (15 females per 78.5 cm2 Q f 97

111 FIG. 4.1 DDT BIOASSAY CORRECTED MORTALITY [% ] _ CORRECTED MORTALITY [% ] 1*8 g/m o -o o*9 g/mz - A 0 *35 g/m^ J g n : T " 4 TIME AFTER T&ATMEfJ T [wee s ] 2 FIG. 4.2 PIRIMIPHOS METHYL BIOASSAY 98

112 FIG. 4.3 PERMETHRIN BIOASSAY CORRECTED MORTALITY ( % ] CORRECTED MORTALITY [ % ]. 4.4 CYPERMETHRIN BIOASSAY 99

113 FIG. 4.5 BIOASSAY WITH BLANK EF 10CH 80- CORRECTED MORTALITY [% ] MORTALITY [ % ] TIME AFTER TREATMENT [weeks] FIG. 4.6 EFFECT OF EXPOSURE TIME 100

114 board space) is much higher than that encountered under normal conditions in the field, and therefore interference and hence disturbance of the resting mosquitoes is likely to be much higher in the funnels. This means that the bioassays might be expected to give lower mortalities than those encountered in the field, especially since an exposure period of one hour is a much shorter time than a mosquito would rest on a hut wall given no disturbance or irritancy. This has resulted in bioassays being carried out in the field by caging wild mosquitoes on a treated hut wall giving lower mortalities than in free-living wild populations in the same hut (Hervy and Sales 1981). However the other effects of the insecticides used in these bioassays must be considered; pirimiphos methyl is an insecticide with a strong vapour action, and this is likely to be much greater in its effect in the enclosed funnel than it would be inside a hut. When DDT and the pyrethroids are used the effects of irritancy become important, which cause a mosquito to leave the treated surface much earlier than it would normally do so (this is discussed in detail in Chapter 5 section 5.6.1), and the confinement of the funnel may therefore result ^ in the mosquitoes remaining in closer proximity to the treated surface, or result in more frequent re-settling on the surface than would occur in the field. For these reasons the use of funnels may result in higher mortalities than would be shown by the same deposits on an identical surface in the field. However it is difficult to assess the relative importance 101

115 of these and the factors mentioned earlier which can be expected to show lower mortalities than field treatments and therefore whether the mortalities indicated here are higher or lower than field mortalities cannot be ascertained with any certainty. The exposure time used in these studies was one hour. This time was selected as it is the time recommended by the World Health Organization in the diagnostic tests for resistance in anopheline mosquitoes to DDT, malathion and permethrin (WHO 1981a,b). The minimum kill required in the WHO Programme for the Evaluation and Testing of New Insecticides is 70%, following exposure (Barlow et at 1982), to the treated surface for one hour and therefore 70% was used here as the threshold below which the insecticide was deemed ineffective. The most commonly used doses of the insecticides used in the field are as follows: DDT - 2 g/m^ (Fontaine 1983), pirimiphos methyl - 2 g/m2 (Rishikesh et al 1977), permethrin mg/m^ (Rishikesh et al 1978) down to 125 mg/m^ (Taylor et al 1981) and cypermethrin 500 mg/m^ (Hervy et at 1982) and 250 mg/m2 (Hervy and Sales 1982). The DDT and pirimiphos methyl were tested up to the commonly used field dose, but it was felt that the field doses of pyrethroids were high and offered scope for dosage reduction. 102

116 4.4.1 Insecticide Effectiveness DDT at 1.8 g/m^ gave excellent mortalities for 22 weeks (Fig. 4.1), with 20 weeks showing over 93% mortality. Hadaway and Barlow (1965) found that DDT as a wettable powder sprayed onto plywood and tested against Anopheles stephensi was effective for over 52 weeks at 2 g/m^; the bioassay procedure being similar to that used here, with one hour exposure, but the storage of the boards being at the lower humidity of 50-55% RH rather than 60-65%. The higher storage humidity used in this experiment is likely to prolong residual activity rather than shorten it (Hadaway and Barlow 1965), and Anopheles stephensi is likely to be similar in susceptibility to Aedes aegypti. Therefore DDT appears to be less persistent as an Electrodyn formulation compared with an similar dose applied as a wettable powder. Despite this, the 22 weeks residual activity obtained is an acceptable result when considering the usefulness of the Electrodyn formulations in a field situation, and these tests do not take into account effects such as irritancy, which may be important in the field and which could be altered by formulation. The irritancy shown by the Electrodyn formulations in the field trials appeared to be similar to that shown shown by the wettable powders (see Chapter 5, section 5.6.1), but there is some evidence that the use of an oil in a formulation does affect irritancy (Nunn 1979), and it is possible that with other anopheline species or different hut surfaces, a 103

117 difference in mortality due to a difference in irritancy may be found. The lower doses of DDT EF (0.9 and 0.35 g/m^) show relatively short persistencies (6 and 7 weeks respectively); however they could still be considered for use in the field if the treated surface is relatively non-porous, but are unlikely to persist for long on mud surfaces. Pirimiphos methyl EF at 1.9 g/m2 also gave acceptable persistence on the plywood surface (Fig. 4.2) being effective (over 70% mortality) for at least 14 weeks, dropping to 60% and then rising again to over 70% up to 22 weeks after treatment. The reason for the mortalities dropping and rising again is unclear, but may have been due to malfunctions of the humidistat in the controlled environment room. Some malfunctions were noted over this period, but were rectified within 1 or 2 days, and would only have affected the stored boards, not the humidity at the time of the bioassay. Pirimiphos methyl in acetone has been tested in the laboratory on plywood at 1 g/m2 and gave 24 weeks mortality of Anopheles quadrimaculatus (Wilson et al_ 1971, 1973), but no details have been given regarding the storage of the boards, and it is possible that environmental conditions were more severe in this study. Contact bioassays with Anopheles aconitus carried out in houses treated with pirimiphos methyl wettable powder at 2 g/m2 in Indonesia showed only 6 and 9 weeks persistence on bamboo and wood surfaces respectively (Shaw et al^ 1979), pirimiphos 104

118 methyl emulsifiable concentrate treatments at 1 g/m^ also in Indonesia gave contact bioassay mortalities against A.aconi tus of 4 and 5 weeks on bamboo and wood respectively (Supalin et ajl 1979). A village treatment of pirimiphos methyl wettable powder at 2 g/m2 carried out in Nigeria gave over 70% contact bioassay mortality against A.gambiae for only 4 weeks on mud and thatch surfaces. Thus the pirimiphos methyl EF in these experiments showed shorter persistencies than the acetone formulation of Wilson et at (1971, 1973), but may have been stored under different conditions, but gave longer persistence than equivalent doses of wettable powders sprayed on a variety of surfaces in the field; however it must be remembered that a field treatment is subject to more variable influences, such as physical contact, smoke deposits, variations in temperature and humidity etc. Thus it is difficult to compare these results with bioassays using other formulations at the same dosage, but when considered alone, the persistence of pirimiphos methyl EF at 1.9 g/m^ on plywood is good enough to make field testing worthwhile. The lower dose of 0.6 g/m^ Was only effective for 5 weeks after treatment (Fig. 4.2), but as with DDT, this may be acceptable in some circumstances on similar surfaces. The permethrin EF bioassays were carried out at 0.35 and 0.2 g/n\2, and these doses gave 18 and 13 weeks satisfactory mortality respectively (Fig. 4.3). This is much less than the field dose tested by Coosemans and 105

119 Sales (1977) and Rishikesh et aj (1978), which was 0.5 g/m2, and bioassays carried out in the second of these two trials using Aedes aegypti showed high mortalities on the mud surfaces for about 10 weeks. Thompson and Meisch (1978) tested permethrin emulsifiable concentrate at dosages between 1.0 and g/m2 and found 10 weeks persistence down to g/m2 on plywood with 1 hour exposure to A.quadrimaculatus. Barlow and Hadaway (1975) carried out bioassays with permethrin WP at 1.0 g/m2 against A.stephensi on plywood and found high kills for over 32 weeks. Permethrin as an Electrodyn formulation therefore gives very good persistence at relatively low dosages, and certainly merits further testing in field trials at these doses. Cypermethrin EF tested at 0.1 g/m^ gave over 70% mortality for up to 22 weeks after treatment, similar to the persistence of DDT EF, and even at 0.05 g/m2 gave over 70% mortality for 18 weeks (Fig. 4.4). Barlow et al (1977) found very low toxicities with cypermethrin WP at 0.25 g/m^ on plywood against A.stephensi, and attributed this to poor pick-up by the mosquitoes of the relatively viscous formulation. In field trials, bioassays with Aedes aegypti of cypermethrin WP sprayed at the much higher dose of 0.5 g/m^ in Indonesia showed over 70% mortality for up to 21 weeks on wood and 24 weeks on bamboo (Barodji et al 1984). Thus cypermethrin EF has 106

120 given good persistence in relation to bioassays with other formulations at higher doses, and seems to be the most promising of the formulations tested with considerable scope for dose reduction, even below the 0.5 g/m2 used in these bioassays. The bioassay procedure with permethrin and cypermethrin does not however, allow for repellent effects which, in the wild, may cause the mosquito to leave the treated surface before it has picked up a lethal dose, and therefore the use of these pyrethroids may well have problems in the field not indicated by high mortalities of the bioassays. The bioassay using a blank solution produced high mortalities when tested on the day after treatment, but then dropped in subsequent weeks to low levels, not significantly different from those of the controls (Fig. 4.5). The toxic effect of solvents to mosquitoes has been shown for some other compounds, notably amyl salicylate, cyclohexyl acetoacetate and ethyl laurate (Hadaway and Barlow 1958), but persistence of toxic effect has not been studied. Clearly in this case the toxic effect disappears within the first week after treatment. The blank formulation used is a mixture of cyclohexanone and white oil, although it is impossible to establish the toxic component(s) from these results. The experiment to look at short exposure times aimed to relate the bioassay procedure to variations in resting time in the field. A deliberately low dose of 107

121 insecticide was chosen (0.5 g/m2 in both cases) since at full dose immediately after treatment the two insecticides (DDT and pirimiphos methyl) would be expected to be extremely effective under a variety of conditions. Thus the dose used was only 25% of the normal field dose. The pirimphos methyl Electrodyn formulation gave excellent mortalities (over 85%) even down to 1 minute's exposure (Fig. 4.6). The DDT EF gave good mortalities (over 70%) down to an exposure time of 5 minutes, but at 1 minute's exposure, mortality levels dropped to less than 10% (Fig. 4.6). The difference in mortality between the two insecticides at the exposure time of 1 minute is probably due to pirimiphos methyl having a strong vapour action which would compound its contact toxicity; DDT has no such fumigant action, and since it is irritant to mosquitoes (Kennedy 1947), it may have caused repeated short flights over the 1 minute period, thus considerably lessening the insecticide pickup by the mosquitoes. Kartmann and Da Silviera (1946) tested mortality of Anopheles gambiae to DDT with short exposure times, and found 95% mortality with an exposure time of 1 minute, but the dose used was 1.35 g/m^, considerably higher than that used here. In general therefore, the Electrodyn formulations gave acceptable persistencies for all the insecticides tested on a plywood surface, and produced high mortalities even with short exposure periods. These results justified further work with the Electrodyn system 108

122 for mosquito control, with particular attention to the behaviour of the formulations on other surfaces, such as mud, and with regard to other factors, such as irritancy. The pyrethroids, particularly cypermethrin, showed excellent persistence at relatively low doses. Barlow et al (1977) suggested that the lowest dose likely to be effective on a variety of surfaces was 100 mg/m2 (0.1 g/m^), because of poor availability for pick-up on plywood of the wettable powders they tested. They suggested that the rapid knock-down action of the pyrethroids occurred before a lethal dose was picked up in many cases. This does not appear to be a problem in these tests, although whether this is due to the formulation increasing availability for pick-up is not clear; however Barlow et aj-_ (1977) were able to improve the toxicity of pyrethroids on plywood by formulating 1 and 5% a.i. (w./v.) solutions of deltamethrin in 55% by volume di-(2-ethyhexyl)phthalate and 45% "Shellsol AB", although this formulation showed poor persistence on plaster. They concluded that _ the pyrethroids are undoubtedly an important group of insecticides for mosquito control but that consideration must be given to formulation to overcome certain disadvantages on some surfaces. Therefore the Electrodyn formulations give very good persistence on plywood and can be expected to perform equally well on similar surfaces, such as wood, thatch and bamboo, however their performance on mud is unknown and requires study. 109

123 CHAPTER FIVE FIELD TRIALS 5.1. EXPERIMENTAL HUT TECHNIQUES The assessment of residual insecticides in the field has presented a great many problems. Village huts in endemic malaria areas vary immensely in size, design and construction materials, and also contain different quantities of furniture, and therefore some standardisation is obviously necessary in order to effectively evaluate the efficiency of an insecticide and its effects on mosquito behaviour. The first experimental huts were built by Haddow (1942) in East Africa and by Senior White and Rao (1943) in India. These were simply purpose-built huts of a standard size and shape, in which mosquitoes could be collected. During the 1940s, several workers independently developed standard huts fitted with window traps to act either as entry or exit traps (Hocking 1947, Muirhead-Thomson 1947). The development of these huts meant that for the first time detailed and quantitative studies could be made on such behavioural phenomena as repellency (Muirhead Thomson 1947) and relative toxicity of formulations. Other experimental hut designs were developed; for example, Hadaway (1950) built circular huts with traps placed at the top of the wall, Wilkinson (1951) built a portable wooden hut with both entry and exit traps, while Davidson (1953) built a square version of Muirhead-Thomson's window trap hut at Taveta, Kenya. 110

124 These huts had concrete floors with water channels outside the hut walls to exclude ants from the huts. Ants can be a major source of error in mortality counts since they will often enter a hut in large numbers and carry off dead mosquitoes from the floor. The water channels failed to exclude ants completely however, as they were still able to enter at the base of the walls and through hairline cracks in the concrete floor (Burnett 1957). When the Taveta site was abandoned in 1955, after the Pare-Taveta Malaria Scheme used large scale dieldrin treatments, new window trap huts were designed and constructed by Rapley (1961) at Magugu in Tanzania. These huts were more permanent structures, being built of burnt brick to which an internal plaster of the mud to be studied could be applied, and renewed for successive experiments. Ant invasion was prevented by raising the hut off the ground by short concrete pillars each of which was surrounded by a water channel. The ant-proof design of these huts has been criticised by Hamon e_t al (1963), who showed a positive correlation between atmospheric humidity and mortality of mosquitoes on mud walls treated with DDT. They claimed that raising the hut of the ground will interfere with the natural migration of water and insecticides through the wall, thus affecting the sorption rate of the insecticide. Smith (1964a) argued that in East Africa, ant prevention was a big enough problem to justify the use of raised 111

125 floors, and that since most mosquitoes in these huts rest on the roof, the behaviour of insecticides on the walls is of lesser importance. The raised floor has the added advantage of preventing ground water entering the huts during periods of exceptionally heavy rain and flooding. Window trap huts were used extensively for screening insecticides and studying mosquito behaviour, but in the light of these and other studies, it became clear that a more efficient type of trap hut was necessary for use with insecticides which exhibit some degree of irritancy. A study made using specially constructed experimental huts with dichlorvos (DDVP), showed that the proportion of the total egress occurring through the eaves increases with overall mortality (Smith 1965b). In the absence of insecticide, 51% of Anopheles gambiae in all gonotrophic states and 19% of recently fed individuals were found to leave the huts each night; of the mosquitoes leaving: 15% and 30% respectively, left through the eaves (Smith 1965b). Huts with corrugated iron roofs resulted in greater egress, with 63% of A.gambiae in all abdominal stages and 34% of blood fed females leaving each night, of which 77% and 92% respectively occurred via the^ eaves (Smith et al_ 1967). The reason for the greater egress in iron roofed huts is not entirely clear; heating of the roof by the sun is not responsible as egress occurs before any significant heating. It has been suggested that egress is simply physically easier in these huts, or that the corrugated 112

126 iron acts as a mirror, reflecting the dawn light rays into the hut, thus stimulating the mosquitoes. It is clear therefore, that under certain conditions, significant eave egress by Anophelines occurs. Egress by Mansonia uniformis (Theo.) is also predominantly via the eaves; studies by Smith (1963b) in the absence of insecticide recorded 72% of recently fed females leaving through the eaves, compared with 10% in the window traps. Obviously, the window trap huts are ineffective under these conditions. A more sophisticated hut was therefore designed and built in Tanzania, the verandah trap hut (Smith 1965a). More recently a cheaper alternative, the louvre trap hut was designed, also in Tanzania (Smith et ajl 1972). The construction of both these huts will be described in the following section. Experimental huts therefore, are a very valuable means of assessing the performance of an insecticide. The disadvantages of using such huts are that they only give a partial indication of the effects of a large-scale spraying operation on the total mosquito population. In addition to this, their design and standardisation results in only partial resemblance to many village huts, and thus the behaviour of mosquitoes in them may be atypical. For example, Smith (1962) found that in local native huts, A.gambiae rested on the roof and walls in approximately equal proportions, while in experimental huts in the same area, resting was largely confined to 113

127 the roof. The reasons for this are complex, but the major factors appear to be the absence in experimental huts of a fire, a rough wall surface and the numerous household articles around the walls. Despite these drawbacks, experimental hut studies are still very important if considered as a preliminary stage to large- scale village trials, in order to be able to interpret the results of a village trial more accurately. 5.2 EXPERIMENTAL SITES Location and Site Description Two field trials were carried out in this study, both in northern Tanzania, with the help of the Tropical Pesticides Research Institute. Tanzania was selected because of the availability of experimental huts at the Institute's outstation. These comprise several specially constructed trap huts on two sites in the Umbugwe area. The Umbugwe area lies in the Mbulu district of the Northern Region of Tanzania (lat 'S and long 'E) at 3,500 ft. above sea level. The two sites comprise a louvre and window trap hut site on the western edge of the largest village, Magugu, and a verandah trap hut site a few miles to the west in a small hamlet, Vibau Vitatu. During the rainy seasons, important mosquito breeding sites are provided by the village rice swamps, located mainly around Vibau Vitatu (see map 1). The area has two distinct wet seasons, the main 114

128 MAP 1 MAGUGU AREA - VILLAGE AND HUT SITES 1 - Louvre trap hut site 2 - Verandah trap hut site.;i Houses 115

129 MAP 2 PLAN OF LOUVRE TRAP HUT SITE M agugu 911 Unused Hut P L Pit Latrine 12m

130 MAP 3 PLAN OF VERANDAH TR AP H U T S IT E Vibau Vatatu N 117 Latrine 0) k_o w Laboratory r^- 2 3 m

131 rains normally occur in March and April, and the short rains between November and January. The hottest months of the year are typically November to March, and the coolest, June, July and August. The two experimental hut sites are areas of cleared grassland. The huts are set at least 14 feet apart, and at least one pit latrine and a laboratory are also present on the site (see maps 2 and 3) Experimental Huts Two types of experimental hut were used in these trials, the verandah trap huts and the louvre trap huts. The older window trap huts on the site were not used due to their poor state of repair, and the fact that they are generally unsuitable for use with irritant insecticides. The basic hut was built of burnt brick on a timber floor, and was 8 X 8 feet (2.4 X 2.4 m) inside, with walls 6 feet (1.8 m) to the eaves (Fig. 5.1). The floor was raised from the ground and supported on 1 foot cubic concrete pillars, around the base of which were water channels to exclude ants (Fig. 5.2). The huts were lined internally with mud plaster, which was stripped off and renewed between successive trials to prevent contamination from the previous trial. one of two locally available types, The mud used was Magugu black mud or Babati red loam, the latter being a much more sorptive mud rich in iron oxide. 118

132 FIG. 5.1 BASIC TRAP HUT DESIGN FIG. 5.2 CONCRETE PILLAR AND ANT PREVENTION CHANNEL 119

133 The hut roof was one of three types: corrugated iron, grass thatch on sisal poles and grass thatch with an internal mud lining. The grass thatch and mud roofs were renewed for each trial to avoid contamination, and the corrugated iron was thoroughly washed down. Specific construction details have been given for these huts by Rapley (19 61). The louvre trap huts were similar to the basic window trap hut but have a set of entry louvres on one wall (Figs. 5.3 and 5.6). In the opposite wall was a 1 foot square window, to which a lobster pot type of exit trap, made of mosquito net on a wire frame, was fitted. A wooden frame with a horizontal row of five 1 foot square openings was set into the west wall, 1 foot below the eaves. Five louvre frames made of plywood were set into this, each frame holding eleven 2 inch wide sheet aluminium louvres, angled at 53 to the vertical and 0.8 in. apart. The louvres and insides of the frames were painted with matt black paint. The louvres were designed to permit entry of mosquitoes but to deter egress by minimising the light coming through them'. This means that mosquitoes are preferentially attracted to the lighter window and thus caught in the window trap. The eaves were completely blocked with 1/16 in. wire mesh, thus allowing no entry or egress. A simple lobster-pot trap of the same type as was in the window was fitted to the central louvre set to give an estimate of any mosquito egress via the louvres, the louvre trap catch 120

134 FIG. 5.3 LOUVRES (OPEN) WITH LOUVRE TRAP IN POSITION FIG. 5.4 VERANDAH TRAP HUT 1 2 1