Reporting Colloquium. Control of nuisant mosquitoes in Mediterranean natural areas. AGRO.M Montpellier

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1 Reporting Colloquium Control of nuisant mosquitoes in Mediterranean natural areas Thursday 27 th march 2003 AGRO.M Montpellier A methodological proposal for the sustainable management of a "Ramsar" site in Languedoc-Roussillon (France) PROGRAMME LIFE-ENVIRONNEMENT Nr LIFE99 ENV/F/ November 15 th March 31 th 2003

2 TO SUMMARIZE IMPROVING, INNOVATING AND LASTING The innovative project from EID Méditerranée (Interdepartmental organisation for the control of mosquitoes on the Mediterranean coast) that the European Commission has selected in its LIFE-Environment programme is an approach which is part of a quality assurance process aimed at conforming to the norms ISO 9000 and The objective was to propose applying a standardised methodology compatible with sustainable management of the chosen site, reconciling respect for biodiversity with control of the nuisance. It consisted in experimenting with innovative practical methods (bio-insecticides, suitable spraying methods) in the particular natural environments of Mediterranean wetlands and in comparing them with current methods and practices, in order to assess their technical efficacy and their economic feasibility and of partially or totally adopting them if they meet the need adequately and provide progress. This project was carried out within the 37,000 hectares of wetlands in the Petite Camargue (in the Gard and Hérault Departments). In 1996, this area was designated as a "Ramsar" site, from the name of the Iranian town where in 1971 an international agreement on the conservation of wetlands was signed. This is not a minor detail: awarding this prestigious designation was made after 35 years of mosquito control and means that the activities of EID Méditerranée in the Petite Camargue have not compromised the quality of the sites, to such a point that the latter have deservedly received international recognition. This project for local authorities, which started in November 1999, has extended over 3 years. It has merited financial support from the European Union of 450,000 euros, equivalent to the EID Méditerranée contribution. It has involved the following 5 tasks: 1- organisation of a standardised qualitative and quantitative recording network to assess populations of adult mosquitoes; measurement and management of the effect of the methodology on the nuisance. 2- extending the computerised ecological cartography of the distribution of the mosquito breeding sites. 3- over the selected areas, using different equipment and approved techniques, whenever necessary, spraying from the air or on the ground of Bacillus thuringiensis ser. israelensis (Bti), biological insecticide. Over the rest of the area, necessary action was taken using the chemical insecticide temephos. 4- comparison of the biological efficacy of the treatments (World Heath Organisation - WHO standardised tests) and the quality of spraying. 5- setting up an evaluation method, in representative biotopes, of the unintentional side effects of the chosen insecticides on aquatic invertebrates. The methods developed or optimised over the last three years will mean the concept of the sustainable management of nuisant mosquitoes can be refined by setting up and evaluating other alternative measures, applied selectively, singly or in combination with Bti. This project opens up horizons in terms of methodological development and raises new questions of a technical nature and also concerning the economic and sociological approach to the control of nuisant mosquitoes. This booklet is a supporting material for the present colloquium. At the end of the colloquium, in addition to the scientific and technical report which will be given to the European Commission (Environment DG), a final instructive document will be published, which will summarize the findings. 1

3 DATA SHEET MOSQUITOES CONTENTS THE TARGET SPECIES Culex pipiens Adult female: Light brown, truncated abdomen with transverse light stripes. Palps shorter than the proboscis. Dark feet, wings with narrow scales. Life cycle: Larval habitat: All types of stagnant water except lakes, lagoons and mesotrophic areas of water: - in the urban environment: rain water drains, cellars and flooded under-floor spaces, abandoned swimming pools, drums, miscellaneous receptacles, etc. - in the suburban environment: lagooning basins and water purification station discharges. - in the natural environment: essentially reed-beds with reeds and bulrushes. Also occupies, above all at the end of summer, more halophilic habitats with sea rushes, awl-leafed rushes and sometimes salicornia. Salinity up to 20 g/l. Seasonal distribution Reference period Monthly average (% du total annuel) 60% 40% 20% The questions, the necessities, the context WHAT Methodological proposal: the LIFE-Environment project - What is this project? WHO The organisation carrying out the project -EID Méditerranée (Interdepartmental organisation for the control of mosquitoes on the Mediterranean coast) WHERE Nuisance from the females: 0% J F M A M J Larval populations Populations larvaires J A S O N D Dispersal: from several hundred metres, in a strictly urban environment, to more than 3 km from suburban breeding sites. Aggressiveness: occurs essentially at night inside inhabited buildings and varies depending on the origin of the female. It is highest for populations from urban larval development sites which are warm, dark and have a high content of organic material of animal origin but practically non existent for females coming from natural habitats in clear water exposed to the sun. Nuisance females Nuisancefrom desthe femelles - curative anti-larval campaigns for urban and suburban publicly owned laying sites (drains, lagooning basins, etc.) or those requiring action beyond the means of private individuals (e.g. under-floor spaces), - preventive anti-larval campaigns by distributing information for designers and managers of public facilities and above all for private individuals:. draining cellars and under-floor spaces,. sealing covers of cisterns and septic tanks and fitting mosquito netting on air vents,. elimination of abandoned containers,. regular draining of rainwater containers. draining of pools each week in summer or introducing gold fish. M E D I T E R R A N E E - L I F E E N V I R O N N E M E N T The Petite Camargue: Ramsar nr 17 HOW The phases of the project RESULTS Management: E I D A methodological proposal for the sustainable management of a "Ramsar" site in Languedoc-Roussillon WHY Laying: eggs deposited on the surface of the water, clumped together in a pod. Hatching occurs 1 to 2 days after laying. Aquatic development: 8 days to 1 month, accelerated by warmth. Period of activity: from April to October for populations of open habitats. The females overwinter. Continuous activity for covered urban laying sites heated during the winter. 29 CONTROL OF NUISANT MOSQUITOES IN MEDITERRANEAN NATURAL AREAS - Culex (Culex) pipiens LINNAEUS, P R O J E C T Assessment of the nuisance Cartography Operational treatments Efficiency and feasibility of the methodology Unintentional effects Discussion and prospects Dissemination of results MOSQUITO DATA SHEETS The target species: Ochlerotatus caspius ; Ochlerotatus detritus ; Culex pipiens ; Anopheles hyrcanus E I D M E D I T E R R A N E E - L I F E E N V I R O N N E M E N T P R O J E C T 2

4 WHY THE QUESTIONS, THE NECESSITIES, THE CONTEXT Ochlerotatus habitat (ex-aedes) the general natural context Because of their biological, landscape and socio-economic value, the latter particularly in regard to tourism, wetland and coastal areas form a heritage zone the preservation of which is a priority. However, these protected or labelled wetland or coastal areas are very favourable sites for the proliferation of numerous insect species which are vectors of viral or parasitic diseases (human and animal arboviroses - malaria - canine filarioses, etc.) or which may be nuisant. These insects, mainly the mosquitoes, affect the quality of life of populations and can represent a health risk. It is therefore essential that there should be balanced sustainable management of these habitats combining respect for the biodiversity with remedies for the vector and pest problems which restrict the socio-economic development of local communities. From the beginning, the task of control of nuisant mosquitoes has been associated with the notion of respecting wetlands (creation of twelve reserves in the coastal development area of Languedoc-Roussillon). Ochlerotatus caspius 3 Only the females bite Ochlerotatus detritus blood enables their eggs to mature (it provides proteins) the context of mosquito control Mosquito control in 5 departments of Languedoc-Roussillon and Provence (the departments of Pyrénées- Orientales, Aude, Hérault, Gard and Bouches-du-Rhône) is a task undertaken by EID Méditerranée for the last forty years. It consists of reducing to a tolerable level the nuisance due to mosquitoes proliferating in the wetlands bordering coastal lakes and lagoons. The possibility cannot be excluded that disease vector problems may appear (cf. recurrence in 2000 of the West Nile virus in Languedoc, which was the origin of equine deaths) conferring on EID Méditerranée an objective in terms of public health. The technique: selective anti-larval control Of the forty or so species of mosquito found in French Mediterranean coastal areas only a few bite humans. EID Méditerranée's strategy is to practice selective control, only targeting the species which are the greatest nuisance to man, in the first place Ochlerotatus caspius and Ochlerotatus detritus. The natural (or rural) environment is mainly composed of marshy areas which are subject to temporary submersion, such areas in the Mediterranean (salt marshes, rush and reed beds) being typically the domain of these halophilic* Ochlerotatus. These lay their eggs on the ground, over several thousand hectares, and develop very rapidly (in 7 days) in the presence of water - rain, flooding from the sea, irrigation etc. - developing in a natural habitat but able to spread *halophilic: species living in salty habitats

5 WHY THE QUESTIONS, THE NECESSITIES, THE CONTEXT for several tens of kilometres, thus threatening the comfort of nearby dense urban populations. Their control involves about 10,000 hectares of wetland habitat, which is treated on average two to three times a year. The decision to act depends on permanent monitoring and identification of the target species. To do this, a special ecological 1/5000 map of the whole of the coastal area is used for rapid and precise localisation in time and space of the mosquito larvae, the presence and abundance of which is related to the type of habitat. Using these methods has enabled anti-larval campaigns to be undertaken. Then comes monitoring of the treatment and of its effectiveness, both in terms of mortality and general reduction of the nuisance due to the adults, and as regards regular monitoring of the sensitivity to insecticides of the natural populations subjected to the treatment, in order to anticipate the risk of resistance appearing. Finally, and this is without doubt one of the most important aspects, the fact must never be under-estimated that mosquito control is above all a job in the field requiring attention to detail, care and a great deal of vigilance. the policy: efficiency - cost - impact The public requirement, in terms of mosquito control, is based on three points: - Optimal efficiency. - Controlled costs. - Control of unintentional impact (quality control action meeting ISO standards). Prospecting Cartography: digitalization the context of regulations The context in which mosquito control takes place has changed. More than ever, two elements are leading together to changes necessary in the means and methods which have been used up until now: * The growing awareness of the biological value of wetland areas is being seen in the adoption of labels and protected status for them which, more and more, is leading to those involved in and those managing the environment participating in decision making; * The revision at European level of the approval for all pesticides intended for agricultural (agrochemicals) and domestic (biocides) uses, under two directives (dir. 91/414/CE and 98/8/CE, respectively). This situation means the methods used by the European mosquito control operators for their basic strategy have to evolve. However, no modification of the strategy is possible unless those applying it are able to adopt a new method effectively and over a sufficient time scale, together with the practical 4

6 WHY RESULTS THE QUESTIONS, THE NECESSITIES, THE CONTEXT DISSEMINATION OF RESULTS Bti This is Bacillus thuringiensis ser. Israelensis, a strain of bacteria commonly found in the soil or in an environment rich in insects, which is remarkably toxic to mosquito larvae. Its use as an insecticide - we are thus talking of a bio-insecticide - is widespread throughout the world, particularly for the larval control of nuisant or vector mosquitoes, because of its very high selectivity relative to other representatives of the aquatic fauna. It is registered in France. Its active ingredient, a complex spore-toxin, acts exclusively by ingestion, requires homogeneous spraying and can be made rapidly ineffective by photodegradation, sedimentation and adsorption in the substratum. Sporulating cell of Bti (JF. Charles, Institut Pasteur) E I D M E D I T E R R A N E E he results will be distributed as a priority to those involved locally, the users and environmental managers, in the hope of encouraging their sustained involvement in planning and carrying out activities: - the local and regional authorities (district, departmental and regional councils). - the Mediterranean Environment Agency (AME Languedoc-Roussillon), - the Regional Environment Agency (ARPE Provence - Alpes - Coast d Azur), - the Regional Environment Departments of Languedoc-Roussillon and Provence - Alpes Côte d'azur (State departments), - the Coastal and Lake Conservancy, - the Etang de l'or Management Association, -the Association for the Protection and Management of the Camargue in the Gard Department, - the Tour du Valet Biological Station, - etc. he final report will include the information necessary for decision makers to become aware of the importance of the project. It will present the calculation of economic feasibility of a mosquito control programme favouring the use of the bio-insecticide Bti, considering the environmental benefits. communication programme has been set up and will grow as the project advances: * via EID Méditerranée's existing organs of information: - "EID info" and the "EID Letter", - information brochure, - special features, Internet, CD Rom, etc, - scientific publications. * via the internal press of local authorities financing EID, the daily press and regional periodicals. * via meetings, conferences or events of a technical or scientific nature: - local theme meetings, - diverse scientific councils, - EDEN (European Association of public mosquito control operators), and EMCA (European Mosquito Control Association) colloquia, - European Sove meeting (Society for vector ecology), Mosquito Control Abatement International Congress (Thessalonique, Grèce), Symposium international de l'eau (Cannes), TEM-TEMCA Fair (Madrid), 81e congrès de l'association générale des hygiénistes et techniciens municipaux (Montpellier), etc. T T A temephos Exclusively a larvicide, temephos is an insecticide belonging to the organophosphorous family (cholinesterase inhibitors), marketed under the name of "Abate", and acts on the mosquito larvae by contact and ingestion. It combines the advantages of not being bioaccumulable and of presenting relatively little risk to mammals (oral LD50 in the rat: 8,600 mg/kg, not classified), birds, fish, aquatic fauna and flora, when its authorised conditions of use are observed - conditions which led to its approval. It has the disadvantage of being less selective for certain aquatic invertebrates, mainly insects, when they are present at the time of treatment of mosquito larval sites. But the strategy adopted by EID minimises this disadvantage to a large extent. Bacillus thuringiensis 5 pamphlets, meetings, events means for its implementation. That implies a period of training, experimentation and application of the data resulting from the study. In other words, there can be no question of making profound changes overnight in all or part of the practices without taking the risk of finding the programme in a blind alley. In more concrete terms, operational agencies are confronted with the following problems: * The withdrawal of certain chemical insecticides, envisaged particularly from an environmental point of view, poses problems of efficiency and feasibility in the short term. What are the concrete alternatives that can be applied straight away or that can be envisaged in the medium term? * The use of the latest generation of insecticides, in particular the bio-insecticides, necessitates adapting equipment and the methods of application and their evaluation. What procedures and what material and financial means can be set up to make this change? * Does satisfying the objective of sustainable development necessitate the use of a single category of products or must it/can it integrate a wider array and under what conditions (integrated campaign)? * Taking into account the environment is inherent to mosquito control activities. Apart from the choice of insecticides, what are the measures to be taken, involving participation by the operational agencies, aimed at better control and minimising unintentional effects? - L I F E E N V I R O N N E M E N T P R O J E C T E I D M E D I T E R R A N E E - L I F E E N V I R O N N E M E N T P R O J E C T 26

7 WHAT METHODOLOGICAL PROPOSAL: THE LIFE-ENVIRONMENT PROJECT Facing to these considerations and the questions raised, a project produced by EID Méditerranée and submitted in 1998 to the LIFE-Environment Community programme received financial support from the European Commission in LIFE-Environment The Financial Instrument for the Environment, supporting development and application of Community environmental policy. This European programme supports innovative action with the intention of promoting sustainable development. "Ramsar" nr 17 site and the area controlled by EID Méditerranée in the Petite Camargue Potential larva breeding sites and protection measures sustainable management This is development which meets current needs without compromising the capacity of future generations to meet theirs. It is a process of evolution during which the use of resources, the direction of investments, the advance in technological development and institutional transformations meet the needs of the future as much as those of the present (Brundtland World Commission on the Environment and Development ). what is this project? This project proposes to demonstrate over a period of three years a methodology on a "real life" scale that makes control of the nuisance due to mosquitoes compatible with preservation of the environment. This approach should contribute to setting up sustainable management for wetland areas and provide answers to questions which are today asked by operators. The "Petite Camargue" in the Gard department, a vast area of wetlands included in the Ramsar inventory as nr 17, has been chosen for carrying out this demonstration. It has been chosen for good reason, because the zone is an integral part of the EID Méditerranée action area and moreover forms in many respects a replica of the Grande Camargue which indeed has no mosquito control. The methodology proposed here is based on using a particularly selective insecticide of biological origin, Bacillus thuringiensis ser. israelensis (or Bti, an entomopathogenic bacterium), assessed in parallel to the synthetic organophosphorous insecticide, temephos. The aim sought was to diminish as much as possible the impact of insecticide spraying on the biocoenoses of mosquito breeding sites, over several hundred or thousand hectares within the Ramsar nr 17 area. This bio-insecticide, which has been known for a long time, was the subject of numerous studies on efficacy and impact on non-targeted fauna within EID Méditerranée in the early '80s. It has only been used up until now on a small scale because of certain technical problems related to its use, inadequate effectiveness and greater cost. But progress concerning the formulation of the bacteria and the adaptation of spraying techniques has recently contributed to providing solutions to these problems. The project will demonstrate how an emphasis can be put on ecological interest and efficiency while combined with new methodology over a vast area where mosquitoes are already controlled. The results are addressed directly to local authorities since it is a question of changing current practices to benefit the environment. Indeed, later, depending on these results the feasibility of extrapolating the scheme to the whole of the controlled area will be assessed. But the project is also interesting because the opportunity has been taken to apply a certain number of innovative methods concerning the GIS (geographical information system) and the recording and use of data, with the aim of improving strategy and knowledge of the phenomena which come into play and finally, in terms of choice and application of insecticides, the traceability of action taken, evaluation of its efficiency and its unintentional effects. Each of these points is covered in more detail in order to make clear the main points of an integrated standardised control methodology, compatible with sustainable management. 6

8 WHO RESULTS THE ORGANISATION CARRYING OUT THE PROJECT DISCUSSION AND PROSPECTS he aim of the project was essentially to demonstrate a methodology for controlling the nuisance due to mosquitoes that is consistent with the policy for managing natural areas in which this control takes place and is compatible with the notion of sustainable development. This involves finding an equilibrium between a sufficient level of satisfaction by reducing the nuisance and the best possible preservation of the environment without exceeding the cost that the local authority is prepared to pay for this. The necessity for perfect control of the nuisance in the particular situation of the Mediterranean coastal region is of course a vital element in the system. But to achieve this the perception level of this nuisance must be correctly defined, be this by the capture of adult anthropophilic mosquitoes or in terms of socio-economic assessments, which present numerous difficulties to which answers must be found. The methodological approach proposed in the Life project is based on the adoption on an operational scale of a bioinsecticide with its advantages and limitations of use. It has provided the opportunity for assessing the capacity to modify the control strategy and demonstrate the necessity if not the advantages of adapting or optimising the methods used to measure these changes. While it is clear that the use of Bti no longer poses real problems, its use on a wider scale requires better quantification of the operational risk. In other words, what might average effectiveness of 85 or 90 % on the larvae mean in terms of the emergence of adults from larvae which have survived and the nuisance thus produced? This question is far from being anodyne if it concerns a breeding site close to dwellings where the number of larvae per square metre is sometimes considerable, the adults emerging from them being capable of flying long distances. This point requires effective continuation of the investigations, all the more so if Bti is to become one of the main methods of control in the next decade. Evaluation of unintentional effects on the environment may however form one of the pragmatic elements of sustainable development. While overall within Europe the large majority of programmes to counteract against mosquitoes organised by local authorities has always taken care to use the least harmful methods, the development of environmental concerns, as well as of regulations is such that cautious use and adaptation of actual control methods is necessary. With the Life project it has been possible to set up a risk evaluation system in situ. The data collected, even though fragmented, add to and shed light on the numerous data supplied in scientific literature. This approach must be continued if not enlarged in order to produce perennial indicators for mosquito control activities. Global evaluation of the unintentional effects of mosquito control on natural sites must therefore be integrated into a multi-disciplinary approach and take into account how the ecosystems concerned function overall. In order to complete the study carried out in this Life project, the following points could be developed: T EID Méditerranée (Interdepartmental organisation for the control of mosquitoes on the Mediterranean coast) ID Méditerranée is a public service organisation created in 1958 by the councils of the French departments of Hérault, Gard and the Bouches-du-Rhône and joined in 1963 by the councils of the Pyrénées-Orientales and Aude departments. The inter-ministerial mission for the development of the Languedoc-Roussillon coastline for tourism (the Racine mission), created in the same year, made mosquito control the prerequisite for any economic development in coastal areas and for tourism in particular. Since 1982, the 5 departments, together with the Languedoc-Roussillon Region have totally financed the organisation. EID Méditerranée is the local authorities' operator in coastal wetland areas. * Mosquito control today involves 215 district council areas between Marseilles and Cerbère. It is a perennial activity since it involves controlling the populations of a few species of mosquito, and therefore their nuisance level, and not their eradication (which is both impossible and not desirable); * EID Méditerranée's action is based on permanent observation of the habitats. Throughout the year a hundred or so operational agents collect data (vegetation, hydraulic, topographic) for mosquito control and also in a partnership agreed for ecological monitoring purposes (Coastal Conservation Unit, Tour de Valat Biological Station, the Gard Camargue Association, etc.). In this respect, EID Méditerranée is actively involved in the management of wetland areas; * EID Méditerranée staff have acquired in depth knowledge of mosquito control which explains the numerous requests for help (expert advice, technical assistance) from beyond its area of activity, both in France and abroad. This expertise extends to the control of other biological pests (black flies, coypu, etc.); * For about fifteen years EID Méditerranée has been investing in a programme restoring the coastal dune cordon, which means both physical control of the mosquitoes (by preventing the flooding of marshy areas behind the dunes) and management and enhancement of the coastal environment; * EID Méditerranée is a founder member (1996) of ADEGE (the National Agency for Mosquito Control and the Management of Mosquito Controlled Natural Areas), with the two other French Inter-Departmental Agencies (of Atlantique and Rhône-Alpes), the Martinique and French Guyana mosquito control services and SIVU in Lauterbourg (Alsace). In 1999, the European association of public authorities and operators involved in mosquito control and the management of natural areas where mosquitoes are controlled, EDEN, was formed, bringing together 15 mosquito control public operators representing about forty French, Spanish, Greek and German local public authorities. E the technical departments Conducting the project was done with the aid of the technical departments of EID Méditerranée. * The Research and Development Department had overall control with the laboratory as spearhead, which has the main tasks of: - research, development and validation of means (mainly insecticides) and methodologies. The criteria for the choice of insecticides are biological efficacy and the greatest possible selectivity. - monitoring operations, checking levels of sensitivity and the possible appearance of resistance phenomena in target species. - participation in improving knowledge of mosquitoes in the areas of biology, ecology, ethology, taxonomy and the habitats where they live. * The Department for the Control of Mosquitoes and other Pest Insects and the operational agencies of Saint-Chamas (Bouches-du-Rhône), Montcalm and Aigues-Mortes (Gard) and Lunel-Viel (Hérault) carried out survey and control activities in the field (prospecting and treatment). * The Environment Department contributed to an internal control focusing on the identification and measurement of the unintentional side effects of mosquito control activities. 7 E I D M E D I T E R R A N E E - L I F E E N V I R O N N E M E N T P R O J E C T E I D M E D I T E R R A N E E - L I F E E N V I R O N N E M E N T P R O J E C T 24

9 WHERE THE SITE OF APPLICATION Sharp rush Salicornia Black bog rush Ramsar Ramsar nr 17 site This is not an acronym but the name of an Iranian town where, on 2nd February 1971, an agreement was signed which makes it compulsory for signatories to designate the areas of their territory to be included in the list of wetlands of international importance. The parties to it undertake to promote conservation and rational use of these wetlands. This convention was ratified by France in the Petite Camargue: Ramsar site nr 17 The project applies to the Petite Camargue. This French Mediterranean coastal area was added to the "Ramsar" inventory in The control of the nuisance caused by mosquitoes has been carried out here for nearly 40 years. The Petite Camargue, composed of that part of the Camargue in the Department of Gard and the Etang de l'or, is a vast wet land of 37,000 hectares, of which a little over 10,000 hectares are natural wetland habitats. It extends from the Petit-Rhône to the west bank of the Etang de l'or, straddling the departments of Gard and Hérault. The combined action of river and sea flooding has contributed to the formation of a mosaic of marshes, lakes and lagoons which form migratory stopping points, areas of reproduction and feeding areas for numerous bird species of which at least 150 are rare: purple heron, common bittern and little bittern, night heron, black-winged stilt, bearded tit etc. Other remarkable residents should be pointed out: the European pond turtle and the endemic species Hydrobia scamandri. The Etang de l'or, an area of 3,000 hectares of water surrounded by a wet area of 2,000 hectares, is a breeding ground for migrating fish: eel, bass, sea bream etc. The flora is also very rich, the Petite Camargue including habitats as varied as reed-beds, pine groves, salt marsh and water meadows. In addition it contains a great many areas protected by regulations or by acquisition of property: - the natural protected site of the Etang de l'or; - the Fromagère and Buissongros marshes; - the departmental areas of Mahistre and Musette; - several properties belonging to the Coastal and Lakeside Conservancy, In the Petite Camargue area, the Association for the protection and management of the Camargue in the Department of Gard and the Association for the management of the Etang de l'or work to protect and develop these rich and fragile habitats. Supported by the Gard and Hérault councils, the State and the European Union, these two management organisations have set up an environment charter combined with a Water Improvement and Management Scheme (SAGE) and a bay contract. These two instruments supplement the action undertaken in conjunction with users and institutional partners. By reconciling the management of the habitat and development of human activities, the aim is to improve water quality and management and conserve the ecological and landscape characteristics of these sites. 8

10 HOW THE PHASES OF THE PROJECT five tasks The project consists of carrying out the following 5 tasks: 1 1- Organisation of a standardised qualitative and quantitative recording network to assess the population of adult mosquitoes; measuring and managing the incidence of the methodology on the nuisance Extension of the computerised ecological mapping of the distribution and functioning of mosquito breeding sites Air or ground spraying, whenever necessary, over the selected areas and using different equipment and approved techniques, of Bacillus thuringiensis ser. israelensis (Bti) (over the rest of the area, action was taken using the synthetic insecticide temephos) Comparison of the biological effectiveness of the treatments (World Health Organisation - WHO - standardised tests) and the quality of the spraying Evaluation of the qualitative and quantitative impact of chosen insecticides on aquatic invertebrates in representative biotopes. To which must be added: the diffusion of the results and increasing awareness of them A standardised data bank of ecological and meteorological parameters was set up and provides a tool for analysing the interrelations existing between them and the data collected during the 5 tasks. 9

11 RESULTS SUMMARY 1 Assessment of the nuisance The trapping techniques Comparison between the two methods Comarative sociological and ethnological investigations: Camargue and Petite Camargue 2 Cartography A thematic cartography of the habitats The geographical information system (or GIS) 3 Operational treatments Surface areas affected, surface areas treated in the ramsar nr 17 zone and the means of spraying used Monitoring of aerial treatment using DGPS 4 5 Efficiency and feasibility of the methodology Larvicidal effectiveness of Bti Are the methods of spraying used effective and efficient? Unintentional effects The experimental site The functioning of this habitat The respective impacts of Bti and temephos on non-targed fauna Discussion and prospects Dissemination of results 10

12 RESULTS ASSESSMENT OF THE NUISANCE Capture on human bait the trapping techniques At the present time, the technique used by EID Méditerranée to assess the nuisance is capture on human bait. The trapper, who acts as bait, exposes his bare forearm or calf to the mosquitoes for 15 minutes at sunrise. With the other hand, he uses a mouth suction trap to capture them when they land on him. From experience we can estimate that there is a nuisance in the rural environment (Oc. caspius) when 5 or more mosquitoes are captured. The choice of this threshold is taken after drawing a parallel between capture results and complaints relating to the nuisance. However, the increasing awareness of the vector role of certain mosquitoes (arbovirus) has spurred us into automating the nuisance evaluation system by mechanical trapping. Basing our decision on different techniques described in the literature, a carbon dioxide trap (CO2) was selected. The carbon dioxide, produced by living organisms, is attractive to mosquitoes. This gas can be packaged and released in small quantities on sublimation of dry ice. The mosquitoes thus attracted are carried by the air current produced by a fan driven by an electric motor and projected into a mosquito netting cage. After a phase perfecting the apparatus (trial of different models of traps in 1999, development and in-house production of the appropriate model in 2000) CO2 trapping was carried out regularly in 2001 and 2002, at the same time as capture on human bait. CO2 trap CO2 trap (détail) 11 comparison between the two methods During these 2 years, CO2 trapping collected 128,116 mosquitoes, representing 18 species and 7 genera (Ochlerotatus, Anopheles, Coquillettidia, Culex, Culiseta, Aedes and Uranotaenia) of the 8 genera present in France (the single species of the 8th genus, Orthopodomyia, is not anthropophilic). However, the diversity demonstrated is not exhaustive because non-nuisant species (not attracted by CO2) and/or those with a restricted distribution were not captured. Trapping on human bait carried out on the same sites as the CO2 traps collected 6,090 mosquitoes. The quantitative difference between the two methods is above all related to the number of captures made by each of them, and to the period of trapping (15 minutes for human baiting, about 15 hours per trapping session with CO2). Both methods revealed 4 species to be in the majority : Ochlerotatus caspius, Culex pipiens, Anopheles hyrcanus and Ochlerotatus detritus. Ochlerotatus caspius is the species most represented by the two methods (39 % in CO2 traps and 54 % with human bait). It is very aggressive and develops in coastal natural habitats which are temporarily submerged (cf. Mosquito information sheets). Another species, Culex pipiens, is present frequently in the

13 RESULTS ASSESSMENT OF THE NUISANCE Monthly adult populations of Oc. caspius captured by CO2 trapping Qualitative and quantitative distribution of species captured by CO2 trapping CO2 traps (31 %) while being poorly represented in the human bait captures (1 %). This difference is due to the trophic behaviour of the species which varies depending on the larval breeding site. In fact, the natural breeding sites in clear water would host ornithophilic populations, certainly attracted by the CO2 but not aggressive towards man, while populations in water heavily laden with organic matter, thus rather more in urban areas, would have a more pronounced anthropophilic behaviour. As the human bait captures were made in rural areas, the Culex pipiens species is logically less strongly represented in the results of this technique. Finally the species Anopheles hyrcanus is a very occasional nuisance both in terms of area affected and in time, close to its larval development areas, mainly in rice field, from which it spreads little and only in August and September. conclusions on trapping techniques The in depth comparison of these two methods on a common site should allow a nuisance threshold to be estimated using only CO2 trapping. In fact, it seems that where human bait trapping captures 5 anthropophilic mosquitoes (nuisance threshold reached), a CO2 trap captures a minimum of forty. However, to date no linear relationship between the two techniques has been demonstrated and this estimation should be statistical confirmed using a greater number of data entries. The CO2 trap has several advantages over trapping on human bait as currently carried out. In the first place it makes it possible to read fully the two peaks of mosquito aggressiveness (evening and morning). Moreover, the capture of rare species not represented in the human bait captures can be interpreted as a view of the quality and/or limits of action in certain areas. Finally, with this method entomological monitoring of vector risks within the action zone is possible. Setting up a CO2 trap network has a relatively high cost both in terms of material (traps, batteries, dry ice etc) and logistics (delivery of dry ice, setting up/dismantling). Validation of the CO2 method of trapping requires working out and applying a study protocol to compare the two methods of trapping very carefully. This study will at the same time make it possible to define by reasoned choice the threshold for declaring mosquitoes of rural origin a nuisance in or at the edge of an urban zone. 12

14 RESULTS ASSESSMENT OF THE NUISANCE Carried out by the DESMID team (Ecological and Social Dynamics in Delta Habitats - URA CNRS 1451), this enquiry recorded some paradoxes: although falling within the life sciences, mosquitoes and mosquito control are rich subjects for sociology and anthropology. They also evoke varied comments and practices from the local people involved, which are apparently contradictory, revealing the complexity of social reality and processes of change. conclusions of the investigation If they are continued, sociological investigations will allow progress in two directions: * The tolerance threshold of populations is subjective and lowers with time and as they become accustomed to mosquito control operations. Sociological analysis helps provide an understanding of the mechanisms which induce this modification and the reasons for the "sensitivity" in order to define, in the long run, acceptable nuisance thresholds. * Numerous methods of investigation exist to quantify intentional and unintentional impact; however, as the mesological, ethological and biological parameters etc. cannot be taken into account entirely, they remain by definition relatively limited. Ecological research does not aim to provide a finite answer but to reduce uncertainty. It is therefore essential to consider together the different scientific, technical and sociological points of view. Sociological studies should thus be continued, in the first instance by enlarging them to all the social groups likely to suffer a nuisance from mosquitoes (e.g. inhabitants and tourists), and secondly by analysing institutional reasoning and debates concerned with this subject. comparative sociological and ethnological investigations: Camargue and Petite Camargue The inhabitants in the Camargue and the Petite Camargue say that they are bothered by mosquitoes, right to the point of being extremely exasperated, while at the same time to a certain extent being accustomed to them, even to having a sort of immunity to the insect's bite. The mosquito is classified as a pest, but also as being useful. These ways of classifying the mosquito do not allow a clear differentiation between two distinct population groups. On the contrary, these two descriptions may both be used by the same individual. Thus we arrive at the paradoxical picture of a "useful pest". From this point of view in particular, the mosquito is depicted both as part of the food chain, essential for ecological equilibrium, and as a disease carrier. Another paradox is that the inhabitants consider mosquito control as progress, while emphasising its possible consequences on the environment; and, following from the two previous points, the inhabitants want mosquito control of inhabited areas, but have reservations as far as mosquito control in natural areas is concerned. Similarly aspirations concerning mosquito control or opposition to it oscillate between the desire for daily well being, demands for recognition giving value to resistance to mosquitoes, the refusal of or a wish for tourist development. Finally, the two sub-populations, those "with mosquito control" versus those "without mosquito control" at the same time show both visible differences and significant common factors. Concerning the perception of nuisance, the statistics demonstrate a large difference between the two areas, those without mosquito control being much more inclined to say that they are bothered than those with mosquito control. However, the question of the relative nuisance reappears in the qualitative investigation, where it can be seen that a low density of mosquitoes can also be the source of discomfort. Moreover, both tend to highlight their relative "immunity". Those with mosquito control use less protection than those without it, but when they do, apart from mosquito netting which is tending to disappear in the Petite Camargue, the same procedures are chosen. Those with and those without mosquito control also have in common their limited knowledge concerning mosquitoes and above all concerning mosquito control. As far as the description of the mosquito is concerned, both populations groups describe it at one and the same time as a pest and as useful. The disadvantage most often cited in the Camargue is the presence of mosquitoes while in the Petite Camargue it is the presence of too many tourists which is put at the top of the list. Too many mosquitoes or too many tourists? The question of mosquito control is not limited to what is sometimes presented as a dilemma and includes the wish for a comfortable life for all. A border-guard or a filtering system, the mosquito is also presented as a weighty matter as far as tourist development is concerned. 13

15 RESULTS CARTOGRAPHY Example of an ecological map a thematic cartography of the habitats EID Méditerranée has chosen to develop two types of mapping, based particularly on differentiated typologies: * the "mosquito" ecological map, produced at a scale of 1/5000, which defines the potential larval development areas using a suitable typology (more than thirty habitats identified on the sandy coasts of the Golfe du Lion). This is a particularly accurate method. With 400 ha mapped, the Carbonnière site is sufficiently representative of the Ramsar nr 17 area. The Enfores site where the impact study was carried out on an area of 29 ha has also been mapped. * the thematic cartography of wetland areas at 1/25,000, the typology (18 habitats defined) of which is close to that of the Habitat Directive (Corine Biotope classification), specially adapted to the problem of Mediterranean wetlands. Natural wetlands represent nearly 30 % of the whole of the Ramsar nr 17 area and are very diverse in nature: reed-beds, rushes, bull rushes, meadows and salt marsh, i.e. salicornia, purslanes, salsolacea etc. to which must be added more than 13,000 ha of salt beds and flooded agricultural land (rice paddies). Example of a thematic map of wetland areas Use of a GIS the geographical information system (or GIS) The geographic area studied in this project, i.e. the Ramsar nr 17 area, includes numerous aspects of various natures (environmental, the interests of tourism etc.). The traceability of operational mosquito control activities in this area is ensured by setting up a GIS. Several preliminary stages have followed one another: * choice of programs: MapInfo 6.0 for the GIS part; Mapbasic 6.0 and its programming language for the special developments; Access 2000 for the database part; TERA VIEW for imaging; the data for the RASTER base: use of the IGN 25 scans already available, supplemented by aerial photography or even by orthophotomap; for the VEC- TOR layers: use of layers in hand (districts ) and production of specific data by EID Méditerranée (EID 1/25000 layer, EID 1/5000 layer); * development of the actual system: creation of the Life database aiming to contain all information relating to treatments and the development of an application in Mapbasic allowing geographical objects and their data to be linked; * training of staff and bringing into service during July In addition, with the system a certain number of requests and analyses can be carried out such as: * analysis of the cover provided by the different 14

16 RESULTS RESULTS OPERATIONAL TREATMENTS CARTOGRAPHY n selected areas within chosen perimeters and as and when required, spraying of a formulation based on Bti (Vectobac 12 AS) is carried out from the air or on the ground. Outside of these areas, treatment is carried out whenever necessary with the registered organophosphate insecticide, temephos. The traceability of aerial treatments is greatly improved by the use of an onboard differential global positioning system (DGPS), allowing positioning of the aircraft and computerised recording of successive swaths. O treatments per product in order to find out in all respects the proportion of product sprayed; * cross-checking of all treatments as a function of the habitat typology concerned and knowledge of the surfaces treated by type of habitat mapped, for statistical use; * obtaining, for a given area, simple statistics on the activities carried out since the beginning of the project particularly for producing reports; * production of a rapid configurable printing module to facilitate the printing of work done by all users, not requiring special knowledge of the MapInfo program in order to print. * analysis of the DGPS treatment record (see page 16). Data and queries surface areas affected, surface areas treated in the Ramsar nr 17 zone and the means of spraying used Rural larval sites and the level of nuisance n the Ramsar nr 17 zone, the hatching of Ochlerotatus spp. covers on average 14,000 ha per year with, for example, for the period , a minimum of 11,070 ha (1998) and a maximum of 19,033 ha (1999). For various reasons, more than 80 % of the larvicide treatment has been carried out by air for many years. The exclusive use of synthetic products up until 1998 meant that a very low volume treatment could be developed (1 to 5 l/ha of mixture) allowing us to get the best out of limited but sufficient aerial equipment (1 aircraft with a load capacity of 300 kg). The combined circumstances of the extension of aerial treatment to other EID Méditerranée areas and above all the wish to include Bti in the range of larvicides has lead to air capabilities being increased. Thus, two aircraft with load capacities of 500 kg and 800 kg respectively, with autonomy of 4 hours in the air, were available for this Life programme. I conclusions Mapping the habitats: The study carried out in the Petite Camargue has confirmed the usefulness of mapping wetlands where mosquitoes are controlled in order to locate larval breeding sites and monitor the effectiveness of their control. The ecological maps produced at 1/5000 remain well suited to this purpose. It is therefore planned to continue to update them. As regards mapping of habitats at a scale of 1/25000, studies based on vegetation surveys over the coastal area are envisaged in order to confirm their typology and continue to completely cover the EID Méditerranée area of action. Afterwards the updated versions of this digital map will be made to match a more accurate background (orthophotoplans). Ground treatment (caterpillar vehicle) monitoring of aerial treatment using DGPS Geographical information system (GIS) The computerised production of these maps depends on high quality reference documents such as the IGN 25 scan maps and more recently orthophotoplans. These maps and their associated data can be used in the GIS and be available for numerous applications. It is therefore planned to develop GIS applications for the whole area of action and to include in them aspects related to the control of water, (Digital Terrain Model or DTM). Analysis of cover from the treatments according to the different products * The system used: This was the AgGPS TrimFlight3 system from the Trimble company, which meets all aspects of the specifications, particularly in terms of precision and autonomy. Entirely designed specifically for aerial spraying, it consists of a receiving aerial, a GPS-DGPS AgGPS 132 receiver, a "strengthened" AgGPS 170 computer allowing adjustment of recording parameters and real time display of spraying. The recording function, working from the aircraft's valve control, makes real time display of the swath possible with the optional addition of map backgrounds (contours of areas of land, topographical landmarks etc.). A luminous diode guidance bar, which can provide assistance during spraying on areas with straight borders completes this system which has been specially authorised for use by the civil aviation authority. The system was installed by an approved company in the PA 36 aircraft. The installation and use of this equipment made operational recordings available from October It will also serve to generalise intra-plot traceability of mosquito control treatment using DGPS. Aerial treatment Cross-checking all treatments as a function of the habitat typology concerned 15 E I D M E D I T E R R A N E E - L I F E E N V I R O N N E M E N T P R O J E C T E I D M E D I T E R R A N E E - L I F E E N V I R O N N E M E N T P R O J E C T 16

17 RESULTS RESULTS OPERATIONAL TREATMENTS CARTOGRAPHY n selected areas within chosen perimeters and as and when required, spraying of a formulation based on Bti (Vectobac 12 AS) is carried out from the air or on the ground. Outside of these areas, treatment is carried out whenever necessary with the registered organophosphate insecticide, temephos. The traceability of aerial treatments is greatly improved by the use of an onboard differential global positioning system (DGPS), allowing positioning of the aircraft and computerised recording of successive swaths. O treatments per product in order to find out in all respects the proportion of product sprayed; * cross-checking of all treatments as a function of the habitat typology concerned and knowledge of the surfaces treated by type of habitat mapped, for statistical use; * obtaining, for a given area, simple statistics on the activities carried out since the beginning of the project particularly for producing reports; * production of a rapid configurable printing module to facilitate the printing of work done by all users, not requiring special knowledge of the MapInfo program in order to print. * analysis of the DGPS treatment record (see page 16). Data and queries surface areas affected, surface areas treated in the Ramsar nr 17 zone and the means of spraying used Rural larval sites and the level of nuisance n the Ramsar nr 17 zone, the hatching of Ochlerotatus spp. covers on average 14,000 ha per year with, for example, for the period , a minimum of 11,070 ha (1998) and a maximum of 19,033 ha (1999). For various reasons, more than 80 % of the larvicide treatment has been carried out by air for many years. The exclusive use of synthetic products up until 1998 meant that a very low volume treatment could be developed (1 to 5 l/ha of mixture) allowing us to get the best out of limited but sufficient aerial equipment (1 aircraft with a load capacity of 300 kg). The combined circumstances of the extension of aerial treatment to other EID Méditerranée areas and above all the wish to include Bti in the range of larvicides has lead to air capabilities being increased. Thus, two aircraft with load capacities of 500 kg and 800 kg respectively, with autonomy of 4 hours in the air, were available for this Life programme. I conclusions Mapping the habitats: The study carried out in the Petite Camargue has confirmed the usefulness of mapping wetlands where mosquitoes are controlled in order to locate larval breeding sites and monitor the effectiveness of their control. The ecological maps produced at 1/5000 remain well suited to this purpose. It is therefore planned to continue to update them. As regards mapping of habitats at a scale of 1/25000, studies based on vegetation surveys over the coastal area are envisaged in order to confirm their typology and continue to completely cover the EID Méditerranée area of action. Afterwards the updated versions of this digital map will be made to match a more accurate background (orthophotoplans). Ground treatment (caterpillar vehicle) monitoring of aerial treatment using DGPS Geographical information system (GIS) The computerised production of these maps depends on high quality reference documents such as the IGN 25 scan maps and more recently orthophotoplans. These maps and their associated data can be used in the GIS and be available for numerous applications. It is therefore planned to develop GIS applications for the whole area of action and to include in them aspects related to the control of water, (Digital Terrain Model or DTM). Analysis of cover from the treatments according to the different products * The system used: This was the AgGPS TrimFlight3 system from the Trimble company, which meets all aspects of the specifications, particularly in terms of precision and autonomy. Entirely designed specifically for aerial spraying, it consists of a receiving aerial, a GPS-DGPS AgGPS 132 receiver, a "strengthened" AgGPS 170 computer allowing adjustment of recording parameters and real time display of spraying. The recording function, working from the aircraft's valve control, makes real time display of the swath possible with the optional addition of map backgrounds (contours of areas of land, topographical landmarks etc.). A luminous diode guidance bar, which can provide assistance during spraying on areas with straight borders completes this system which has been specially authorised for use by the civil aviation authority. The system was installed by an approved company in the PA 36 aircraft. The installation and use of this equipment made operational recordings available from October It will also serve to generalise intra-plot traceability of mosquito control treatment using DGPS. Aerial treatment Cross-checking all treatments as a function of the habitat typology concerned 15 E I D M E D I T E R R A N E E - L I F E E N V I R O N N E M E N T P R O J E C T E I D M E D I T E R R A N E E - L I F E E N V I R O N N E M E N T P R O J E C T 16

18 RESULTS OPERATIONAL TREATMENTS * Results: Of the 91 treatments carried out using the PA36 aircraft, on the Ramsar nr 17 area during of the period , 35 of them were recorded i.e %. The records obtained, superimposed over the borders of areas required to be treated by the pilot, were analysed using Mapinfo, in terms of cover, with the help of an application developed in MapBasic. This method showed up the "useful" cover of the swaths, expressed as a percentage. The results of these analyses feed a database which forms an important component for traceability. How the DGPS works Record of the spraying (23/03/2001) Example of analysis of a DGPS recording conclusions The proportion of Bti sprayed compared with synthetic larvicides has grown from a few % in 1999 to more than 20 % in The results have been satisfactory within the framework established but extension of its use on an operational scale to the whole of the area of action still remains limited by: * the irregularity of results which do not allow its routine use to be envisaged for larval biotopes situated near urban areas, * the volume of mixture to be sprayed per hectare, which seriously complicates spraying when action is required more than 30 or 40 km away from the refilling site. For extending this project it is therefore essential to: * develop new directions for research concerning new formulations approved at the end of 2002, such as VectoBac WG, with the prospect of low volume treatments; * test, over a complete season, the exclusive use of Bti in a sector of 150 to 200 ha, little exposed to external infestation and which up until now has been controlled using a synthetic product and is close to an urban area. A trapping system, which is as dense as possible should allow the results to be evaluated both on the treatment sites and within the built-up areas. The possible perception of the change of larvicide could be recorded from a survey among the population. 17

19 RESULTS EFFICIENCY AND FEASIBILITY OF THE METHODOLOGY T emephos treatment is highly effective but the slight variability of the data does not permit reliable statistical analysis. For this reason only the influence of the parameters concerning Bti is given here. Evaluation in plots of the efficacy of a larvicide formulation Sensitivity test (WHO) larvicidal effectiveness of Bti Over the whole of the period studied, 35 Bti treatments (23 from the air, 12 on the ground) were analysed. Bti had a mean efficiency of 81 ± 8.7 % from aerial treatment and 92 ± 16 % from ground treatment. Bti therefore appears to be more effective via ground treatment than via air treatment. Statistical analysis of air treatment shows that: * Bti has greater efficiency on first instar larvae than second instar larvae. Individual studies carried out during the project in 2000 and 2002 confirmed the wide variations of sensitivity for each stage of development using VectoBac 12AS (L2 instar larvae are 27 times more sensitive than L4 instar larvae). Whatever the quality of the application, a considerable increase in the dose of VectoBac 12AS is necessary if there is a significant presence of L4 instar larvae. Consequently, it is obviously preferable to treat the young instars to optimise Bti based treatment; * when the depth of water increases, the efficiency of Bti increases. This can be explained by the fact that sedimentation of the Bti toxic crystals is all the longer the greater the depth of water, which means that the time that the product is available to the larvae is extended; * when the temperature of the water increases, the efficiency of Bti increases. In fact, when the temperature falls, the larvae feed less and ingest less Bti. A certain number of observations were carried out in order to monitor the efficiency of Bti treatment (qualitative and quantitative inventory of larvae and fauna in several characteristic spots in the breeding sites: sensitivity tests on larvae sampled in the field using the World Health Organisation (WHO) standardised method, counting the number of insecticide droplets collected on siliconised slides and estimation of the quantity of the product which has reached the water surface). Graph representation of the mean diameter and number of drops of insecticide product collected on a slide conclusions At present the efficacy assessment of a treatment is qualitative. In order to develop a more quantitative standardised evaluation method for effectiveness, a tool for estimating the abundance of larvae (abacus) has been created. It avoids manual counting of sampled larvae and makes it possible to take a large number of samples in a relatively short period (1:30-2 hours for 120 samples). Its use can be combined with spatial analytical techniques (krigging type and DTM). The statistical analyses made until now studied the influence of parameters on efficacy without taking into account interactions and possible correlations between the parameters. Continuing the collection of data while enlarging the scale of observation beyond the Ramsar nr 17 area and 18

20 RESULTATS EFFICIENCY AND FEASIBILITY OF THE METHODOLOGY using tools such as the abacus will mean several objectives can be met, including better knowledge of larval behaviour, better understanding of how representative larval samples are and an increase in the variability of the data collected to confirm trends observed and make more refined analyses (generalised linear models and Hill and Smith analysis) enabling study of the interactions between the parameters (qualitative that quantitative). are the methods of spraying used effective and sufficient? The decision to adapt low volume spraying by aircraft, used until now for chemicals, to the use of Bti has proved to be satisfactory. The volume of mixture per hectare being greater using Bti (3.9 l/ha as against 1.4 l/ha on average for temephos), the average yield (surface treated per hour) considering the two aircraft together has decreased by about 21 % compared with spraying synthetic products. The choice between the two aircraft was optimised on an ad hoc basis, depending on the volume to be sprayed, to reduce the number of returns for refilling. Naturally, it can be seen that the yield increases with the load capacity. While the aircraft available allowed operational application under satisfactory conditions, they also demonstrated their limitations concerning extending Bti applications, as the two aircraft were being used for other treatment during the same time period. Their activities in the Ramsar nr 17 zone thus represented respectively 37.7 % (2000), 49.8 % (2001) and 38.7 % (2002) of their total flight time over the whole EID Méditerranée area of action. This shows the limits concerning extending Bti application, the two aircraft being used for other treatment during the same period of time. 19 As from the 2003 campaign, it is planned to increase significantly the area treated with Bti. With this in mind, from what we have learned from the project, means of increasing the productivity of the aircraft are under consideration. This should take the form of the addition of a third aircraft with a payload of more than 1500 kg. It remains to be seen how this aircraft will be viewed in terms of disturbance, given that it will cause less than a helicopter (which has a low carrying capacity and requires considerable support for resupplying in open country). These proposals also fall with the plan decided in 2000 by EID Méditerranée's board of management which predicted a significant increase in treatment with Bti. Financial commitments have been made therefore, to allow 15 % of all treatments to be made using Bti in 2003, reaching 25 % in 2007, proportions which may be reviewed upwards depending on the progress established from the results, from spraying improvements and the cost of products, the large scale use of which necessitates financial control of costs. conclusions

21 RESULTS UNINTENTIONAL EFFECTS The study was undertaken by external consultants (Laboratory of ecology of continental and Mediterranean waters, UAM III., Saint Jérôme, University of sciences and Techniques,Marseilles), with the EID Méditerranée laboratory nevertheless making its contribution in terms of logistics (choice of sites, sampling, sorting). The aim of this study was to calibrate a method for evaluating the unintentional impact of different insecticide products, which is easily applicable by any user. Aerial view of the Enfores the experimental site The study site chosen was at the location known as the Enfores (Department of the Bouches-du-Rhône). Composed of three marshy depressions (commonly known as the Wolf, the Sheep and the Lamb), it is a representative Ochlerotatus sp. biotope that had never been subjected to against mosquitoes insecticide treatment. Physical and chemical analyses of the soil and water (surface and ground water) have shown that the three pre-selected depressions of the Enfores experimental site correspond to three independent morpho-pedological units, which function in noticeably different ways. The Sheep depression, the biggest, which is subjected to salinisation due to upward leaching, has more impermeable substrata which encourage surface water being maintained. The method of artificial flooding enhances these differences because the flooding occurs via drains which first feed the Wolf, then the Sheep and finally the Lamb. This order certainly determines a humidity gradient between the three depressions. In addition, preliminary studies have brought to light a heterogeneity in terms of fauna between the three depressions: the Sheep appears to be richer and more diverse than the other two depressions, with greater hatching of Ochlerotatus caspius eggs. The impact study was therefore carried out in this depression. Ground water control Sampling aquatic fauna Physico-chemical measurements of surface water (ph, temperature, salinity, conductivity, etc.) the functioning of this habitat The first year of the study was concentrated on monitoring aquatic invertebrate population dynamics (macro invertebrates and micro crustacea). Sampling occurred every 15 days as follows: the operator sampled the fauna by making three passes of the Langeron net (15 cm diameter), each of a metre length. The operation was repeated two more times to provide three samples per sampling points. The samples were then returned to the laboratory where they were sorted, and where the fauna were counted. Different taxa were thus identified: Diptera, Ephemeroptera, Heteroptera, Coleoptera for the macro invertebrates and Cladocera, Copepoda and Ostracoda for the micro crustacea. The analyses relating to the taxonomic richness also showed three successive phases of colonisation of the habitat: * the first phase was characterised by the development of Ochlerotatus spp. and endogenous taxa. It was 20

22 RESULTS UNINTENTIONAL EFFECTS Defining a biological identity card Defining a biological identity card the Digital Terrain Model (DTM) The DTM is a collection of data forming a network of points which generate altimetric georeferenced information concerning a geographical area. The points (X, Y and Z) are read using a tacheometer. From this file of points spatial statistical methods are used to extrapolate the altitude values to all the points included in the study area. On the Enfores site, a sensor continually measuring the water level is placed at the lowest point of the Sheep depression. These measurements fed into the model allow the extent of the water area to be seen. On adding to this a GIS layer showing the ecological zones it is possible to represent the ecological zones in relief, and to calculate the volumes of water involved for each of these zones. 21 marked by a rapid replacement of the dominant species (the Ochlerotatus sp. reappear during a flooding or a rise in water level following a dry period); * the second phase was more stable: it was characterised by the presence of other endogenous taxa and the appearance of exogenous taxa, leading to increased richness; * the last phase was characterised by a decline in diversity where only a few sedentary species survive with a strong contingent of carnivorous taxa. Once the population dynamics were clearly established, it was possible to start the impact study strictly speaking and to begin application of insecticides. In fact, observing the colonisation of temporary habitats in phases (juvenile phase, mature phase and senescent phase) allows disruption suffered by colonising populations to be assessed. Factorial correspondence analysis (FCA) is the method used to evaluate natural disturbances and those linked to the impact of pesticides. These FCAs, carried out on the populations of treated and control areas, show the effects of disturbances due to drying out and to the insecticides, in as far as these two factors have a determining effect. They equally allow the length of the disturbance or disturbances and the recolonisation time to be assessed. FCAs, carried out from taxa easily identifiable by EID Méditerranée's technical staff, can form a reference system for treated habitats. This "biological identity card" may in time be a means of appreciating the stability of habitats relative to natural conditions and the impact of insecticide treatment. the respective impacts of Bti and temephos on non-target fauna The Sheep depression was prepared for treatment: it was divided into three strips (50 x 200 m) having the same ecological zones. The most westerly strip was treated with temephos (Abate 500 E), the most easterly strip with Bti (VectoBac 12AS), the central strip served as a control. In each of these three strips, 5 sampling points were selected and marked with a piezometric tube, each in a different ecological zone. Sampling of fauna was carried out before and after treatment. Sampling of fauna was carried out before and after treatment at 5 previously marked sampling points in each strip. The insecticide concentrations were deliberately modified relative to operational doses, in order to determine the impact threshold. The numbers of taxa collected during these 5 periods of study were recalculated relative to the volume of water estimated using a digital model of the terrain, in order to analyse the density of individuals. It appears that when the concentrations are equal to or greater than the recommended doses (125 g of active substance per ha), temephos has a significant but short-term impact on Ochlerotatus spp., Gammaridae, Chironomidae, Anopheles

23 RESULTS UNINTENTIONAL EFFECTS Marking the edges of the treatment strips in Sheep depression Chaoborus sp. (Diptera) Chironomus sp. (Diptera) and Calopterygidae. On the other hand, when the concentrations are lower than the recommended doses (68.3 and 70 g/ha in May and July 2002), temephos shows no marked toxicity including for Ochlerotatus spp. For concentrations generally greater than the recommended doses (0.8 l/ha), Bti has a significant impact on the larvae of Culex sp. and Anopheles sp., but on the other hand, the Chironomidae and other taxa are not affected. In order to refine the results and provide details on the persistence of the insecticides, the residues of Bti (in cooperation with the Entomopathogenic bacterial and fungi laboratory of the Institut Pasteur in Paris) and temephos were analysed. The results indicated no or little Bti spore persistence in the four ecological zones after two treatments and that the strains of Bt, in particular Bti were rare in this temporary ecosystem. But these results were obtained after only a single season and it will be interesting to monitor potential persistence over several years. Concerning temephos, the results of the analysis of residues showed that there had not been an accumulation of the insecticide, neither in the ground, nor in ground water, during the period considered (January 2001 to November 2002). The action of Bti was therefore more specific than that of temephos and the persistence of action of these two products, of short duration, does not cause noticeable modification in the populations studied at least in the medium term (equivalent to the years of monitoring). The qualitative and quantitative study of taxa, generally not very numerous during treatment, and of their numbers, throughout five treatment campaigns, shows no significant loss of biodiversity or biomass. In addition, it is clear that drying out has a greater disturbance effect than the use of the insecticides. These habitats have a large capacity for natural recovery. conclusions Reproducibility of the method in all habitats The sampling technique may be considered as validated since in the course of the study it allowed the population of aquatic invertebrate fauna to be reliably calculated. Moreover, the results of this project highlight the fact that this methodology, based on comparing the impact of a synthetic insecticide with that of a bio-insecticide, is applicable whatever type of insecticide preparation is used. Nevertheless, before monitoring another product, it is necessary to carry out bibliographical research on the action of the product concerned and its ecotoxicity, as well as on the possible taxa living in our habitats which are sensitive to it (laboratory tests). The method can also be transposed to other habitats: it has indeed begun to be tested on the Ramsar nr 17 site, but the lack of data necessitates continuing the investigations. 22