IOBC / WPRS

Working Group „Pesticides and Beneficial Organisms“

OILB / SROP

Groupe de Travail „Pesticides et Organismes Utiles“

Proceedings of the meeting

at

Berlin, Germany

10th –12th October 2007

Editors: Heidrun Vogt, Jean-Pierre Jansen, Elisa Vinuela & Pilar Medina

IOBC wprs Bulletin Bulletin OILB srop Vol. 35, 2008

The content of the contributions is in the responsibility of the authors

The IOBC/WPRS Bulletin is published by the International Organization for Biological and Integrated Control of Noxious Animals and Plants, West Palearctic Regional Section (IOBC/WPRS)

Le Bulletin OILB/SROP est publié par l‘Organisation Internationale de Lutte Biologique et Intégrée contre les Animaux et les Plantes Nuisibles, section Regionale Ouest Paléarctique (OILB/SROP)

Copyright: IOBC/WPRS 2008

The Publication Commission of the IOBC/WPRS:

Horst Bathon Luc Tirry Julius Kuehn Institute (JKI), Federal University of Gent Research Centre for Cultivated Plants Laboratory of Agrozoology Institute for Biological Control Department of Crop Protection Heinrichstr. 243 Coupure Links 653 D-64287 Darmstadt (Germany) B-9000 Gent (Belgium) Tel +49 6151 407-225, Fax +49 6151 407-290 Tel +32-9-2646152, Fax +32-9-2646239 e-mail: [email protected] e-mail: [email protected]

Address General Secretariat:

Dr. Philippe C. Nicot INRA – Unité de Pathologie Végétale Domaine St Maurice - B.P. 94 F-84143 Montfavet Cedex (France)

ISBN 978-92-9067-209-8 http://www.iobc-wprs.org Pesticides and Beneficial Organisms IOBC/wprs Bulletin Vol. 35, 2008

Preface

This Bulletin contains the contributions presented at the meeting of the WG “Pesticides and Beneficial Organisms” held in Berlin, 10 - 12 October 2007. The meeting was hosted by the Federal Biological Research Centre for Agriculture and Forestry (BBA), Institute for Ecotoxicology and Ecochemistry in Plant Protection, in Berlin-Dahlem (now Julius Kuehn Institute (JKI), Federal Research Centre for Cultivated Plants, Institute for Ecological Chemistry, Plant Analysis and Stored Product Protection). According to the change from BBA to JKI (since 1st January 2008) the addresses of authors and participants from former BBA institutes have been actualised in this Bulletin. The meeting was attended by 60 participants from 15 countries. Subjects of 24 oral and 5 poster presentations were methodical aspects of laboratory, semi-field and field side effect tests, effects of plant protection products and biocontrol agents (entomopathogenic fungi and nematodes) on beneficials in diverse crops as well as effects of reduced pesticide rates on field populations of beneficials, the establishment of crop specific selecivity lists and regulatory issues. A highlight of the meeting was the film “Fascination Insect Microcosm”, which illustrates in captivating macro images the biology of beneficial insects and mites, their hunting and parasitizing behaviour. The producer himself, Prof. Dr. Urs Wyss, University Kiel, live commented the film excellently. During the meeting, after 10 years of my convenorship, Dr. Jean-Pierre Jansen, Centre wallon de Recherches Agronomiques (CRA-W), Département Lutte biologique et Ressources Phytogénétiques - Unité de zoologie, Gembloux, Belgium, was elected as my successor and subsequently endorsed by the IOBC/wprs Council. He has many years of experience in ecotoxicological studies with beneficial arthropods and their integration in integrated plant management and has an excellent affiliation with the WG due to his regular attendance of the meetings. Furthermore, he is involved in the evaluation of the “Pesticide Registration Dossier – Part: Beneficial Arthropods” for the Belgian Health and Environment Federal Public Service and thus knows very well about the needs and open questions in side-effect testing. The WG meeting 2007 had a very good ambience with intensive discussions and scientific exchange. The participants enjoyed the agreeable atmosphere of the conference site and the best care and attention by the local organizer team. A half day ecxursion included the visit of the famous “Sanssoucis” palace in Potsdam and sight seeing of Potsdam with detailed information about its history. After the excursion, everybody enjoyed the nice dinner buffet. On behalf of all attendants I express my gratitude and thanks to the local organizer, Barbara Baier, and her colleagues for the perfect organization. The meeting was supported by IOBC wprs and the Bundesministerium für Ernährung, Landwirtschaft und Verbraucherschutz BMVEL (Federal Ministry of Food, Agriculture and Consumer Protection), Bonn/Berlin. With the election of my successor, I take my leave from convenorship. I have enjoyed this task a lot and I thank all those who supported me and who were highly dedicated to the WG, including all colleagues who helped in reviewing the manuscripts for the Bulletins of the meetings. The WG has always been an important forum for pesticide side-effect issues and the enhancement of integrated pest management by preserving beneficial organisms. I am convinced that the WG will remain of vital importance and I wish the New Convenor, Jean Pierre Jansen, successful and fruitful work together with the WG.

Heidrun Vogt (Former Convenor) Dossenheim, April 2008 ii Pesticides and Beneficial Organisms IOBC/wprs Bulletin Vol. 35, 2008

Dr. Heidrun Vogt – 10 years successful convenorship of the IOBC Working Group “Pesticides and Beneficial Organisms”

Barbara Baier1 & Udo Heimbach2

Julius Kuehn Institut, Federal Research Centre for Cultivated Plants, 1Institute for Ecological Chemistry, Plant Analysis and Stored Product Protection, Königin- Luise-Str. 19, D-14195 Berlin. e-mail: [email protected] 2Institute for Plant Protection in Field Crops and Grassland, Messeweg 11/12, D-38104 Braunschweig. e-mail: [email protected]

At the meeting in Tunis, 1997, Dr. Heidrun Vogt was elected as new convenor of the working group “Pesticides and Beneficial Organisms” (as successor of Dr. Sherif A. Hassan) and thereafter endorsed by the IOBC/WPRS Council. From 1998 to 2007 she was convenor of the working group. In these 10 years she organized 8 working group meetings in 6 European countries. Spain, Italy and Portugal were the first South European countries and Poland was the second East European country to hold the meeting. The meetings took place every year until 2003 and from then on every second year. Each meeting was approached by approximately 80 participants from 16 mostly European countries, but by a growing number from Canada, Japan, and the USA. The participants presented 27 papers and posters in average dealing with various aspects of side effects of pesticides on beneficial organisms. The presentations contained necessary basic information for integrated pest management and the protection of beneficial organisms. Most of them were published in the IOBC Bulletin. Heidrun Vogt reviewed every single manuscript, partially assisted by other group members. She was managing editor of 7 IOBC Bulletins with high-quality contributions. Joint Pesticide Testing Programme was continued with the 8th and 9th programmes during her convenorship. 20 pesticides registered in at least one of the WPRS member countries were tested, each on up to 24 beneficial organisms. The results of the ninth programme finished this work in 2003. The working group decided together with the IOBC Commission on “IP-Guidelines and Endorsement” to compile the data of all Joint Pesticide Programmes and from contributions published in the IOBC Bulletins to make them more easily available – especially for farmer organisations, who need these information for the implementation of integrated pest management, but also for extension service and scientists. Ms Vogt had a great share in this work. The results for more than 170 pesticides are now available on the internet at http:www.iobc.ch/news.html (IOBC Toolbox: Selectivity of pesticide). As convenor of the IOBC working group she supported and intensified the co-operation with the EU, EPPO, BART, OECD and SETAC Europe. This co-operation concentrated mainly on the development and validation of test guidelines to identify effects of pesticides on specified non-target organisms with respect to the registration of pesticides. She headed a ring test group to develop and validate a laboratory test for Chrysoperla carnea and was member of a ring test group to develop a field test for Phytoseiidae in vine and fruit orchards. Furthermore, she supported the work of the other eight ring test groups. In 2000, the results of 11 validated test methods were published with Heidrun Vogt as one of the responsible authors

iii iv

in the IOBC booklet „Guidelines to evaluate side-effects of plant protection products to non- target arthropods - IOBC, BART and EPPO Joint Initiative“. In the year 2000 she was a member of the organisation committee and of the editorial board of the ESCORT 2 workshop in Wageningen, which discussed the role of non-target arthropod testing in registration. The agreements made at this workshop have been published as a guidance document and serve as basic document for the implementation of the revised EPPO/CoE risk assessment scheme and the EU guidance document on terrestrial ecotoxicology. It is due to her work and her commitment as convenor of the working group in the last ten-years, that the working group continuously was an important forum for bringing together experts on side-effect issues, to discuss new questions and to search for solutions, to set standards for regulatory requirements on side effect testing on non-target organisms, and to support protection and enhancement of beneficial organisms within integrated pest management. Therefore we want to express our thanks to Dr. Heidrun Vogt and give our appreciation, also in behalf of all members of the working group, for her considerable and valuable work as convenor from 1998 to 2007. We are glad that she will stay active in the group also in future.

Left: Heidrun Vogt and the local organizer of the Berlin meeting, Barbara Baier and (right) with her successor Jean-Pierre Jansen.

v

Participants of the Meeting in Berlin, 10-12 October, 2007 vi

List of Participants

Ali, Ali Cavaco, Miriam Humboldt University Berlin Direccao-Geral de Agricultura e Desenvolvimente Faculty of Agriculture and Horticulture, Institute Rural for Horticulture Science, Section Phytomedicine Quinta Do Marques Lentzeallee 55-57 2780-155 Oreias, Portugal 14195 Berlin, Germany [email protected] [email protected] Chaton, Pierre-Francois, Dr. Baier, Barbara, Dr. Agence Française de Sécurité Sanitaire des Julius Kuehn Institute (JKI), Federal Research Aliments Centre for Cultivated Plants, Institute for Direction du Végétal et de l’Environnement Ecological Chemistry, Plant Analysis and Stored 10, rue Pierre Curie Product Protection, Königin-Luise-Str. 19 94 704 Maison-Alfort Cedex, France 14195 Berlin, Germany [email protected] [email protected] Damos, Petros Barić, Božena, Dr. School of Agriculture, Faculty of Agriculture Aristotle University of Thessaloniki, Department of Agricultural Zoology Laboratory of Applied Zoology and Parasitology Svetosimunska cesta 25 54124 Thessaloniki, Greece 10 000 Zagreb, Croatia [email protected] [email protected] Dinter, Axel, Dr. Bartels, Anja, Mag. DuPont Crop Protection, AGES DuPont de Nemours (Germany) GmbH Austrian Agency for Health and Food Safety DuPont Straße 1 Spargelfeldstraße 191 61352 Bad Homburg, Germany 1226 Wien, Austria [email protected] [email protected] Donka, András Barth, Markus Humboldt University Berlin BioChem agrar GmbH Faculty of Agriculture and Horticulture, Institute Kupferstraße 6 for Horticulture Science, Section Phytomedicine 04827 Gerichshain, Germany Lentzeallee 55-57 [email protected] 14195 Berlin, Germany

Bostanian, Noubar, J., Dr. Freier, Bernd, Prof. Dr. Agriculture and Agri-Food Canada, Julius Kuehn Institute (JKI), Federal Research Institut des Sciences de l’ Enviroment, Centre for Cultivated Plants, Institute for Strategies 430 Boulevard Gouin and Technology Assessment in Plant Protection St. Jean-sur-Richelieu, Stahnsdorfer Damm 81 Québec, Canada J3B 3E6 14532 Kleinmachnow, Germany [email protected] [email protected]

Broufas, Georgios Gobin, Bruno, Dr. Laboratory of Agricultural Entomology & pcfruit – zoology Zoology, De Brede Akker 13 Faculty of Agricultural Development, 3800 Sint – Truiden, Belgium Democritus University of Thrace [email protected] Pantazidou 193 68 200 Orestiada, Greece [email protected]

vii

González Núñez, Manuel, Dr. Jansen, Jean-Pierre, Dr. Instituto Nacional de Investigación y Tecnología Walloon Agricultural Research Centre Agraria y Alimentaria (INIA) Dept. Biological Control – Zoology unit Laboratorio de Entomologia Agroforestal Chemin de Liroux 2 Carretera de La Coruña km 7,5 5030 Gembloux, Belgium 20040 Madrid, Spain [email protected] [email protected] Jilesen, Claudia Güven, Bilgin, Msc. Plant Protection Service, the Netherlands Bornova Zirai Mücadele Arastirmy Enstitüsü P.O.Box 9102 Gençlik Cad. No.6 6700 Wageningen, Netherlands 35040 Bornove/Izmir, Turkey [email protected] [email protected] Juan, Delphine Halsall, Nigel, Dr. ENIGMA Syngenta Hameau de St. Véran Jeallott’s Hill International Research Centre 84190 Beaumes de Vénise, France Bracknell, Berkshire, RG42 6EY, [email protected] United Kingdom [email protected] Katz, Peter, Dr. Katz Biotech AG Hargreaves, Natalie An der Birkenpfuhlheide Syngenta 15837 Baruth, Germany Jeallott’s Hill International Research Centre [email protected] Bracknell, Berkshire, RG42 6EY, United Kingdom Laborie, Benedicte [email protected] Bayer CropScience 16 Rue Jean-Marie Leclair Helsen, Herman, Dr. 69009 Lyon, France Applied Plant Research, Wageningen UR [email protected] PO box 200 6670 AE Zetten, Netherlands Lehmhus, Jörn, Dr. [email protected] Eurofins-Gab GmbH Eutinger Straße 24 Hoffmann, Sebastian 75223 Niefern-Öschelbronn, Germany Springborn Smithers Laboratories (Europe) joern.lehmhus@eurofins-GAB Seestraße 21 9326 Horn, Switzerland Lerche, Sandra [email protected] Humboldt University Berlin Faculty of Agriculture and Horticulture, Hommes, Martin, Dr. Institute for Horticulture Science, Julius Kuehn Institute (JKI), Federal Research Section Phytomedicine Centre for Cultivated Plants, Institute for Plant Lentzeallee 55-57 Protection in Horticulture and Forests 14195 Berlin, Germany Messeweg 11/12 [email protected] 38104 Braunschweig, Germany [email protected] McCormac, Alexandra, Dr. Mambo- Tox Ltd. Jäckel, Barbara, Dr. 2 Venture Road, Chilworth Science Park Official Bureau of Plant Protection Berlin Southampton SO16 7NP, Mohriner Allee 137 United Kingdom 12347 Berlin, Germany [email protected] [email protected] Mead-Briggs, Mike, Dr. Jans, Daniela, Dr. Mambo- Tox Ltd. Bayer CropScience AG, Ecotoxicology 2 Venture Road, Chilworth Science Park Industriepark Höchst, H872 Southampton SO16 7NP, 65926 Frankfurt am Main, Germany United Kingdom Daniela.Jans@bayercropscience. com [email protected] viii

Medina, Maria Pilar, Dr. Schenke, Detlef, Dr. Unidad de Protección de Cultivos, Julius Kuehn Institute (JKI), Federal Research E.T.S.I.Agrónomos, Centre for Cultivated Plants, Institute for Universidad Politécnica de Madrid Ecological Chemistry, Plant Analysis and Stored Ciudad Universitaria s/n Product Protection, Königin- Luise Str. 19 28040 Madrid, Spain 14195 Berlin, Germany [email protected] [email protected]

Moll, Monika, Dr. Schmitt, Günther, Dr. IBACON GmbH Schmitt Faunistic Studies Arheilger Weg 17 Friedenstraße 23 64380 Rossdorf, Germany 18190 Sanitz, Germany [email protected] [email protected]

Neumann, Nadine Schumacher, Kerstin Department of Crops Science Agricultural Julius Kuehn Institute (JKI), Federal Research Entomology, University of Goettingen Centre for Cultivated Plants, Institute for Strategies Grisebachstraße 6 and Technology Assessment in Plant Protection 37077 Goettingen, Germany Stahnsdorfer Damm 81 [email protected] 14532 Kleinmachnow, Germany [email protected] Nienstedt, Karin, Dr. Springborn Smithers Laboratories (Europe) Scott-Brown, Alison, Dr. Seestraße 21 Royal Botanical Gardens Kew 9326 Horn, Switzerland Jodrell Laboratory, RBG Kew [email protected] Richmon, Surrey,TW9 3AB, United Kingdom Olszak, Remigiusz, Dr. [email protected] Research Institute of Pomology and Floriculture Pomologizna 18 Sekrecka, Malgorzata 96-100 Skierniewice, Poland Research Institute of Pomology [email protected] and Floriculture Pomologizna 18 Pappas, Maria, Dr. 96-100 Skierniewice, Poland School of Agriculture [email protected] Aristotle University of Thessaloniki, Laboratory of Applied Zoology and Parasitology Sermann, Helga, Dr. 54124 Thessaloniki, Greece Humboldt University Berlin [email protected] Faculty of Agriculture and Horticulture, Insitute for Horticulture Science, Peusens, Gertie Section Phytomedicine pc-fruit Lentzeallee 55-57 De Breede Akker 13 14195 Berlin, Germany 3800 Sint-Truiden, Belgium [email protected] [email protected] Sharples, Amanda Rodrigues, J. Raul, Prof. Dr. Covance Laboratories Ltd Dep. Ciéncias da Planta e do Ambienta Otley Road Escola Superior Agrária de Ponte de Lima Harrogate, North Yorkshire HG3 IPY, Convento de Refóios United Kingdom Refóios do Lima [email protected] 4990-706 Ponte de Lima, Portugal [email protected] Sterk, Guido Biobest NV Röhlig, Uta Ilse Velden 18 BioChem agrar GmbH 2260 Westerlo, Belgium Kupferstraße 6 [email protected] 04827 Gerichshain, Germany [email protected] ix

Talebi, Khalil, Dr. Volkmar, Christa, Prof. Dr. University of Tehran Martin Luther-University Halle-Wittenberg Department of Plant Protection, University Faculty of Natural Sciences III, Institute of College of Agriculture & Natural Resources Agricultural and Nutritional Sciences Karaj 3158711167, Iran Ludwig-Wucherer-Straße 2 [email protected] 06108 Halle/Saale, Germany [email protected] Ulber, Bernd, Dr. Department of Crops Science, Agricultural Waibel, Jutta Entomology, University of Goettingen Bayer CropSciences AG Grisebachstraße 6 Ecotoxicology 37077 Goettingen, Germany Industriepark Höchst, H872 [email protected] 65926 Frankfurt am Main, Germany [email protected] Viñuela, Elisa, Prof. Unidad de Protección de Cultivos, Warmers, Christian E.T.S.I.Agrónomos, Eurofins-Gab GmbH Universidad Politécnica de Madrid Eutinger Straße 24 Ciudad Universitaria s/n 75223 Niefern-Öschelbrunn, Germany 28040 Madrid, Spain [email protected] [email protected] Wyss, Urs, Prof. Dr. Vogt, Heidrun, Dr. Christian-Albrechts-University Julius Kuehn Institute (JKI), Federal Research Institute of Phytopathology Centre for Cultivated Plants, Institute for Plant Hermann Rodewald-Straße 9 Protection in Fruit Crops and Viticulture 24118 Kiel, Germany Schwabenheimerstraße 101 [email protected] 69221 Dossenheim, Germany [email protected]

Pesticides and Beneficial Organisms IOBC/wprs Bulletin Vol. 35, 2008

Contents

Preface...... i

Dr. Heidrun Vogt – 10 years successful convenorship of the IOBC Working Group “Pesticides and Beneficial Organisms” Baier, B. & Heimbach, U...... iii

List of participants...... vi

Contents...... xi

Residues of acetamiprid in leaves of Aesculus hippocastanum and effects on the horse chestnut leaf miner (Cameraria ohridella) following trunk spraying Schenke, D., Jäckel, B. & Schmidt, H...... 1-9

Who benefits from low-input pesticide use within the tritrophic system: crop – aphid – predator? Schumacher, K. & Freier, B...... 10-17

Impact of low-input pesticides usage on spider communities with special regard to accumulated effects Volkmar, Ch., Schumacher, K. & Müller, J...... 18-25

Effects of different control measures against the olive fruit fly (Bactrocera oleae (Gmelin)) on beneficial arthropod fauna. Methodology and first results of field assay González-Núñez, M., Pascual, S., Seris, E., Esteban-Durán, J. R., Medina, P., Budia, F., Adán, Á. & Viñuela, E...... 26-31

Impact of Success Bait (a.i. spinosad) against Rhagoletis cerasi on insect fauna in field test (Abstract)Barić, B., Pauković, M., Bertić, D. & Pajač, I...... 32

Effects of bait sprays to control the European cherry fruit fly (Rhagoletis cerasi L.) on aphid predators (Abstract) Vogt, H. & Köppler, K...... 33-34

Earwigs in fruit orchards: phenology predicts predation effect and vulnerability to side-effects of orchard management Gobin, B., Moerkens, R., Leirs, H. & Peusens, G...... 35-39

Side effects of pesticides on the European earwig Forficula auricularia L. (Dermaptera: Forficulidae)Peusens, G. & Gobin, B...... 40-43

About the presence and abundance of beneficials in overwintering sites of Anarsia lineatella (Lepidoptera: Gelechiidae) in peach orchards of northern Greece Damos, P. & Savopoulou-Soultani, M...... 44-50

xi xii

Is the use of some selected insecticides compatible with two noctuid endoparasitoids: Hyposoter didymator and Chelonus inanitus? Medina, P., Morales, J.J., González-Núñez, M. & Viñuela, E...... 51-59

The extended laboratory test guideline for Aphidius rhopalosiphi: some areas of debate relating to the methodology Mead-Briggs, M...... 60-65

Pesticides selectivity list to beneficial arthropods in four field vegetable crops Jansen, J.P., Hautier, L., Mabon, N. & Schiffers, B...... 66-77

Concerns and solutions in non-target arthropod regulatory risk assessment of plant protection products Chaton, P.F., Vergnet, Ch. & Alix, A...... 78-84

Toxicity of certain pesticides to the predatory mite Euseius finlandicus (Acari: Phytoseiidae) Broufas, G.D., Pappas, M.L., Vassiliou, G. & Koveos, D.S...... 85-91

Side effects of pesticides used in vineyards in the Aegean region on the predatory mite Typhlodromus perbibus Wainstein&Arutunjan (Acari: Phytoseiidae) under laboratory conditions Göven, M.A. & Güven, B...... 92-95

Effects of ten pesticides to Anystis baccarum (Acari: Anystidae) Bostanian, N.J. &. Laurin, M.-C...... 96-100

Influence of some insecticides and acaricides on beneficial mites and on Coccinella septempunctata (Coleoptera; Coccinellidae) larvae Olszak, R. W. & Sekrecka, M...... 101-108

Effect of the entomopathogenic fungus Lecanicillium muscarium on the predatory mite Phytoseiulus persimilis as a non-target organism Donka, A., Sermann, H. & Büttner, C...... 109-112

Effects of Beauveria bassiana, Heterorhabditis bacteriophora, H. megidis and Steinernema feltiae on the Mediterranean fruit fly Ceratitis capitata and the very sensitive braconid Psyttalia concolor in the lab Medina, P., Corrales, E., González-Nuñez, M., Smagghe, G. & Viñuela, E...... 113-121

Aged-residue method for evaluating toxicity of plant protection products to Stethorus punctillum (Weise) (Coleoptera: Coccinellidae) Nienstedt, K.M. & Miles, M...... 122-127

Chlorantraniliprole (DPX-E2Y45, DuPont™ Rynaxypyr®, Coragen® and Altacor® insecticide) - a novel anthranilic diamide insecticide - demonstrating low toxicity and low risk for beneficial insects and predatory mites Dinter, A., Brugger, K., Bassi, A., Frost, N.-M., Woodward, M.D...... 128-135

Influence of organic matter on bio-availability of two pesticides and their toxicity to two soil dwelling predators (Abstract) Hautier, L., Mabon, N., Schiffers, B. &. Jansen, J.-P...... 136

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Different methods of application – Different laboratory test strategies (Abstract) Norr,C., Baier, B. & Schenke, D...... 137

Assessment of side-effect of water-soluble nitrogen fertilizers applied as foliar spray on the parasitic wasp Aphidius rhopalosiphi (DeStefani-Perez) (Hym.; Aphidiidae) Dantinne, D. & Jansen, J.P...... 138-142

Field toxicity of four acaricides on the predatory mites Amblyseius andersoni (Chant) and Euseius stipulatus (Athias-Henriot) (Acari: Phytoseiidae) in apple orchard at Northwest of Portugal (Abstract) Rodrigues, J. R. & Torres, L. M...... 143

Influence of teflubenzuron residues on the predation of thrips by Iphiseius degenerans and Orius laevigatus (Abstract) Scott Brown, A.,Simmonds, M. & Blaney, W...... 144-145

Study on the side-effects of three pesticides on the predatory mite, Phytoseius plumifer (Canestrini & Fanzago) (Acari: Phytoseiidae) under laboratory conditions Noii, S., Talebi, K., Saboori, A., Allahyari, H., Sabahi, Q. & Ashouri, A...... 146-151

Special Topic

The need for taxonomists of pest and beneficial organisms - results of an inquiry at the meeting of the IOBC working group “Pesticides and Beneficial Organisms” in Berlin in October 2007 Schmitt, G...... 152-156

Pesticides and Beneficial Organisms IOBC/wprs Bulletin Vol. 35, 2008 pp. 1-9

Residues of acetamiprid in leaves of Aesculus hippocastanum and effects on the horse chestnut leaf miner (Cameraria ohridella) following trunk spraying

Detlef Schenke1, Barbara Jäckel2 and Heinz Schmidt1 1 Julius Kuehn Institute, Federal Research Centre for Cultivated Plants, Institute for Ecological Chemistry, Plant Analysis and Stored Product Protection, Königin-Luise-Str. 19, D-14195 Berlin; 2 Official Bureau of Plant Protection Berlin, Mohriner Allee 137, D-12347 Berlin; [email protected]; [email protected]

Abstract At the beginning of flowering in 2006, the neonicotinoid acetamiprid was sprayed on trunks of twenty and eighty-year-old horse chestnut trees at different application rates. Sampling took place at the end of the first and second leaf miner generations. To estimate mortality, efficiency and parasitation, living, dead and parasitized leafminers were counted. Acetamiprid residues were only found in the first samples taken after application, which showed high deviations. All variants showed low efficiency of acetamiprid independently of the application rate. The parasitation rate of chalcidoid wasps was generally higher in the treated variants than in untreated trees.

Key words: Aesculus hippocastanum, Cameraria ohridella, acetamiprid, trunk application, residue, chalcidoid wasp

Introduction

During the past years, the horse chestnut leafminer has spread over most of Europe. Experts’ opinions (Heitland, 2006) suggest that there is currently no risk of the white Aesculus hippocastanum to die out due to the leafminer. Damage rather results in an early fall of leaves and clearly reduces net primary production. Normally important functions of trees at urban sites such as dust filtering and balancing the temperature amplitude, CO2 fixation and oxygen formation can be lost. All these impediments make tree nurseries tend to choose tree species other than Aesculus hippocastanum for new plantings in urban places. Some biological and chemical control measures against the horse chestnut leafminer Cameraria ohridella have shown different results (Jäckel et al., 2006, 2007). The trunk application of systemic pesticides is an alternative method with mitigating drift and leaching. There is particularly little knowlegde about the fate and behaviour of insecticides in case of trunk application of plant protection products. Pesticide concentration in leaves is important from the ecotoxicological point of view in order to assess the effects on beneficial organisms and the decomposition of fallen leaf litter. In this investigation, the efficiency of acetamiprid against Cameraria ohridella, side effects on chalcidoid wasps and residues in leaves of the horse chestnut tree were studied after trunk spraying.

1 2

Material and methods

Substances The formulation AF with the active substance (a. s.) of acetamiprid was provided by Scotts Celaflor GmbH & Co. KG for the trunk spraying of trees. Acetamiprid (C 10013000), imidacloprid (C 14283700) as surrogate and imidacloprid D4 (C 14283710) as internal standard were purchased from Dr. Ehrenstorfer (Augsburg, Germany).

Test site and weather The studies with acetamiprid were performed with young and old trees at different sites (Table 1). The trees differed in their circumferences and, importantly, the thickness of their barks. Figure 1 shows the weather conditions in Berlin in 2006.

Table 1. Test sites.

Variant AF 40-60 AF100 Location in Berlin Priesterweg Eggersdorfer Str. Age [years] 20 >80 Trunk circumference [cm] 40-60 >100 Canopy height [m] 1.5 10 Canopy diameter [m] 2 6 Flowering 18th calendar week Disposal of leaf litter Nov 05 Nov 05

150 40 [mm] [°C] 120 30 [h] 90 20

60 10

30 0

0 -10 JanMarMayJulSep

rain [mm] sunshine [h] min.temp [°C] max.temp [°C]

Figure 1. Weather data, Berlin 2006. 3

Application The neonicotinoid acetamiprid was sprayed on trunks in all variants at the beginning of flowering in 2006, BBCH 61 (Stauss et al., 1994). The choice of the application date was connected with the egg deposition of the moths for the development of the first leaf miner generation. This is also the time of the most important growth stage for the horse chestnut trees, which lasts until the end of June. Trees of both age groups were sprayed either with the 100% formulation or the 10% mixture (Table 2).

Table 2. Application of acetamiprid.

Variant AF 40-60 AF100 10% 100% 10% 100% Application date 3 May 2006 9.30-11.00 a.m. No. of sprayed trees 6 6 4 4 No. of untreated trees 4 4 4 4 Nozzle, pressure [bar] flat fan, 2-3 bar Spray band height [m] 2 Concentration of mixture [%] 0.05 0.5 0.05 0.5 Volume rate per tree [mL] 113 111 390 366 Application rate a. s./tree [g] 0.05 0.55 0.19 1.83 Weather condition Temperature [°C] 8-17 Wind [m/s] 0.3-1.5 First rain after application [mm] 18 May 2006 [9]

Residues Leaves for residue analyses were taken from all four directions within the canopy of horse chestnut trees between 2.5 and 3 m height. The field samples of approximately 500 g/tree were packed in light-tight bags and stored at 4°C for max. 24 h. They were then homogenized, mixed and frozen at -20°C until analysis. For the analytical procedure, 10 g were taken for the extraction in acetone/water-solution (3/1) with an Ultra Turrax®. After cleanup with a Chem Elut-Column® the ethyl acetate extract was reduced to dryness and then refilled with the internal standard in acetonitrile. Samples were measured by liquid chromatography coupled with mass spectrometer as a detector (Table 3).

Effects and side effects Four leaves were taken from all four directions of each tree between 2.5 and 3 m height. Mines were dissected in 25 randomly selected pinnate leaves. Living, dead and parasitized mines were distinguished from each other and counted. Efficiency was calculated as ratio of the difference between living leaf miners in untreated leaves and treated ones to living leaf miners in untreated leaves multiplied by 100 (Abbott). Parasitation is the ratio of parasitized leaf miners to the total number of leaf miners per tree multiplied by 100.

4

Results and discussion

Validation of the analytical procedure The quality of the analytical method was verified in recovery tests (Table 4). The recovery of added substances was sufficient at all concentration levels. The accuracy of the field sample analyses was evidenced by a 96% surrogate recovery (added to each sample before the beginning of the analytical procedure) as well as with an acceptable variation of 9.4%.

Table 3. Parameters of the LC/MS/MS-method.

Device HPLC – MS API 2000 Applied Biosystems Pump PE Series 200 Micro Pump Injector PE Series 200 Autosampler Injection volume 10 µL Column Phenomenex Gemini C18 150x3mm, 5µm Column thermostat 30°C Ionisation ESI Solvents A: methanol+0.1% acetic acid /B: water+0.1% acetic acid Gradient 0 minÆ 10 min; 5% AÆ 95% A Flow rate: 0,6 mL/min Mass acetamiprid imidacloprid imidacloprid-D4 Q1 222.9 256.1 260.1 amu Q3 126.0 209.0 213.1 amu Dwelltime 500 ms

Table 4. Results of recovery of acetamiprid and surrogate imidacloprid in horse chestnut leaves (n. d.: not detectable).

Analyte: acetamiprid Surrogate: imidacloprid Recovery samples Field samples Fortification 0 0.01 0.1 1.0 0 0.01 0.1 1.0 0.05 [µg/sample] Number of tests 4 6 6 6 4 6 6 6 64 Recovery [%] n. d. 96.0 97.1 79.5 n. d. 107 94.0 80.1 96.2 Variation [%] 7.6 7.1 5.7 10.4 8.5 5.3 9.4 LOD [mg/kg] 0.0001 – 0.005 Internal standard 500 pg/µL imidacloprid-D4

Application In urban areas, where spraying the foliage is hardly feasible and not tolerated, other application methods have been developed for the transport of the a. s. through the trunk to the canopy. The control of the horse chestnut leaf miner with soil injection of neonicotinoids leads to good results (Lohrer et al., 2003, Malinowski, 2006). The trunk application on horse chestnut seems to be rather beneficial, due to the fact that this tree species has a ring porous 5

system for the water transport (Langner, 2003). The invasive trunk application methods like trunk injection or infusion (Krehan, 1997a; Łabanowski, 2003; Heidecke, 2005, 2006) and stem implantation (Scholz und Wulf, 1998; Scholz, 1997; Krehan, 1997a) present alternatives (Schnee et al., 2006) but imply the hazard of phytotoxic reactions (Jäckel et al., 2007). As far as trunk infusion with acetamiprid was concerned, phytotoxicity similar to wilting was observed. The reasons were attributed to the solvents in the formulation or a possible over- dosing (Krehan, 1997b). Different results of the control of leaf miners were traced back to the unequal transport of the a. s., without providing analytical evidence. Acetamiprid is transported in the xylem as a systemic substance. Specific properties are its solubility in water (4.25 mg/L), its stability at a pH-range from 4 - 7 sufficient for the pH 5 like in the xylem liquid, and its stability to daylight (BCPC, 2004-05). Trunk spraying with acetamiprid suggests a non-invasive alternative for the control of the leaf miner. In contrast to the trunk infusion with 40-80 g acetamiprid per tree (20 m height, diameter in 1 m of height: 30-50 cm) (Krehan, 1997b) only 0.05 g - 2 g per tree were sprayed on the trunk. Heidecke und Schmidt (2005) stated that some research was still required into what amount of a. s. is necessary for a sufficient control of leaf miners on trees of various heights. The best way to protect the leaves is the application before flowering. However, the ability of transporting substances in the xylem before and during flowering might be too low (Schmidt und Roloff, 2004). For this study, the application (Table 2) was a compromise, performed at the beginning of flowering and oviposition by swarming moths.

Residues Residues in leaves after trunk spraying with pesticides have not been investigated yet. The choice of suitable sampling dates is difficult. On the one hand, Heidecke (2006) claims the rate of xylem flow to be of a wide range from 50 to 200 cm/h, whereas Wulf and Siebers (1992) detected phosphamidon (miscible with water, stable in neutral and acid media) on the other, even 7 days after trunk-injection in leaves of lime, being a diffuse-porous tree. In contrast to the injection, the a. s. must penetrate through the bark in case of spraying. Consequently, the first sampling date was determined on the sixth day after application. Leaves were sampled for analysis in all variants for four times until the end of September. Figure 2 shows the residues in leaf samples collected six (9 May) and sixty-two (4 July) days after application. Each triangle represents the residue of one tree. In the first samples taken after application, acetamiprid residues were found with high deviations in each variant. The leaves were sampled from the trees in a uniform manner from each direction and mixed for analysis. The distribution of residues in the canopy was not investigated. The highest residues were surprisingly found in the variant AF 40-60, where young trees had been sprayed with the 10% low-concentrated formulation. Residues above the detection limit could only be found in leaves of old trees sprayed with high concentrated mixture 62 days after application. In samples taken after another 55 (28. / 29. August) and respectively 83 (25. / 26. September) days, no residues were detected. Acetamiprid residues in horse chestnut leaves show no clear pattern depending on the application rate and the age of the trees. This requires a careful interpretation. An attempt was made to estimate the fraction of acetamiprid which was transported in the leaves. Maximum residue data of each variant were multiplied with the leaf mass of trees on the assumption of a uniform distribution in the canopy (Table 5). With the exception of the values in the AF 40-60 (10%)-variant, the uptake of acetamiprid in the leaves was rather marginal.

6

1.000 xx AF 40-60 AF 100 0.100

0.010

0.001 acetamiprid in leaves [mg/kg]

0.000 662662 662 662 days after application 56 90 sampling for efficiency control

Figure 2. Residues of acetamiprid in horse chestnut (Aesculus hippocastanum) leaves (fresh weight), 6 and 62 days after trunk spraying (10%: grey; 100%: black).

Table 5. Preliminary estimate of the fraction of applied acetamiprid analysed in horse chestnut leaves (dry mass of leaves: 27%).

Variant AF 40-60 AF100 10% 100% 10% 100% Application rate a. s./tree [g] 0.05 0.55 0.19 1.83 Leaf mass per tree (dry mass) [kg] 7 24 Maximum residue (6 d after appl., dry mass) [mg/kg] 2.637 0.082 0.026 0.233 Uptake of a. s. in leaves per tree [mg] 18.46 0.57 0.62 5.56 Fraction of the applied a. s. in the canopy [%] 37 0.1 0.3 0.3

Effects The weather conditions were favourable for the development of the first leaf miner generation in 2006. The rainy 20th calendar week (15.-21.05.) was followed by a long period of hot and dry weather (Figure 1). The medium infestation in the first generation with 17-33 mines per pinnate leaf was also observed in the leave damage in the canopy (turning brown). 30-40 mines per pinnate leaf were counted for the second generation. Leaf samples for the estimation of the effects were taken shortly before the moth hatched at the end of the first (56 d after spraying) and second leaf miner generations (90 d after spraying). The third generation was not considered, since the influence of the leaf miners on the tree development was low at that time. A sufficient control of Cameraria ohridella is usually achieved at efficiency rates of approx. 60%. Under these circumstances, horse chestnut trees appear green and do not feature any brown canopy areas. Figure 3 illustrates the efficiency of trunk spraying to the control of the leaf miner. No effects were observed in the variants with the low concentration independently of the age of the trees. Only poor efficiency was found in some 100% variants. 7

As for the trunk injection with a high concentration of acetamiprid according to Krehan (1997a) it was only in the top regions of the canopy where the infestation rate was reduced by slightly more than fifty per cent. In this study, none of the tests showed a sufficient efficiency, for which reason the acetamiprid concentration may very well be assumed to be too low in all cases.

100

AF 40-60 AF 100 80

60

40

20 Efficiency (Abbott) [%]

000 0 0 56 90 56 90 days after application

1st 2nd 1st 2nd end of generation

Figure 3. Efficiency of acetamiprid against horse chestnut leaf miner (Cameraria ohridella) 56 (end of first generation) and 90 days (end of second generation) after trunk spraying (10%: grey; 100%: black).

Side effects

For the period between 2002 and 2006, the average parasitation rate was estimated to be 2.4% (+ 0.88%) for the first leaf miner generation, 3.7% (+ 2.2) for the second generation, and 5.5% (+ 5.4%) for the third generation in Berlin. (Jäckel et al., 2007). The performed tests did not identify any effects by the application of acetamiprid compared with the untreated variants. Parasitation rates largely achieve the natural level, independently of any application whatsoever (Figure 4). One exception is the slightly increased parasitation rate in the old horse chestnuts applied with the concentrated acetamiprid mixture. This side effect was also observed by Nejmanova et al., (2004) in trees treated with the neonicotinoids acetamiprid, imidacloprid and thiacloprid. A feasible hypothesis is a potential sedation of the leaf miner pupae, which makes it easier to penetrate the larvae by parasitoids.

8

100

AF 40-60 AF 100 80

60

40 Parasitation [%] Parasitation

20

0 56 90 56 90 days after application 1st 2nd 1st 2nd end of generation

Figure 4. Parasitation of horse chestnut leaf miner (Cameraria ohridella) 56 (end of first generation) and 90 days (end of second generation) after trunk spraying (untreated trees: white; 10%: grey; 100%: black).

Acknowledgements

The authors would like to thank B. Große, T. Koch, S. Korber and I. Stachewicz for their dedication and commitment throughout the project.

References

BCPC (ed.) 2004-05: The e-pesticide Manual, Volume 3.1, 13th Edition by Tomlin, C.D.S. Heidecke, C. und Roloff, A. 2005: Wirksamkeit systemischer Insektizide gegen Ross- kastanien-Miniermotte (Cameraria ohridella). Jahrbuch der Baumpflege 216-219 (24) Heidecke, C. 2006: Optimierung der Stammapplikation systemischer Pflanzenschutzmittel auf der Grundlage baumbiologischer und holzanatomischer Aspekte. Contrib. Forest Sciences 26 Eugen Ulmer GmbH Co., Stuttgart, ISBN 3-8001-5428-5 Heitland, W. 2006: Controcam – Control of Cameraria. Nachrichtenblatt. Deut. Pflanzenschutzd. 58: 251-252. Jäckel, B., Grabenweger, G., Koch, T., Schmolling, S., Hopp, H., & Balder, H. 2006: Untersuchungen zur Bekämpfung der Kastanienminiermotte in Berlin, Abschlußbericht, Projektnummer 10700UEP/WÜ5. Jäckel, B., Balder H., Grabenweger, G., Hopp, H., Koch, T., Schmolling, S. 2007: Integrierte Konzepte zur Bekämpfung der Rosskastanien-Miniermotte in Berlin. In: Jahrbuch der Baumpflege, ISBN 978-3-87815-222-4: 93-105. Krehan, H. 1997a: Roßkastanienminiermotte – Vergleich der Bekämpfungsverfahren. Forstschutz Aktuell 19/20: 2-6. 9

Krehan, H. 1997b: Erste Erfahrungen mit Bauminfusionen gegen die Roßkastanien- miniermotte. Forstschutz Aktuell 21: 26. Langner, T. 2003: www.baumprüfung.de/statik des baumes/kernbildung. Łabanowski, G., Soika, G. 2003: Macro-injection system against the horse-chestnut leafminer (Cameraria ohridella) in Poland. Scientific works of the Lithuanian Institute of Horticulture and Lithuanian University of Agriculture Horticulture and Vegetable Growing 22: 512-517. Lohrer, T., Gerlach, W. W.P., Fischer, P., Fuchsbichler, G. und Eichinger, H.-M. 2003: Untersuchungen zur Laub- und Kompostbelastung nach einer Bodenapplikation mit Imidacloprid zur Bekämpfung der Kastanienminiermotte Cameraria ohridella (Lepido- ptera, Gracillariidae) Nachrichtenblatt. Deut. Pflanzenschutzd. 55: 240-241. Malinowski, H. 2006: Systemic insecticides and the posibility of their use in the protection of the horse-chestnut trees against the horse-chestnut leafminer (Cameraria ohridella Deschka & Dimić). Sylwan 1: 48-57. Nejmanová, J., Cvačka, J., Hrdý, I., Kuldová, J., Muck, A. and Svatoś, A. 2004: Residues of diflubenzuron on horse chestnut leaves and efficacy of insecticides against the horse chestnut leafminer (Cameraria ohridella) with notes on its parasitization. Proceedings of 1st International Cameraria Symposium, IOBC Prague: 36. Schnee, H., Dittrich, S., Pfüller, R. 2006: Bekämpfung der Kastanienminiermotte durch Insektizidanwendungen. Mitt. Biol. Bundesanst. Land-Forstwirtsch. 400: 155. Scholz, D. 1997: Zur Anwendung von Baum-Implantaten im Pflanzenschutz unter besonderer Berücksichtigung baumbiologischer und mykologischer Aspekte. Dissertation der Georg- August-Universität Göttingen. Scholz, D. und Wulf, A. 1998: Ansätze zur selektiven Bekämpfung von Baumschädlingen im öffentlichen Grün und im Forst mittels Stammapplikation systemischer Pflanzenschutz- mittel. Gesunde Pflanze 50: 1-6. Stauss, R., Bleiholder, H., van den Boom, T., Buhr, L., Hack, H., Hess, M., Klose, R., Meier, U., Weber, E. 1994: Einheitliche Codierung der phänologischen Entwicklungsstadien mono- und dikotyler Pflanzen. Ciba-Geigy AG, Basel Wulf, A. und Siebers, J. 1992: Zum Transport von Pflanzenschutzmitteln in Bäumen nach Stamminjektion. Nachrichtenblatt. Deut. Pflanzenschutzd. 44: 43-46.

Pesticides and Beneficial Organisms IOBC/wprs Bulletin Vol. 35, 2008 pp. 10-17

Who benefits from low-input pesticide use within the tritrophic system: crop – aphid – predator?

Kerstin Schumacher, Bernd Freier Julius Kuehn Institute (JKI), Federal Research Centre for Cultivated Plants, Institute for Strategies and Technology Assessment in Plant Protection, Stahnsdorfer Damm 81, 14532 Kleinmachnow, Germany

Abstract: Effects of low-input pesticide use on the tritrophic system crop – aphid – predator were investigated in field and laboratory studies. The field study was carried out in a conventional farm of the high-input crop protection area Magdeburger Boerde between 2004 and 2006. The field was divided into two halves during the whole period of investigation representing low- and high-input variants. One half was characterized by 50% reduced pesticide doses and the other one by good plant protection practice (100%). The crop rotation of this field was spring wheat (2004), winter wheat (2005) and peas (2006). Before and after insecticide application densities of aphids and their predators on plants (counts) as well as activity densities and diversity of carabids on ground (pitfall trappings) were investigated. Aphids were insufficiently reduced by insecticides in the low-input variant. In all three years significantly more aphids were found in the low-input variant in comparison to the high- input variant. The abundance of aphid specific predators, e. g. adults and larvae of coccinellids and syrphid larvae, was positively affected by the low-input pesticide use. In all years significantly more aphid predators were observed in low-input-field than in high-input-field. But no clear effect of reduced insecticides use on abundance, structure of dominance, and diversity of carabids was observed. It is concluded, that the potential of natural regulation was enhanced by reducing the insecticide input but the regulation itself was not improved. Thus, aphids were benefited to a greater extent than their predators from reduced insecticide use. The laboratory studies were carried out in climate chambers investigating the tritrophic system weed – aphid – predator by applying different doses of insecticides. In contrast to the field study aphids could be sufficiently reduced by low-input insecticide doses. The doses of insecticide could be reduced even more by utilization of the predator potential to receive a good pest control. But its difficult to transfer the results of laboratory studies to field conditions. It could come to an overestimation of the potential of natural regulation by a predator.

Key words: aphids, predators, reduced insecticide dose, carabids, tritrophic systems

Introduction

In Germany 29,000 t pesticides were used yearly (Schmidt, 2003). Under ecological point of views the use of pesticides is critical, because each application strongly influences the ecosystem. Not only pest populations were affected but also non-target arthropods, e.g. beneficials organisms, as well as species richness declined in general. Thus, natural regulatory mechanisms of pests are reduced. A national Chemical Plant Protection Reduction program (2004) was established to reduce the pesticide input into the crop farming. It is expected that a remarkable reduction results in positive effects not only on the preventive consumer protection but also on the ecological situation in agricultural landscapes. Several studies have shown that a renunciation of chemical pesticides, e.g. in organic farming, improved regulatory mechanisms and the biodiversity in the fields (e. g. Kromp, 1990; Clark, 1999; Letourneau & Goldstein, 2001; Schmidt et al., 2005). However, other studies have found no effects on

10 11

farming systems on species richness of e.g. carabids (Melnychuk et al., 2003; Purtauf et al. 2005). Several studies have shown that diversity and abundances of plants and non-target arthropods increased when intensity of pesticide use decreased (Büchs et al., 1997; Langmaack et al., 2001). In reference to national reduction programs, long-term investigations are needed to detect sustainable ecological effects of a permanently reduced usage of pesticides within conventional arable farming. The aim of the study was to determine ecological effects of a low-input plant protection strategy characterized by a permanently 50% reduced dose of applied pesticides compared to a conventional plant protection strategy in sense of good plant protection practice (100%). Besides laboratory studies with different pesticide doses on tritrophic systems were performed, particularly to investigate the effects on pest-beneficial interactions. The question was: will the potential of danger of pesticide for beneficial organisms be reduced by using 50% pesticide doses, so that the natural regulation improves? It was assumed that 50% doses of pesticides, particularly insecticides, permanently conserves beneficials, improves natural control and enhances bio-diversity in fields.

Materials and methods

Field study The study was carried out on a field of a conventional farm of the high-input crop protection area Magdeburger Boerde between 2004 and 2006. All three years, the field was divided into two halves during the period of investigation, representing a 50%-variant and a 100%-variant. The 100%-variant was characterized by high-input plant protection strategy in accordance to good plant protection practice and the pesticides were applied in recommended doses. The 50%-variant was characterized by a permanent low-input plant protection strategy and exactly half-dose of each pesticide (insecticide, herbicide, fungicide) compared to the doses used in high- input plant protection was applied. Tillage and using of fertilizer and growth regulators were the same for both variants. The field-pair had following sizes (100 %/50 %): 16.1/7.0 ha. The crop rotation was: spring wheat (2004), winter wheat (2005) and peas (2006) and was realistic for the area. Densities of aphids and their predators on plants were determined before and after insecticide application by visual counts at five sampling points (n=5). In wheat, each tiller of a 2m sowing row was investigated. The counts were performed at end of flowering before insecticide spraying and two and three weeks after insecticide spraying. The predators were calculated as predator units (Freier et al., 1998). In peas, 20 or 50 plants were checked for infestation and non-infestation by Acyrthosiphum pisum at each counting point. The occurrence of aphid predators was additionally assessed on a 5-m drill row at each sampling point and also calculated as predator units. Pitfall trapping was conducted to determine the activity densities and diversity of epigeic carabids. Six pitfall traps (10 cm in diameter filled with 2 % formaldehyde and detergent) were set in a row in both variant with a distance of 20 m to each other. The pitfall traps were emptied weekly. The period of examination was 4 to 5 weeks in June and July. The carabids were determined to species level (Trautner & Geigenmüller, 1987) and dominance structure as well as Shannon-Weaver-Index and Evenness were calculated (Wetzel, 2004). The pesticides used were analyzed regarding to their potential of danger for predators known from literature.

12

Laboratory study To analyze the phenomenon of adaptation of tritrophic systems on different insecticide systems, model tests were conducted. The following tritrophic system was tested: Vicia faba L. (bean) – Aphis fabae Scop. (aphid) – Chrysoperla carnea Steph. larva (predator) applying different insecticides doses: 0%, 25%, 50% and 100% (=150 g/ha Trafo WG and 7.5 g/ha λ- cyhalothrin, respectively). The trials were carried out in climate chambers (22 ± 2 °C, 65 ± 5% rel. humidity). Two treatments were performed. One treatment was carried out without predator and the other one with two Chrysoperla carnea larvae as predators. The plants were grown for 15 days, with one plant per pot, before eight aphids were put on each plant. After five days for developing a realistic structure of an aphid population, the insecticide Trafo WG (λ-Cyhalothrin) was applied with the help of a laboratory sprayer (Schachtner, Spray lab, offen, Ludwigsburg). The amount of spray mixture applied was equivalent to 324 l/ha. Immediately after that the lacewing larvae were put on the plants in the trials with predators. For each dose, the density of aphids was counted every 1-3 days after insecticide application and the survival of the predator was observed for 14 days. Thereby the number of individuals at the time of insecticide application was equated with 100%.

Statistical analysis Data were analyzed with the program SAS version 9.1 for Windows (SAS Institute Inc., Cary, NC, USA). The data of the field study were compared between the high-input and the low- input variant. Densities of aphids (wheat) and predators as well as pitfall trap catches were analyzed with a Student´s unpaired t-test between both variants. Data of infested plants (peas) were analyzed with χ2-test. In all statistical calculations, a significance level of 5% was used.

Results and discussion

Field study Analyzing the potential of danger for predators of the pesticides used during the 3-year study, it came out that insecticides affected mainly predators. In spring wheat (2004) and winter wheat (2005), there were no differences of cereal aphid densities (Sitobion avenae, Metopolophium dirhodum, Rhopalosiphum padi) between both variants before insecticide application (Fig. 1). Herbicides and fungicides used before seemed not to affect the aphid densities. After insecticide application, there were significantly more aphids in the 50%-variant than in the 100%-variant in both years. The high-input plant protection strategy gave a good aphid control whereas the effectiveness of the low-input strategy was not sufficient to prevent a high aphid density. There were also no differences of predator densities between the two variants before insecticide application in both years (Fig. 1). After application of insecticide, significantly more predator units were found in the low- input variant. In 2004, syrphid larvae made up to over 90% of the predators, whereas the coccinellids were affected by λ-Cyhalothrin mostly. But in 2005 in contrast to Karate the insecticide Pirimor (Pirimicarb) affected syrphid larvae mostly. Thus, the insecticides had different effects on different predator fractions (Poehling, 1988; Jansen, 2000). In peas 2006, nearly all plants were infested by pea aphids (Acyrthosiphon pisum) in both variants before insecticide application (Fig. 1). The infestation was significantly reduced by the high-input strategy whereas in the 50%-variant the decline was lesser. 13

Aphids Predators 100% Insecticide dose Spring wheat 2004 50% Insecticide dose *** 70 λ-Cyhalothrin λ-Cyhalothrin 90 (0.075 l/ha) (0.075 l/ha) *** 60 80 70 2 50 22 20 40 18 16 30 14 ** 12

Aphids per tiller ** 20 10

n.s. Predator units per m 8 n.s. 6 10 4 2 0 0 BBCH 65 BBCH 75 BBCH 83 BBCH 65 BBCH 75 BBCH 83

Winter wheat 2005

25 Pirimicarb 12 Pirimicarb (300.0 g/ha) (300.0 g/ha) *** n.s. 10 20 ** 2

8 15 * 6 *** 10

Aphids per tiller 4

n.s. Predator units per m 5 2

0 0 BBCH 71/73 BBCH 75/77 BBCH 83 BBCH 71/73 BBCH 75/77 BBCH 83

Peas 2006 Pirimicarb + λ-Cyhalothrin Pirimicarb + λ-Cyhalothrin (300.0 g/ha + 0.038 l/ha) (300.0 g/ha + 0.038 l/ha) n.s. 18 100 *** *** 16 n.s. 14

80 2 *** n.s. 12 60 ** 10

8 40 6 Infested plants (%) plants Infested 4 Predator units per m 20 n.s. 2

0 0 BBCH 65 BBCH 67/69 BBCH 75 BBCH 77/79 BBCH 65 BBCH 67/69 BBCH 75 BBCH 77/79

Figure 1. Effect of high-input (100%) and low-input (50%) plant protection strategy on aphids and their predators in wheat and peas. Application rate of insecticide was given for the 100% variant (50% variant: exactly the half dose). Mean ± standard deviations (n=5). Student´s unpaired t-test and χ2-test (peas: aphids); n.s. not significant (p>0.05), * p<0.05, ** p<0.01, *** p<0.001. 14

Again after the insecticide application significantly more predators survived in the 50% variant than in the 100%variant. Thus, insecticide treatments had a strong effect on aphids and predators. More aphids and predators survived at 50% insecticide treatment than at 100% insecticide treatment. Positive effects on predators of 50% treatment could result from both higher remaining aphid population and lower toxicity to predators. So the predators were conserved by the reduced insecticides doses, but were not sufficient to control aphid populations. The data of the pitfall catches showed different results. In spring wheat (2004), there were no differences of carabids per pitfall trap and week between both variants before insecticide application (Fig. 2).

Spring wheat 2004 Winter wheat 2005

λ-Cyhalothrin 30 (0.075 l/ha) 50 n.s. n.s. Pirimicarb 21 JUNE 2004 (300.0 g/ha) 25 24 JUNE 2005 40 n.s. 20 n.s. 30 n.s. n.s. 15

20 ** 10 * *

10 5 Carabids per pitfall trapweek and Carabids per pitfall trap and week and trap pitfall per Carabids

0 0 01.-08. June 08.-15. June 15.-22. June 22.-29. June 29. June-06. July 08.-15. June 15.-22. June 22.-29. June 29. June-06. July

Peas 2006

Pirimicarb + λ-Cyhalothrin 100 (300.0 g/ha + 0.038 l/ha) 100% Insecticide dose 15 JUNE 2006 50% Insecticide dose n.s. 80

60

40

n.s. n.s. * 20 Carabids per pitfallCarabids per trapand week

0 08.-15. June 15.-22. June 22.-29. June 29. June-06. July

Figure 2. Effect of high-input (100%) and low-input (50%) plant protection strategy on epigeic carabids in wheat and peas. Application rate of insecticide was given for the 100% variant (50% variant: exactly the half dose). Mean ± standard deviations (n=6). Student´s unpaired t-test; n.s. not significant (p>0.05), * p<0.05, ** p<0.01.

After insecticide application significantly more individuals were found in the 50%- variant than in the 100%-variant. The structure of dominance of carabids was similar in both 15

variants (Tab. 1). Carabus auratus, Anchomenus dorsalis and Poecilus cupreus were the three most dominant species. They had an overall proportion of about 80%. In winter wheat 2005 there was a different result (Fig. 2). After insecticide application significantly more individuals were catch in the 100%-variant than in the 50%-variant. But once again the structure of dominance very similar in both variance (Tab. 1). In peas (2006), there were no differences in the activity densities in both variants (Fig. 2). Only in the last week of trapping there were significantly more individuals in the high-input- variant than in the low-input-variant. Again there were only minor changes in diversity indices, whereas the structure of dominance varied to a greater extent (Tab. 1).

Table 1. Effect of high-input (100%) and low-input (50%) plant protection strategy on the structure of dominance, number of species and diversity indices of epigeic carabids in wheat and pea. Number of Shannon- Dominance (%) Evenness Year/ species Weaver-Index Crop 100% 50% 100% 50% 100% 50% 100% 50% 2004 Carabus auratus 45.5 Carabus auratus 31.3 17 15 1.63 1.80 0.57 0.67 Spring Poecilus cupreus 20.3 Anchomenus dorsalis 28.3 wheat Anchomenus dorsalis 19.0 Poecilus cupreus 17.9

2005 Carabus auratus 58.1 Carabus auratus 42.3 21 15 1.43 1.68 0.47 0.62 Winter Harpalus rufipes 15.6 Harpalus rufipes 21.5 wheat Anchomenus dorsalis 14.9 Anchomenus dorsalis 19.1

2006 Carabus auratus 36.2 Anchomenus dorsalis 42.2 17 17 1.57 1.54 0.55 0.54 Peas Poecilus cupreus 32.3 Carabus auratus 29.8 Anchomenus dorsalis 17.5 Poecilus cupreus 14.3

From this, it follows that there were no clear effects of reduced pesticide doses on carabids. Higher activity densities did not occur and the dominance structure as well as diversity did not change. Therefore carabids do not seem to be suitable indicators for low- input plant protection. It is concluded that aphids benefited from reduced insecticide doses more than their predators. The increased predator potential did not lead to better natural control because of the higher relative survival rate of aphids in the 50% dose strategy. Besides no accumulative effects were observed over the 3 years.

Laboratory study There was a good control of aphids at higher insecticide doses in the treatment without a predator (Fig. 3). Only at the 25% doses the population of aphids increased noticeable. With Chrysoperla carnea larvae as predators, aphid populations were each time lower than without predators, for the same insecticide concentrations. So a further reduction of insecticide dose was possible by utilization of a predator potential.

16

Without predator With Chrysoperla carnea larvae

1400 14006001400 0% Insecticide dose 1200 1200 25% Insecticide dose 1200500 50% Insecticide dose 1000 75% Insecticide dose 600 600400 100% Insecticide dose 800 300 400 600 400 Aphid density (%) Aphid (%) density 200 Aphid density (%) Aphid 400 (%) density Aphid 200200 200 100

0 00 01234567891011121314 0123456789101112131401234567891011121314 Days after insecticide application DaysDaysDays after afterafter insecticide insecticideinsecticide application applicationapplication

Figure 3. Relative aphid densities days after insecticide application (λ-Cyhalothrin) with different doses treated; without a predator and with Chrysoperla carnea larvae as predators, respectively.

It is concluded that in contrast to the field study aphids could be sufficiently reduced by low-input insecticide doses. The dose of insecticides could be reduced even more by utilization of the predator potential to receive a good pest control. But it is difficult to transfer the results of laboratory studies to field conditions. It could come to an overestimation of the potential of natural regulation by a predator.

Acknowledgements

The authors thank the Deutsche Bundesstiftung Umwelt for supporting this project. They also thank Birgit Schlage, Andreas Schober and Ute Mueller (BBA) who provided valuable assistance with on-the-site data collection and insect determination.

References

Büchs, W., Harenberg, A. & Zimmermann, J. 1997: The Invertebrate Ecology of Farmland as a Mirror of the Intensity of the impact of man? – An approach to interpreting results of field experiments carried out in different crop management intensities of a sugar beet and an oil seed rape rotation including set-aside. In: Entomological Research in Organic Agriculture. Kromp, B. Meindl, J. & Oxon,J. (eds.). Biol. Agric. Hortic. 15: 83-107. Clark, M. S. 1999: Ground beetle abundance and community composition in conventional and organic tomato systems of California´s Central Valley. Appl. Soil Ecol. 11: 199-206. Freier, B., Möwes, M., Tritsch, H. & Rappaport, V. 1998: Predator units - an approach to evaluate coccinellids within the aphid predator community in winter wheat. IOBC/wprs Bull. 21 (8): 103-111. Jansen, J. P. 2000: A three-year field study on the short-term effects of insecticides used to control cereal aphids on plant-dwelling aphid predators in winter wheat. Pest Manag. Sci. 56: 533-539. 17

Kromp, B. 1990: Carabid beetles (Coleoptera, Carabidae) as bioindicators in biological and conventional farming in Austrian potato fields. Biol. Fert. Soils 9: 182-187. Langmaack, M., Land, S. & Büchs, W. 2001: Effects of different field management systems on the carabid coenosis in oil seed rape with special respect to ecology and nutrional status of predacious Poecilus cupreus L. (Col., Carabidae). J. Appl. Entomol. 125: 313- 320. Letourneau, D. K. & Goldstein, B. 2001: Pest damage and arthropod community structure in organic vs. conventional tomato production in California. J. Appl. Ecol. 38: 557-570. Melnychuk, N. A., Olfert, O., Youngs, B. & Gillott, C. 2003: Abundance and diversity of Carabidae (Coleoptera) in different farming systems. Agr. Ecosyst. Environ. 95: 69-72. Poehling, H. M. 1988: Influence of Cereal Aphid Control on Aphid Specific Predators in Winter Wheat. Entomol. Gener. 13: 163-174. Purtauf, T., Roschewitz, I., Dauber, J., Thies, C., Tscharntke, T. & Wolters, V. 2005: Land- scape context of organic and conventional farms: Influences on carabid beetle diversity. Agr. Ecosyst. Environ. 108: 165-174. Schmidt, K. 2003: Ergebnisse der Meldungen für Pflanzenschutzmittel und Wirkstoffe nach § 19 des Pflanzenschutzgesetzes für die Jahre 1999, 2000 und 2001 im Vergleich zu 1998. Nachrichtenbl. Deutsch. Pflanzenschutzd. 55: 121-133. Schmidt, M. H., Roschewitz, I., Thies, C. & Tscharntke, T. 2005: Differential effects of landscape and management on diversity and density of ground-dwelling farmland spiders. J. Appl. Ecol. 42: 281-287. Trautner, J. & Geigenmüller, K. 1987: Tiger Beetles Ground Beetles. Illustrated Key to the Cicindelidae and Carabidae of Europe. Verlag Josef Margraf, Aichtal. Wetzel, T. 2004: Integrierter Pflanzenschutz und Agrarökosysteme. 2. überarb. und erw. Aufl. Steinbeis-Transferzentrum, Pausa/Vogtland.

Pesticides and Beneficial Organisms IOBC/wprs Bulletin Vol. 35, 2008 pp. 18-25

Impact of low-input pesticides usage on spider communities with special regard to accumulated effects

Christa Volkmar 1, Kerstin Schumacher 2, Julia Müller1 1 Martin-Luther-University Halle-Wittenberg, Institute of Agricultural and Nutritional Science III, 06108 Halle, Germany 2 Federal Research for Agriculture and Forestry, Institute of Integrated Plant Protection, 14532 Kleinmachnow, Germany

Abstract: Reduction programs are designed to reduce chemical plant protection to a minimum. The intensity of plant protection can be measured by the treatment frequency index (BI). This study focused on the ecological impact of a long-term reduction of chemical plant protection in commercial crop cultures and investigated the effects of a 50% reduction on Araneae at three fields in Ochtmers- leben (Saxony-Anhalt). Each field was divided into two halves representing a 100% and a 50% application variant. The analysis was based on qualitative and quantitative parameters such as activity density, species diversity, pattern of dominance as well as other computed parameters. The results indicate that the impact of insecticides on the spider population was the strongest when the insecticides were applied the earliest. There were no significant shifts in dominance structures between both variants. Some endangered spider species were only sampled in the 50% variant. In summary, a 50% long-term reduction of chemical plant protection has positive effects on spider coenoses.

Key words: reduction program, plant protection, indicators, Araneae, low-input pesticide usage

Introduction

Chemical plant protection agents are designed to manage pests, but they also harm non-target organisms. The intensity of plant protection can be measured by the treatment frequency index (BI). The BI is defined as the amount of applied pesticides referred to admitted expenditure amount and size of the cultivation area. This study focused on reducing pesticide applications in order to protect useful organisms such as predators. This could open up new potential for natural pest regulation. It tries to understand the ecological impact on spider communities of a long-term reduction of chemical plant protection (50%) in commercial crop cultures at an operational level (Backhaus et al., 2005).

Material and methods

The aim of the study was to investigate the effects of a 50% reduction of plant protection strategy on Araneae at three fields in Ochtmersleben (Saxony-Anhalt). Each field was divided into two halves during the whole period of investigation. One half was characterized by 50% reduced pesticide dose (50% variant) and the other one by good plant protection practise (100% variant). The crop rotation of the fields were (2003-2006): field 1 winter wheat - peas - winter wheat – winter barley; field 2 spring wheat – winter wheat – peas – winter wheat; field 3 sugar beet – spring wheat – winter wheat – peas. Insecticides were applied from 2004 till 2006 (fig. 1). The treatment frequency index for each variant is shown in table 1. The treat- ment frequency index includes the treatment with herbicides, insecticides and fungicides (Anonymus, 2004; Schumacher, 2007).

18 19

Spiders were collected using pitfall traps over a four-week-catch period (T1-T4) in June and July in 2003 - 2006. In each variant, six pitfall traps were placed in a row in a distance of 20m to each other. The sampling fluid was 2% formalin solution. The pitfall traps were emptied weekly. Spiders were determined according to specific keys (Heimer & Nentwig, 1991; Roberts, 1985, 1987) and classified on the basis of nomenclature by Platnick (1993). The analysis of results was based on qualitative and quantitative parameters such as activity density, species diversity, and pattern of dominance. To analyse the differences between the variations we used a student’s unpaired t-test and the statistics software SAS 9.0. In all statistical calculations, a significance level of 5% was used [n.s. not significant (p>0.05); * p<0.05; ** p<0.01; *** p<0.001].

Table 1. Overview about the insecticides applied on all three fields from 2003-2006 as well as treatment frequency indices of the 100% and 50% variants (sb: sugar beet, sw: summer wheat, wb: winter barley, ww: winter wheat).

Field 1 (ww-peas-ww-wb) Field 2 (ww-ww-peas-ww) Field 3 (sb-sw-ww-peas)

Year Insecticides Treatment indexInsecticides Treatment indexInsecticides Treatment index applied 100% 50%applied 100% 50%applied 100% 50% 2003 / 3.5 1.9 / 4.6 0.8 / 5.4 2.7

Karate Zeon 2004 Karate Zeon 2.9 1.4 Decis 5.4 3.3 5.8 4.0 + Decis

Pirimicarb + 2005 / 3.8 2.1 4.1 2.2Pirimicarb 6.25 3.3 Karate Zeon

Pirimicarb + Pirimicarb + 2006 Decis 5.2 3.4 4.5 3.4 3.8 1.9 Karate Zeon Karate Zeon

Results and discussion

In 2003-2006, 20,511 spiders belonging to 69 species and 15 families have been documented (table 3). The intensity of plant protection influenced the species diversity in all cases, with more species in the 50% variant than in the 100% variant in 2 fields out of 3 and the contrary for the last one (table 2). In final, when all results were compiled, more spiders’ species were found in the 50% variant than in the 100%.

Table 2. Numbers of species (Araneae) caught in the 100% and 50% variant 2003-2006 in all three fields.

Number of species (2003-2006) Variant Field 1 Field 2 Field 3 Sum 100% 43 29 33 44

50% 38 40 37 50 20

Table 3. Species list of Spiders, Ochtmersleben 2003–2006 (sb: sugar beet, sw: summer wheat, wb: winter barley, ww: winter wheat).

notice: The family lines always give sums 2003 - 2006 Field 1 Field 2 Field 3

4 year crop rotation Î sw peas ww wb ww ww peas ww sb sw ww peas sum 100% 50% 100% 50% 100% 50% Agelinidae 0 0 0 1 0 1 2 Araneidae 2 4 0 1 1 1 9 Araneidae juv 0 1 0 0 0 1 2 Mangora acalypha (WALCKENAER) 2 3 0 1 1 0 7 Clubionidae 1 0 0 0 0 0 1 Clubiona reclusa O. P. CAMBRIDGE 1 0 0 0 0 0 1 Dictynidae 2 0 0 3 2 2 9 Argenna patula (SIMON) 0 0 0 0 0 1 1 Argenna subnigra (O. P.-CAMBRIDGE) 2 0 0 3 2 1 8 Gnaphosidae 8 7 4 3 8 9 39 Drassodes lapidosus (WALCKENAER) 0 0 0 0 1 1 2 Drassyllus lutetianus (L. KOCH) 6 5 4 2 4 5 26 Drassyllus pusillus (C. L. KOCH) 1 0 0 0 0 0 1 Drassyllus praeficus (L. Koch) 0 1 0 0 0 0 1 Gnaphosidae 0 1 0 0 1 2 4 Micaria pulicaria (SUNDEVALL) 1 0 0 0 1 1 3 Zelotes spec. 0 0 0 1 1 0 2 Linyphiidae 3346 4796 2727 3728 1842 3119 19558 Araeoncus humilis (BLACKWALL) 112 123 80 41 21 38 415 Bathyphantes gracilis (BLACKWALL) 27 14 23 20 10 11 105 Diplocephalus picinus (BLACKWALL) 0 0 0 2 0 0 2 Diplostyla concolor (WIDER) 3 5 1 1 1 0 11 Erigone atra BLACKWALL 1080 1707 541 769 176 475 4748 Erigone dentipalpis (WIDER) 256 493 194 195 59 254 1451 Erigonella himalis (BLACKWALL) 0 0 2 0 0 0 2 Lepthyphantes tenuis – Gruppe 70 117 78 70 31 52 418 Linyphiidae 105 106 149 151 187 201 899 Mecynargus foveatus (F. DAHL) 0 0 0 1 0 1 2 Meioneta rurestris (C. L. KOCH) 87 124 76 77 52 86 502 Micrargus herbigradus (BLACKWALL) 1 0 0 0 0 1 2 Micrargus subaequalis (WESTRING) 0 0 0 1 0 0 1 Microlinyphia pusilla (SUNDEVALL) 1 1 1 0 0 0 3 Milleriana inerrans (O. P. CAMBRIDGE) 2 0 1 2 0 0 5 Mioxena blanda (SIMON) 0 2 0 1 0 2 5 Oedothorax apicatus (BLACKWALL) 1558 2035 1534 2350 1281 1960 10718 Oedothorax fuscus (BLACKWALL) 3 2 0 1 2 5 13 Oedothorax retusus (WESTRING) 1 0 2 1 0 0 4 Ostearius melanopygius (O. P. CAMBRIDGE) 23 26 1 0 0 1 51 Pelecopsis parallela (WIDER) 0 1 0 0 0 1 2 Pocadicnemis juncea LOCKET & MILLIDGE 0 0 0 2 0 5 7 Porrh. microphthalmum (O. P. CAMBRIDGE) 16 37 43 42 21 26 185 Savignia frontata (BLACKWALL) 0 0 1 0 0 0 1 Tiso vagans (BLACKWALL) 0 1 0 0 0 0 1 Troxochrus scabriculus (WESTRING) 0 1 0 1 0 0 2 Walckenaeria alticeps (DENIS) 0 0 0 0 1 0 1 Walcken. atrotibialis (O. P. CAMBRIDGE) 1 1 0 0 0 0 2 21

Table 3. contd notice: The family lines always give sums 2003 - 2006 Field 1 Field 2 Field 3

4 year crop rotation Î sw peas ww wb ww ww peas ww sb sw ww peas sum 100% 50% 100% 50% 100% 50% Lycosidae 185 163 80 89 78 58 653 Alopecosa cuneata (CLERCK) 2 0 0 0 0 0 2 Alopecosa pulverulenta (CLERCK) 2 2 0 0 0 0 4 Lycosidae 61 42 3 8 33 2 149 Pardosa agrestis (WESTRING) 20 9 1 1 7 0 38 Pardosa amentata (CLERCK) 9 3 1 2 0 1 16 Pardosa lugubris (WALCKENAER) 1 1 6 0 1 0 9 Pardosa palustris (LINNAEUS) 6 16 6 10 8 9 55 Pardosa prativaga (L. KOCH) 55 69 51 48 21 34 278 Pardosa pullata (CLERCK) 8 1 0 1 1 2 13 Pardosa spec. 3 3 1 3 0 1 11 Pirata hygrophilus THORELL 2 5 3 7 2 2 21 Pirata latitans (BLACKWALL) 2 1 0 2 2 5 12 Trochosa ruricola (DE GEER) 10 8 6 3 2 0 29 Trochosa spec. 0 2 0 0 0 0 2 Trochosa terricola THORELL 1 1 1 4 0 0 7 Xerolycosa miniata (C. L. KOCH) 3 0 1 0 1 2 7 Philodromidae 0 0 0 1 0 0 1 Tibellus oblongus (WALCKENAER) 0 0 0 1 0 0 1 Pisauridae 1 0 0 1 1 0 3 Pisaura mirabilis (CLERCK) 1 0 0 1 1 0 3 Saliticidae 0 0 0 0 3 0 3 Euophrys frontalis (WALCKENAER) 0 0 0 0 2 0 2 Euophrys petrensis C. L. KOCH 0 0 0 0 1 0 1 Tetragnathidae 48 47 36 23 10 9 173 Pachygnatha degeeri SUNDEVALL 46 46 36 23 10 9 170 Tetragnatha extensa (LINNAEUS) 2 0 0 0 0 0 2 Tetragnatha pinicola L. KOCH 0 1 0 0 0 0 1 Theridiidae 2 7 3 3 0 5 20 Achaearanea riparia (BLACKWALL) 0 2 0 1 0 1 4 Enoplognatha mordax THORELL 1 3 2 0 0 1 7 Enoplognatha thoracica (HAHN) 0 1 0 1 0 0 2 Robertus neglectus (O. P. CAMBRIDGE) 0 1 0 1 0 2 4 Theridion bimaculatum (LINNAEUS) 1 0 0 0 0 0 1 Theridion impressum L. KOCH 0 0 1 0 0 1 2 Thomisidae 4 10 4 5 2 8 33 Ozyptila trux (BLACKWALL) 1 0 0 1 0 0 2 Thomisidae 0 0 2 0 0 0 2 Xysticus kochi THORELL 3 10 2 4 1 7 27 Xysticus ulmi (HAHN) 0 0 0 0 1 1 2 Zodariidae 0 0 0 0 2 3 5 Zodarion rubidium SIMON 0 0 0 0 2 3 5 Zoridae 2 0 0 0 0 0 2 Zora spinimana (SUNDEVALL) 2 0 0 0 0 0 2 Individuals 3601 5034 2854 3858 1949 3215 20511 Species 43 38 29 40 33 37 69 Families 11 7 6 11 10 10 14

22

Activity density was higher in the 50% variant on all fields than in the 100% variant (field 1: 5,034, field 2: 3,858, field 3: 3,215 spiders). Annual variations of field 3 are illustrated by figure 1.

3500 100% pesticide dose 3000 50% pesticide dose

2500

2000

1500

1000 Araneae of Numbers

500

0 2003 2004 2005 2006 Sum

Figure 1. Activity density of Spiders in field 3 from 2003 – 2006 in the 100% variant and 50% variant (n=6).

Pirimor Winter wheat 2005 24. June 2005 Peas 2006

80 180 100% pesticide dose Pirimor + Karate Zeon 15. June 2006 *** 50% pesticide dose 70 n.s. 160 n.s. 140 60 120 50 100 40 * 80 30 n.s. *** 60 ** 20 40 * 10 Araneae per ptifall trap and week Araneae per ptifall trap and week 20

0 0 8.-15. June 15.-22. June 22.-29. June 29. June - 6. July 8.-15. June 15.-22. June 22.-29. June 29. June - 6. July

Figure 2. Effect of high-input (100%) and low-input (50%) plant protection strategy on epi- geic Araneae in winter wheat 2005 and peas 2006 on field 3. Mean ± standard deviations (n=6). Student´s unpaired t-test; n.s. not significant (p>0.05), * p<0.05, ** p<0.01, *** p<0.001.

23

In 2005 and 2006 on field 3, Araneae activity was significantly higher in the 50% variant than in the 100% variant even before insecticide applications started (fig. 2). This could be interpreted as a hint to accumulated effects of long-term reduction programs. The application of herbicides (100% /50%) did not have a significant impact on the weed level in both variants. The typical open-land inhabitants of Linyphiidae had the biggest share in the spider population. From this family Oedothorax apicatus, Erigone atra and Erigone dentipalpis were the species with the highest activity in all years and all variants during the sampling data period, in June. Both sexes of O. apicatus showed this high activity, but only the males of E. atra did provide reliable data. Both spider species did show notably reactions when exposed to insecticides. In field 1, O. apicatus activity declined by 23% and E. atra by 34% within four years. In field 2 and 3, the decline amounted to 44% and 19% for O. apicatus and 44% and 63% for E. atra (fig. 3). The family of Lycosidae could not be employed as indicator, since its activity is generally low in June.

425 400 100% pesticide dose 375 50% pesticide dose 350 325

220 200 180 160 140

120 Total numbers 100 80 60 40 20 0 T1 T2 T3 T4 T1 T2 T3 T4

Figure 3. Activity density of Erigone atra (total individuals per date) on field 3 in 2003-2006

(n = 6 pitfall traps).

A further indicator was derived from the test results on Red-Data-Book species. Specimen of Clubionidae, Gnaphosidae and Theridiidae families are more frequently found in extensively treated crop cultures (Al Hussein, 2004). Within the test period of 2003-2006 the endangered species A. riparia, and R. neclectus were only collected in plots with reduced insecticide treatment whereas another endangered species, E. mordax, was found in both variants (table 3). Recent studies on agro-ecosystems in Central Germany name these species as typical for the habitats (Volkmar & Freier, 2003; Zöphel & Kreuter, 2001). Their specific ecological needs can also be met in a varied agro-ecological environment. There were no significant shifts in dominance structures between the variants (table 4).

24

The four-year data indicate that the impact of insecticides on the spider population was the strongest the earliest insecticides were applied. These findings underline the propositions of Krause et al. (1993) and Dinter (1995) about lethal and sub-lethal effects of insecticides on open-habitat species. A 50% reduction of insecticide treatments showed positive effects on spider activity over 4 vegetation periods. The potential for natural pest regulation was in all cases higher in the 50% variants. Furthermore, the results on species diversity indicate that a 50% reduction led to a stable pattern of >15 spider species. Within that pattern, O. apicatus and the species of Erigone were the dominant species of the spider coenoses.

Table 4. Dominance and relation of male to female for the three most frequent species in catches of 2006 in both variants.

100 % 50 % Dominance Relation Dominance Relation (%) ♂♂ to ♀♀ (%) ♂♂ to ♀♀ Field 1 (summer wheat) Erigone atra 41.9 7.9 : 1 47.1 6.7 : 1 Oedothorax apicatus 32.1 1.3 : 1 28.5 1.1 : 1 Erigone dentipalpis 7.9 161.0 : 1 10.4 44.0 : 1 Field 2 (winter wheat) Oedothorax apicatus 49.4 1.1 : 1 61.3 1.0 : 1,3 Erigone atra 17.7 7.0 : 1 19.4 6.9 : 1 Erigone dentipalpis 7.2 18.5 : 1 5.1 32.0 : 1 Field 3 (peas) Oedothorax apicatus 61.0 3.7 : 1 56.1 1.2 : 1 Erigone atra 6.0 22.0 : 1 14.5 91.5 : 1 Erigone dentipalpis 14.6 61.0 : 1 Meioneta rurestris 7.5 17.0 : 1

In summary, a 50 % long-term reduction of chemical plant protection did have positive accumulated effects (e.g. on field 3) on spider coenoses.

References

Anonymus, 2004: Reduktionsprogramm Chemischer Pflanzenschutz. BMVEL (Hrsg.). Backhaus, G.F. Beer, H. Gutsche, V. Freier, B. 2005: Beiträge der Biologischen Bundes- anstalt zum Reduktionsprogramm chemischer Pflanzenschutz des Bundesministeriums für Verbraucherschutz, Ernährung und Landwirtschaft. Nachrichtenbl. Deut. Pflanzenschutzd. 57: 45-48. Dinter, A. 1995: Untersuchungen zur Populationsdynamik von Spinnen (Arachnida: Araneae) in Winterweizen und deren Beeinflussung durch insektizide Wirkstoffe. Cuvillier-Verlag Göttingen. Krause, U. Pfaff, K. Dinter, A. Poehling, H.-M. 1993: Nebenwirkungen von Insektiziden, vor allem Pyrethroiden, auf epigäische Spinnen bei der Bekämpfung von Getreideblattläusen. Agrarökologie, 1-147. 25

Heimer, S. & Nentwig, W. 1991: Spinnen Mitteleuropas. Verlag Paul Parey, Berlin und Hamburg. Platnick, M. J. 1993: Advances in spider taxonomy 1988-1991. Entomol. Soc. & Am. Mus. Nat. Hist., New York. Roberts, M. J. 1985: The spiders of Great Britain and Ireland. 1. Atypidae to Therodio- somatidae. Harley Books, Martins, Great Horkesley. Roberts, M. J. 1987: The spiders of Great Britain and Ireland. 2. Linyphiidae. Harley Books, Martins, Great Horkesley. Schumacher, K. 2007: Effekte einer reduzierten Dosis von Pflanzenschutzmitteln auf tritrophische Systeme im Ackerbau. Dissertation Mathemematisch-Naturwissenschaftliche Universität Potsdam. Volkmar, C. & Freier, B. 2003: Spinnenzönosen in Bt-Mais und nicht gnetisch veränderten Maisfeldern. Z. Pflanzenkrankh. u. Pflanzensch. 110: 572-582. Zöphel, B. & Kreuter, T. 2001: Nachwachsende Rohstoffe (Hanf, Flachs, Salbei und Kamille) Anbau und Bedeutung für den Lebensraum Acker in Sachsen. Sächs. Landesamt für Landw., Sächs. Landesamt f. Umwelt u. Geologie (Hrsg.): Sonderh.: 64 S. Pesticides and Beneficial Organisms IOBC/wprs Bulletin Vol. 35, 2008 pp. 26-31

Effects of different control measures against the olive fruit fly (Bactrocera oleae (Gmelin)) on beneficial arthropod fauna. Methodology and first results of field assays

Manuel González-Núñez1, Susana Pascual1, Elena Seris1, José R. Esteban-Durán1, Pilar Medina2, Flor Budia2, Ángeles Adán2, Elisa Viñuela2 1 Dpto. Protección Vegetal, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), Carretera de La Coruña Km 7,5, 28040 Madrid, Spain e-mail: [email protected] 2 Unidad de Protección de Cultivos. Escuela Técnica Superior de Ingenieros Agrónomos. Universidad Politécnica de Madrid (UPM). Ciudad Universitaria, s/n. 28040 Madrid, Spain

Abstract: Beneficial arthropod fauna was monitored in test plots of an olive grove in Madrid under four different control strategies against the olive fruit fly: trichlorfon bait sprays (trichlorfon + protein hydrolysate Nu-Lure®), spinosad bait sprays (Spintor Cebo®), kaolin sprays and mass-trapping (Easy- trap® + Nu-Lure®). A beating method was used to sample arthropods from the five plots along 2005. Bait treatments (spinosad or trichlorfon) and mass-trapping did not cause significant effects on populations of beneficial arthropods, but a reduction of parasitoids and predators was observed in samples from plots sprayed with kaolin. However, a longer time period of study will be necessary to confirm the effect of the different control strategies on the evolution of arthropod populations.

Key words: Bactrocera oleae, beneficial arthropods, olive, kaolin, mass-trapping, spinosad, predators, parasitoids, side effects, bait sprays

Introduction

Olive crop is generally characterized for being a well balanced agroecosystem, because crop protection treatments have traditionally been applied at a very low intensity, compared to other tree crops. This explains why, in spite of having 225 potential damaging species described, only twelve of them are able to cause economically important damage (Haniotakis, 2005). This gives an idea of the importance of preserving auxiliary fauna, which naturally controls these high numbers of phytophagous insects, otherwise probably becoming olive crop pests. The olive fruit fly (Bactrocera oleae (Gmelin) Dipt., Tephritidae) is the most important pest in olive groves, causing every year important quantitative and qualitative losses in the main olive crop areas. In olive IPM programs bait sprays and mass trapping are recommended against this pest (Haniotakis, 2005; Johnson et al., 2006). Bait sprays, also called adulticide or preventive treatments, consist of the application of an attractant and insecticide mixture on the foliage to reduce the number of adult flies before they lay eggs on fruits. Protein hydrolysates are usually the attractants, and insecticides commonly used are organophosphates (dimethoate, trichlorfon, malathion...). To minimize the ecological impact, only a small part of the foliage is treated (aerial treatments in bands or land treatments in "spots" on the south face of the canopy) and also other active ingredients less harmful to the environment are being introduced in bait sprays, such as the microbial insecticide spinosad (Ruiz Torres et al., 2004). However, it is necessary to study how these treatments damage beneficial arthropods in the field.

26 27

Mass trapping consists of traps placed at a high density to attract and kill the highest possible number of adults (females preferred), reducing in this way the attack levels until acceptable values. There are different devices applied to capture B. oleae, using normally food attractants (protein hydrolysates from different sources and ammonium salts) (Ros et al., 2003). There is a growing interest in developing this method because of its environmental advantages, so it is almost the only control method available for this pest which fulfils the requirements established for the Organic Production of olive oil (CEC, 1991; Lentini et al., 2005). However, mass trapping devices can also attract and kill many non-target arthropods, but this negative effect must be evaluated. Finally, protective barriers of kaolin (calcined, purified and fine-grained clays) have been tested against olive pests in the last few years with good results. (De la Roca Ranz, 2003; Saour & Makee, 2004). These studies seem to demonstrate that kaolin has an important repellent effect on egg laying by olive fruit fly and olive moth (Prays oleae (Bernard)), reducing the number of affected fruits. Kaolin has the important environmental advantage of being an inert product and non toxic to vertebrates but its effects on beneficial fauna have not been assessed yet in olive groves. Field assays are carried out in an olive grove in Madrid to investigate the effects of bait sprays, mass trapping and protective barriers of kaolin on beneficial arthropods. In this paper we describe the methodology we used and the results obtained during the first year of the field trial.

Material and methods

Test plots The field trial was conducted on test plots of about 0,8 ha (80 trees) under four different pest control strategies against the olive fruit fly: trichlorfon bait sprays, spinosad bait sprays, kaolin sprays and mass-trapping, plus a control plot without any control measure. No differences between plots were observed before treatment applications in the parameters measured and then it was possible to use a single plot per treatment. Thus the plot size was big enough to reduce the effect of insect movements between plots (treatments). The olive grove used is placed in Villarejo de Salvanés (Central Spain, Madrid) and the main olive variety is “Manzanilla”.

Treatments Trichlorfon bait spray, the standard control method for B. oleae in this area, was applied following the regular practice of local olive growers: spray applied on 2-3 m2 spots on the south-eastern face of the tree, using two component mixture in water: Dipagrex 80® (trichlorfon 80%, DEQUISA, Paterna, Valencia, Spain) at 5 g/l plus the protein hydrolysate Nu-Lure® (Miller Chemical & Fertilizer Corp., Hanover, Pensylvania, USA) at 10 g/l. These spray treatments are only applied when pest levels reach treatment thresholds according to the Spanish monitoring web of olive pests (“Red DACUS”) of the "Program for the improvement of the olive oil quality" (Civantos & Caballero, 1993). In the year of study (2005) only one treatment was necessary, at the beginning of October. Spinosad bait spray was applied using Spintor Cebo® also named GF-120® (Dow AgroSciencies, Indianapolis, USA) a special formulation developed for bait sprays against fruit flies, with its own food attractant. Spintor Cebo®, was sprayed diluted in water (1:6), in 70-100 cm diameter spots on the south-eastern face of the tree and in wide-size drops (5 mm in diameter approximately) to increase persistence. One application was done at the same time as the trichlorfon bait spray. Surround WP® (kaolin 95%, Engelhard Corp., Iselin, New Jersey, USA) was sprayed as a suspension in water at 30 g/l, covering the total foliage of olive trees. To ensure covering of 28

olives during the whole campaign two applications were done: the first one at the beginning of summer, when olives begin to be susceptible of attack by B. oleae, and the second one in September, when an important decrease of coating was detected due to rain and the growth of olives. In the mass-trapping plot one Easy-trap® (Utiplas, S.L., Móstoles, Madrid, Spain) was installed per tree, from the beginning of July to the end of November, the period when olives are vulnerable to B. oleae attack. Traps were baited with an aqueous solution of the protein hydrolysate Nu-Lure® (9%) and borax (3%) as a preservative and pH stabilizer. This mixture was replaced every other week. In the control plot, no measures against B. oleae were applied. No other insecticides were utilized, except for one treatment with Bacillus thuringiensis, applied to all test plots when the populations of Prays oleae (Bernard) reached the treatment threshold.

Monitoring predators and parasitoids To compare abundance of predators and parasitoids in the different test plots, arthropod fauna of the canopy of olive trees was monitored periodically using a beating method similar to those proposed by Morris & Campos (1996) or Müther & Vogt (2003)). It consisted of tapping strongly the branches with a stick and collecting the fallen organisms in a bag by means of a 47 cm diameter funnel. Sampling was normally done every other week in summer and autumn and monthly in spring and winter. In every sampling four olive trees were randomly chosen per plot and four branches per tree (one in each orientation) were beaten three times. Arthropods fallen from each tree were collected in a bag and placed in a portable refrigerator and afterwards, in the laboratory, the bags were kept in the freezer. Then the samples were cleaned and captured specimens were classified in four groups: phytophagous on olive trees, predators, parasitoids and other.

Statistical analysis Numbers of predators plus parasitoids captured in each sampling date from the five treatment plots were compared using one-way analysis of variance (ANOVA) and a Tukey test to find significant differences (P≤0.05). Statistical tests were performed using the software ® Statgraphics Centurion XV (StatPoint, 2005).

Results and discussion

The evolution of numbers of predators plus parasitoids sampled along the year from the different treatment plots is shown in Fig. 1. Specimens belonging to groups of predators and pasasitoids have been considered as a whole because the number of species registered was extremely high and statistical analysis on data of single species was impracticable. Bait treatments (spinosad and trichlorfon) were applied once late in the season and did not cause significant effects on populations of beneficial arthropods. Also no differences were observed between the mass-trapping and the control plots. However in the plot treated with kaolin a significant reduction in numbers of beneficial arthropods was registered at one date in autumn. Toxicity of spinosad to many beneficial insects has been reported (Cisneros et al., 2002; Williams et al., 2003). However, in laboratory studies this insecticide formulated as a bait caused negligible mortality of important predators, such as coccinellids (Michaud, 2003; Medina et al., 2004) or chrysopids (Contreras et al., 2005). Although parasitoids seem to be more sensitive to spinosad, Stark et al. (2004) found that the parasitoids of tephritid fruit flies Fopius arisanus (Sonan) and Pysttalia fletcheri (Silvestri) did not feed on the bait. Also, it is advantageous for natural enemies that when spinosad is applied in the field, its toxic effects 29

are short lived due to its low persistence (Miles, 2006). Furthermore, bait sprays only affect a small part of the foliage, so most of beneficial arthropods can survive and recolonize treated spots. In our field tests no significant difference in captures was found between control, spinosad bait and trichlorfon bait, but, due to the unusually low level of B. oleae populations, only a single bait treatment was applied this year, whereas two or three applications are normally needed per year.

Mass trapping

nº of predators + parasitoids ¹ Date

Figure 1. Mean numbers of predators plus parasitoids captured along 2005 in different treatment plots. Arrows indicate treatment applications and the mass-trapping period is also shown. A significant difference with the control plot is indicated () (ANOVA, Tukey, P ≤ 0.05).

Mass-trapping devices capture mainly flying arthropods and some of them, such as Tachinidae, Neuroptera and Hymenoptera, usually play important roles regulating pest populations. However, in this study no differences in the number of natural enemies sampled in control and mass-trapping plots were found. This can be explained because beating is not the best method for monitoring flying insects (Müther & Vogt, 2003), so a direct assessment of arthropods captured by traps is necessary. The difference between numbers of predators plus parasitoids captured in October in control trees (15,75 ± 4,13 specimens per tree) and in trees after two kaolin sprays (2,5 ± 0,65 specimens per tree) could indicate a very drastic reduction in populations in the kaolin plot. But at the end of the crop season this difference disappears. In apple orchards, Knight et al. (2001), Markó et al. (2006) and Sackett et al. (2007) also found that some groups of beneficial arthropods were significantly affected by kaolin. However, in the case of predators, it was not clear, if this decrease in predator abundance in kaolin trees was caused by a reduction in prey numbers or by kaolin directly affecting the arthropod or its predation ability. In our case, a longer time period of study will be necessary to confirm the effect of kaolin on populations of beneficials and to investigate its possible relation with changes in populations of phytophagous species. Random variation in the field trial was very high, resulting in high values of standard deviations, which could prevent us from finding treatment effects. To solve this inconvenience an increase in the number of samples per sampling date would be desirable, 30

although it would then be necessary to reduce the number of sampling dates. Also a deeper knowledge on the side effects of treatments could be gained by analyzing separately data from the most relevant species of predators and parasitoids.

Acknowledgements

This study was supported by the Spanish Ministry of Education and Culture by means the research projects AGL2004-07516-C02-01/AGR and AGL2004-07516-C02-02/AGR.

References

CEC (Council of the European Communities), 1991. Council Regulation (EEC) No. 2092/91 on organic production of agricultural products and indications referring thereto on agricultural products and foodstuffs. Official Journal of the European Communities L 198, 22/07/1991, pp. 1-15. Cisneros, J., Goulson, D., Derwent, L. C., Penagos, D. I., Hernández, O. & Williams, T. 2002: Toxic effects of spinosad on predatory insects. Biol. Control 23: 156-163. Civantos, M. & Caballero, J.M. 1993: Integrated pest management in olive in the Mediterranean area. Bulletin OEPP/EPPO Bulletin 23: 367-375. Contreras, G., Medina, P., Adan, A., Zapata, A. N., Viñuela, E. 2005: Effects of modern bait formulated pesticides on larvae and adults of Chrysoperla carnea under extended- laboratory conditions. IOBC/wprs Bull. 28(7): 245-250. De la Roca Ranz, M. 2003: Surround®, crop protectant: la capa protectora natural para cultivos como el olivar. Phytoma España 148: 83-85. Haniotakis, G. 2005: Olive pest control: Present status and prospects. IOBC/wprs Bulletin 28 (9): 1-9. Johnson, M.W., Zalom, F.G. Van Steenwyk, R., Vossen, P., Devarenne, A.K., Daane, K.M., Krueger, W.H., Connell, J.H., Yokoyama V., Bisabri, B., Caprile, J. & Nelson, J., 2006: Olive Fruit Fly management guidelines for 2006. Uc. Plant Protection Quarterly 16 (3): 1-7. Available online: www.uckac.edu/ppq/Issues.htm Knight, A.L., Christianson, B.A. & Unruh, T.R. 2001: Impacts of seasonal kaolin particle films on apple pest management. Can. Entomol. 133: 413-428. Lentini, A., Delrio, G. & Foxi, C. 2005. Experiments for the control of olive fruit fly in organic agriculture. IOBC/wprs Bulletin 28 (9): 73-76. Markó, V., Blommers, L.H.M., Bogya, S. & Helsen, H. 2006: The effect of kaolin treatments on phytophagous and predatory arthropods in the canopies of apple trees. Journal of Fruit and Ornamental Plant Research 14 (Suppl. 3): 79-87. Medina, P., Pérez, I., Budia, F., Adán, A. & Viñuela, E. 2004: Development of an extended- laboratory method to test novel insecticides in bait formulation. IOBC/wprs Bull. 27(6): 59- 66. Michaud, J.P. 2003: Toxicity of fruit fly baits to beneficial insects in citrus. J. Insect. Sci.. 3(8): 9 pp. www.insectscience.org/papers/2003/ Miles, M. 2006: The effects of Spinosad on beneficial insects and mites used in integrated pest management systems in greenhouses. IOBC/wprs Bulletin 29(10): 53-59. Morris, T.I. & Campos, M. 1996: A hybrid beating tray. Entomologist 15: 20-22. Müther, J. & Vogt, H. 2003: Sampling methods in orchard trials: a comparison between beating and inventory sampling. IOBC/wprs Bulletin 26(5): 67-72. Ros, J.P., Castillo, E. & Blas, P. 2003: Estudio de la eficacia atractiva de diferentes sustancias y mosqueros hacia la mosca del olivo Bactrocera oleae Gmel. Bol. San. Veg. Plagas 29: 405- 31

411. Ruiz Torres, N., Madueño Magdalena, C. & Montiel Bueno, A. 2004: Efectividad de trata- mientos cebo terrestres con Spinosad e Imidacloprid contra la Mosca del Olivo (Bactrocera oleae, Gmel., Diptera: Tephritidae). Resultados preliminares. Bol. San. Veg. Plagas 30: 415-425. Sackett, T. E., Buddle C. M. & Vincent, C. 2007: Effects of kaolin on the composition of generalist predator assemblages and parasitism of Choristoneura rosaceana (Lep., Tortricidae) in apple orchards. J. Appl. Ent. 131(7): 478–485. Saour, G. & Makee, H. 2004: A kaolin-based particle film for suppression of olive fruit fly Bactrocera oleae Gmelin (Dipt., Tephritidae) in olive groves. J. Appl. Ent. 128: 28-31. Stark, J. D., Vargas, R., & Miller, N. 2004: Toxicity of spinosad in protein bait to three economi- cally important tephritid fruit fly species (Diptera: Tephritidae) and their parasitoids (Hymenoptera: Braconidae). J. Econ. Entomol. 97(3): 911-915. StatPoint 2005: The user’s guide to Statgraphics® Centurion XV. StatPoint, Inc. USA. www.statgraphics.com Williams, T., Valle, J. & Viñuela, E. 2003: Is the naturally-derived insecticide Spinosad com- patible with insect natural enemies? Biocontrol Sci. Tech. 13: 459-475.

Pesticides and Beneficial Organisms IOBC/wprs Bulletin Vol. 35, 2008 p. 32

Impact of Success Bait (a.i. spinosad) against Rhagoletis cerasi on insect fauna in field test

Božena Barić¹, Marijana Pauković², D. Bertić¹, Ivana Pajač¹ ¹ Faculty of Agriculture, Department of Agricultural Zoology, 10 000 Zagreb, Svetošimunska 25. Croatia, E-mail: [email protected] ² student of Faculty of Agriculture

Abstract: Cherry fruit fly is the most important pest of cherries in Croatia. Pest control against the cherry maggot in commercial orchards is difficult because of cherry fruits ripening time and long life of insecticides. In Croatia organophosphate insecticides and synthetic pyrethroids are recommended for the control of Rhagoletis cerasi. In the frame of IOBC definition for Integrated Production, priority should be given to ecologically safer methods, minimizing the undesirable side effects to enhance the safeguards to the environments and human health. Spinosad is one of the Naturalytes that is recommended against Vine moths in Croatia. Plant protection against fruit flies in IPP uses bait and kill technique as ecologically safer method. Succes Bait contains 0.24 g/l a.i. spinosad and 99.76 g/l inert ingredient (water, sugar, bait) is registered for control of Med fly (Ceratitis capitata), in citrus orchards and olive fruit fly (Bactrocera oleae), on olives in Croatia. We wanted to investigate the efficacy of Success Bait against Rhagoletis cerasi and its advantages in comparison with common insecticides against European cherry fruit fly. The investigation was carried out in a commercial sour cherry orchard of Borinci near Vinkovci, Slavonia County in Croatia. The experimental plot treated with Success Bait was 0.5 ha in size and consisted of eight rows of sour cherry trees. Success Bait was applied with a hand-operated knapsack sprayer at a rate of 1 liter per hectare diluted in 20 l/ha water. Around the plot, one branch of each tree was treated, in the middle of the plot the same treatment was done on each tree every other row. The first application took place on 24th of May, the second on the 1st of June. The other part of the cherry orchard (25 ha) was treated with organophosphate insecticides (a.i. dimethoate) (OP) and synthetic pyrethroids (a.i. lambda-cyhalotrin). The first treatment took place on 25th May (a.i. dimethoate) and the second on 8th of June (a.i. pyrethroid). Efficacy was calculated by examination of 300 cherries per plot. In the untreated sour cherry orchard, percentage of infested fruits ranged from 10% to 20%. In the plot treated with insecticides and in the experimental plot treated with Success Bait no infested fruits were detected. The fauna observation was carried out in both plots eight days after treatment by branch-beating methods. One sample consisted of thirty-three beats. One sample per plot was collected. More beneficial insects were present in the part where Success Bait was used than in the part where OP insecticide and pyrethroid were used. In experimental plot with Success Bait we found 47 insects from 9 families (Coccinellidae, Ichneumonidae, Braconidae, Formicidae, Curculionidae, Scolytidae, Cicadidae, Corticariinae, Forficulidae). In the orchard part where insecticides were used only five insects were trapped from five families (Coccinellidae, Elateridae, Cicadidae, Psyllidae and Braconidae). The investigation has to repeated next year on larger plots and with much more fauna examinations.

32 Pesticides and Beneficial Organisms IOBC/wprs Bulletin Vol. 35, 2008 pp. 33-34

Effects of bait sprays to control the European cherry fruit fly (Rhagoletis cerasi L.) on aphid predators

Heidrun Vogt & Kirsten Köppler

Julius Kuehn Institute (JKI) - Federal Research Centre for Cultivated Plants, Institute for Plant Protection in Fruit Crops and Viticulture, Schwabenheimer Straße 101 69221 Dossenheim, Germany. E-mail: [email protected]

Abstract: The European cherry fruit fly (Rhagoletis cerasi L.) is the most serious pest of cherries in Europe. For long years broad spectrum and acute toxic insecticides, mainly organophosphates, have been used for its control. In search of environmentally safer control methods, good results have been obtained in first experiments using bait sprays (Köppler, Storch & Vogt 2006; Köppler, Vogt & Storch 2008; Köppler, Kaffer & Vogt 2008). They consist of a mixture of food baits (proteins, sugar) with small amounts of insecticides and are only applied to parts of the tree canopy. To study the effects of these bait sprays on beneficial insects, we carried out laboratory, extendend laboratory and semi-field tests, according to IOBC methodology. We used a 20% solution of GF-120™ Naturalyte Fruit Fly Bait (a.i. 0.02 % spinosyn A and D; water, sugars, proteins 99,98%) and a self made Neem bait, containing 5 % NeemAzal®-T/S (a.i. 10g/l Azadirachtin), mixed in a sugar-brewers yeast solution (4:1:7). Test organisms were Chrysoperla carnea and Coccinella septempunctata. In laboratory tests (exposure on glass arenas to 5 x 5 µl bait trops, food ad libitum), the effects (according to Abbott) on lacewing larvae were low, whereas survival and development of coccinellid larvae to healthy adults was reduced by 50% in the GF-120 treatment and by 100% in the neem treatment. Survival of adult lacewings, exposed for 3 days to 5 x 5 µl bait trops and provided with food supply, was reduced by 40 % in the GF-120 treatment, whereas no mortality was observed in the neem treatment. Both bait treatments did not affect adult coccinellids within 3 days, when these were exposed in the same way as the lacewings adults. In order to imitate more realistic conditions, extended lab tests with bean plants were carried out. Trays with 24 bean plants, 6 of these treated with the bait spray were used as test arena. The bean plants were infested with pea aphids (Acyrthosiphon pisum) as food supply. The experiment was run in a greenhouse. GF-120 reduced larval development of C. carnea to healthy adults by 44 %, the neem bait by 40 %. Both treatments resulted in lower egg production: neem exposed adults produced 39%, GF-120 exposed adults 20% less fertile eggs. The same test with coccinellid larvae resulted in very harmful effects of the neem bait (98% Abbott), whereas GF-120 was harmless. When the number of bean plants treated with neem bait was reduced to 1, 2, or 3 plants of 24, Abbott mortalities of the coccinellids amounted to 77.4, 88.7 and 96.2 %, respectively. A further test with coccinellid larvae following this scheme was run as semi-field test under partial exposure to outdoor conditions in July. In this test, we used neem baits with lower concentration of the insecticide, i.e. 0.5 %, 1 % in comparison to the 5% mixture, and only 2 bean plants of 24 were treated. Still, the neem bait caused high mortalities > 90 %.

References

Köppler, K., Storch, V. & Vogt, H. 2006: Bait Sprays – an alternative to control the European cherry fruit fly Rhagoletis cerasi? In: 12thInternational Conference on Cultivation Techniques and Phytopathological Problems in Organic Fruit Growing. Proceedings to the Conference from 31st

33 34

January to 2nd February 2006 at Staatliche Lehr- und Versuchsanstalt für Wein- und Obstbau Weinsberg, Germany, 61-66. Köppler, K.,Vogt,H. & Storch, V. 2008: Bait Sprays to Control the European Cherry Fruit Fly Rhagoletis cerasi. IOBC Meeting “Integrated Plant Protection in Stone Fruit Orchards”, Balandran, France, 2-4 October 2006. In press: IOBC/wprs Bulletin 37. Köppler, K., Kaffer, T. & Vogt, H. 2008: Bait sprays against the European cherrry fruit fly Rhagoletis cerasi: Status quo & perspectives. In: 13th International Conference on Cultivation Techniques and Phytopathological Problems in Organic Fruit Growing. Proceedings to the Conference from 19th to 20th Feb. 2008 at Staatliche Lehr- und Versuchsanstalt für Wein- und Obstbau, Weinsberg, Germany, 102-108 Pesticides and Beneficial Organisms IOBC/wprs Bulletin Vol. 35, 2008 pp. 35-39

Earwigs in fruit orchards: phenology predicts predation effect and vulnerability to side-effects of orchard management

Bruno Gobin1, Rob Moerkens², Herwig Leirs² and Gertie Peusens1 1 Zoology Department, pcfruit, Fruittuinweg 1, B-3800 Sint-Truiden, Belgium 2 Evolutionary Biology Group, University of Antwerp, Groenenborgerlaan 171, B-2020 Antwerp, Belgium

Abstract: Earwigs, Forficula auricularia, are important natural enemies of a variety of orchard pests. In recent years, numbers of earwigs have declined in both organic and IPM orchards. To understand what factors affect earwig population dynamics, we conducted a detailed phenological survey of earwigs in orchards. Earwigs were counted in artificial refuges in orchard trees. Earwigs appear in the trees from the beginning of June onwards as third instar nymphs, moult into fourth instar nymphs 3 weeks later and reach adulthood early July. At this point, earwigs show an inexplicable reduction in population. Adults remain present until end of October. In most orchards, a small second brood is produced in summer, and this has a positive impact on population size in fall. Comparison of earwig and pest phenologies show that earwigs play important roles in controlling summer pests rather than spring pests. Earwigs are at risk for side-effects of foliar spray applications from June to October, and for soil management or herbicide treatments in winter and early spring.

Key words: Forficula, life cycle, beneficial arthropod, pest control, apple, pear

Introduction

Earwigs are generalists that feed on a variety of plant material, mosses or fungi, and small arthropods (Phillips, 1981). They are important predators of a number of top fruit pests, such as aphids, (Buxton and Madge, 1976; Noppert et al., 1987; Phillips, 1981), Psyllids (Lenfant et al., 1994; Phillips, 1981), scale insects (Karsemeijer, 1973; McLeod and Chant, 1952), and spider mites (Phillips, 1981). These studies clearly demonstrated predatory effects in the laboratory, while only for four of these orchard pests, some studies attempted to demonstrate efficacy in more practically relevant semi-field or field trials. The best documented predatory effect of earwigs is that toward the woolly apple aphid Eriosoma lanigerum, a major pest in apple orchards with integrated or organic pest management. Excluding earwigs leads to woolly aphid proliferation which is negatively correlated with the number of earwigs still present or released on the tree (Mueller et al., 1988; Nicholas et al., 2005). Likewise, earwigs consume high numbers of Psyllid eggs when confined in sleeves on branches in a semi-field setting (Lenfant et al., 1994). Field control of three more pest species was not clearly demonstrated: the same research team found contradictory results in controlling the green apple aphid Aphis pomi (Carroll and Hoyt, 1984; Carroll et al., 1985), while a small scale field study on the apple-grass aphid Rhopalosiphum insertum and the spider mite Panonychus ulmi could not demonstrate any effect of earwig presence (Phillips, 1981). Earwigs have a single generation per year, which makes them especially vulnerable to repetitive disturbance during orchard management. Nevertheless, during their phenology, earwigs move in different strata of the orchards (tree vs. soil), each with specific risks of side- effects. Females lay eggs in an underground nest during late winter or early spring, expel the males and provide broodcare to eggs and the first nymph stage. Females abandon the nests

35 36

when nymphs become second instars, these nymphs will disperse shortly thereafter and move to weeds, shrubs or trees. Especially around early summer (end of june, early july) abundance of late instar earwigs is present in orchard trees. Adults are found from July onwards and males and females form pairs in fall and hibernate as pairs in underground nests. Although the general life cycle of earwigs is well understood, most quantitative studies on earwig presence in orchards are limited to summer occurrence only, as earwigs are most abundant in July. To understand what limits the presence of earwigs in orchards, we conducted a detailed survey of earwig presence covering the whole season. To manage a univoltine species such as F. auricularia a good knowledge of its life history is indeed required. The species was recently identified with molecular techniques as a complex of at least two sibling species, named species A and B (sensu Wirth et al., 1998). These species have distinct life history strategies, having either a single or two broods, and likely a slightly different timing of onset of egg- laying in winter. During this survey, it became evident that, although earwigs are great predators, their phenology does not always co-incide with that of damaging pests. We discuss their potential as a natural enemy by comparing pest phenologies to earwig phenologies. On the other hand, as earwigs move into the trees quite late in the year, they are less at risk from side-effetcs of foliar treatments. Knowledge on the exact timing of appearance can be helpful in deciding ultimate application timings of crop protection agents with potential side-effects (see Peusens and Gobin, p. 40-43 this issue, for details on side-effect monitoring).

Material and methods

Earwig counting During daytime, earwigs readily hide in artificial shelters. Shelters consisted of corrugated cardboard rolls inserted in a Styrofoam coffee cup for rain protection and attached horizontally to a strong branch with iron wire. We counted earwig numbers from each of two shelters placed in 10 to 20 trees per orchard. Nymphal stages were differentiated by size and antennal segments; male and female adults based on sexual dimorphism in cerci. Once counted, earwigs were released on the assessed tree and the shelter was returned to the same location within the tree. Earwigs were counted at least once a week, starting upon appearance in the trees (start of June) until adults migrate to the soil (end of October). Sampled orchards were a mix of IPM and organic orchards. Pest phenologies Pest phenology data were counted as averages from the 50-year database of the Gorsem Fruit Research Centre (now pcfruit). Each year, appearance dates for most life stages of orchard pests are noted, either from direct searches in orchards or from systematic trap monitoring.

Results and discussion

Earwig phenology Earwig larvae appear in the fruit trees from the end of May onwards. We observed very few L2 nymphs in the cardboard shelters in the trees. Once earwigs reach the third nymphal stage, they gradually became more abundant in the trees (Figure 1). The discrepancy between the peak of 3rd and 4th nymphal stages suggests that younger nymphs still dwell on the ground. Addition of shelters on the soil indeed enhanced the capture of 3rd instar nymphs. The fourth nymphal stage is the most abundant in apple and pear. A sharp, and as yet unexplainend, decline in numbers was observed at the timing of moulting of L4 into adults, 37

around mid July. Adults appear end of July. In most orchards, earwigs produce a second brood, with a rather small but positive contribution to the earwig population (Figure 1). The orchards lacking a second brood – thus clearly having only the single brood species – show a gradual decline towards fall. It is as yet unclear whether the other orchards have a mixture of both single and double brood species or solely the double brood species. Earwigs are thus at risk of foliar spray applications from june onwards. All earwigs are likely at risk from some of the organophosphate, pyrethroid and carbamate insecticides (Epstein et al. 2000, Peusens and Gobin 2008). Juveniles are prone to disruption by IGR’s such as diflubenzuron (Ravensberg 1981). The months June and July pose the highest risk with the large peak of nymphs in this period. We have to point out that for a univoltine species such as the earwig, even the smallest side-effect affects the population for the rest of the year.

35 L3 30 L4 adult total 25

20

15

10 number of earwigs per tree earwigs per of number

5

0 6-6 4-7 7-8 4-9 12-6 19-6 26-6 10-7 17-7 25-7 31-7 14-8 22-8 29-8 13-9 20-9 3-10 17-10 30-10 Figure 1: Average numbers of 3rd and 4th instar nymphs (L3, L4) and adults in apple and pear orchards in Belgium in 2006

Predation potential To estimate the predation potential of earwigs on the main orchard pests, we roughly correlated earwig phenology with pest phenology as was observed at the Gorsem research station for the past 50 years (Table 1). The phenology of earwigs is ideal to help control summer and fall generations of E. lanigerum in apple and C. pyri in pear. Exclusion experiments in apple orchards indeed showed that these pests increase when earwigs are absent (Mueller et al., 1988; Nicholas 2005). Earwigs are also likely to assist in controlling summer generations of leafrollers and various Lepidopteran pests. The potential earwig impact on codling moth (C. pomonella) will be minor, as only eggs and recently hatched larvae are exposed. Once the codling moth larvae penetrated the fruit skin, it is well protected from earwig predation within the fruit, and be only at risk again in a late stage when the bore hole is large. Earwigs come too late in the trees to control most aphids, but might in some 38

years assist in cleaning up colony remnants in June. This explains the variability in earwig control of green apple aphids described from successive field trials (Carroll & Hoyt, 1984; Carroll et al., 1985). Even when earwigs can consume red spider mite eggs (Phillips 1981), we believe the impact of earwigs on that pest to be minor compared to its control with predatory mites, the most proliferate beneficial in the majority of belgian apple orchards.

Table 1: The control potential of F. auricularia for major top fruit pests in Belgium, estimated by linking earwig and pest phenologies.

Main Top Fruit Pest Presence Potential Forficula control and remarks Pest species (damaging stage) Various aphids March to June weak Nymphs (L3) might assist cleaning up colony remnants in June Dysaphis plantaginea March to May no Earwigs still underground Eriosoma lanigerum Resume activity: March no Earwigs still underground, unknown whether they feed on root colonies Main migration: Mid May no Earwigs come too late to prevent migration Summer population yes Earwigs assists Aphelinus in controlling Eriosoma by mid July and throughout August Fall population yes Earwigs are main natural enemy at this point Scale insects Lepidosaphis ulmi Year-round, migration likely Migration is too early, but earwigs can May likely feed on settled scales Psyllids Year-round yes Mainly summer generations Cacopsylla pyri 2nd generation: May-June weak Earwigs too late to prevent massive egg- laying and hatching, but L4 feed on larval stages 3d generation onwards yes Earwigs can feed on eggs and eclosed larva until October Lepidopteran pests Year-round partly Summer generations of caterpillars on leaves Adoxophyes orana 1st generation: April no Earwigs still underground 2nd generation: August yes only adult earwigs, low numbers of nymphs Cydia pomonella May to August weak Presence coincides but likely only eggs and recently hatched larvae predated by earwigs

Acknowledgements

This research was funded by IWT Agronomic Research grant N° 04-667. 39

References

Buxton, J.H., & Madge D.S. 1976: The evaluation of the European Earwig (Forficula auricularia) as a predator of the damson-hop aphid (Phorodon humuli). I. Feeding experiments. Ent. exp. & appl. 19: 109-114. Carroll, D.P., & Hoyt S.C.. 1984: Augmentation of European earwigs (Dermaptera: Forficulidae) for biological control of apple aphid (Homoptera: Aphididae) in an apple orchard. J. Econ. Entomol. 77: 738-740. Carroll, D.P., J.T.S. Walker, & Hoyt S.C. 1985: European earwigs (Dermaptera : Forficulidae) fail to control apple aphids on bearing apple trees and woolly apple aphids (Homoptera : Aphididae) in apple rootstock stool beds. J. Econ. Entomol. 78: 972-974. Epstein, D.L.; Zack, R.S.; Brunner, J.F.; Gut, L. & Brown, J.J. 2000: Effects of broad spectrum insecticides on epigeal arthropod biodiversity in Pacific Northwest apple orchards. Environ. Entomol. 29(2): 340-348. Karsemeijer, M.M.D. 1973: Observations on the enemies of the oyster shell scale, Lepidosaphes ulmi, on apple in The Netherlands. J. Plant. Pathol. 79: 122-124. Lenfant, C., A. Lyoussoufi, X. Chen, F.F. D'Arcier, & Sauphanor B. 1994: Potentialités prédatrices de Forficula auricularia sur le psylle du poirier Cacopsylla pyri. Entomol. Exp. & Appl. 73: 51-60. McLeod, J.H., & Chant D.A. 1952: Notes on the parasitism and food habits of the European earwig, Forficula auricularia L. (Dermaptera: Forficulidae). Can. Entomol. 84: 343-345. Mueller, T.F., L.H.M. Blommers, & Mols P.J.M. 1988: Earwig (Forficula auricularia) predation on the woolly apple aphid, Eriosoma lanigerum. Entomol. exp. & appl. 47: 145-152. Nicholas, A.H., R.N. Spooner-Hart, & Vickers R.A. 2005 Abundance and natural control of the woolly aphid Eriosoma lanigerum in an Australian apple orchard IPM program. BioControl 50: 271-291. Noppert, F., J.D. Smits, & Mols P.J.M. 1987: A laboratory, evaluation of the European earwig (Forficula auricularia L.) as a predator of the woolly apple aphid (Eriosoma lanigerum Hausm.). Mededeling Faculteit Landbouwwetenschappen Universiteit Gent 52: 413-422. Peusens, G. & Gobin, B. 2008: Side effects of pesticides on the European earwig Forficula auricularia L. (Dermaptera: Forficulidae). IOBC/wprs Bull. 35: 40-43. Phillips, M.L. 1981: The ecology of the common earwig Forficula auricularia in apple orchards. PhD Thesis, University of Bristol: 1-241. Ravensberg, W.J. 1981: The natural enemies of the woolly apple aphid, Eriosoma lanigerum (Hausm.) (Homoptera: Aphididae), and their susceptibility to diflubenzuron. Med. Fac. Landbouww. Rijksuniv. Gent. 46: 437-441. Wirth, T., R. Le Guellec, M. Vancassel, and M. Veuille. 1998: Molecular and reproductive characterization of sibling species in the European earwig (Forficula auricularia). Evolution 52: 260-265. Pesticides and Beneficial Organisms IOBC/wprs Bulletin Vol. 35, 2008 pp. 40-43

Side effects of pesticides on the European earwig Forficula auricularia L. (Dermaptera: Forficulidae)

Gertie Peusens and Bruno Gobin pcfruit Gorsem, Zoology Department, Fruittuinweg 1, B-3800 Sint-Truiden, E-mail: [email protected]

Abstract: Earwigs are key predators of orchard pests, but show large inter-orchard differences in population dynamics and numbers. In apple and pear orchards, only sufficiently large earwig populations can contribute to pest control. As earwigs have a single generation per year, a potential effect of pest management is likely to influence population dynamics. In an extended lab test the side effect of dried residue of 37 registered plant protection products (twenty-seven insecticides, 1 biological insecticide, 2 oils, 1 surfactant, 1 protectant and 1 herbicide) were evaluated on adult F. auricularia L.. Classified upon their Mode of Action (MoA) 9 of the 17 tested classes of insecticides proved to be harmless, 7 slightly and 1 moderately harmful. For various test products sub-lethal effects such as reduced co-ordination, spastic behaviour were noticed shortly after application. Depending on the active ingredient the earwigs either recovered or died eventually. We still need to verify the validity of our results in a replicated trial and hope to extend the test to juvenile stages and field test.

Key words: Forficula auricularia, insecticides, beneficial

Introduction

In integrated and organic pest management of fruit crops the use of selective plant protection products has become mandatory. To obtain a registration of a compound in Belgium side effect studies of pesticides on non-target arthropods (beneficial organisms) are required and need to be conducted according to standard guidelines (Candolfi et al., 2000; Hassan et al., 1985). European earwigs are important predators of orchard pests, they are capable of keeping the infestation degree of several orchard pests below economic threshold levels, like scale insects (Karsemeijer, 1973; McLeod and Chant, 1952), the apple aphid (Carroll and Hoyt, 1984), codling moth (Glenn, 1977) and spider mites (Phillips, 1981). These results could not always be confirmed however under field circumstances except for some studies on woolly apple aphid Eriosoma lanigerum (Mueller et al., 1988; Nicholas et al., 2003) and pear sucker Cacopsylla pyri (Sauphanor et al., 1994). Perhaps one of the reasons why the biological control isn’t always successful could be a cumulative effect of, even slightly, harmful side effects of subsequent applications of pesticides, especially when the beneficial organism is a univoltine species as the earwig. Knowledge of the influence of the active ingredients on the earwig is very important in order to be able to choose the best selective product. As these data are only available for a few compounds an extended lab test was executed to test the side effects of registered pesticides.

Material and methods

Male and female adult earwigs were collected from organic fruit orchards at the end of summer, mixed together in a large rearing container with cardboard refuges. Earwigs were

40 41

reared in a climatic growth chamber (temperature 12°C, humidity 60% RH, photoperiod 12/12 h light/dark) with ad libitum access to food (crushed cat food) and water. Test units were prepared by filling petri-dishes with a thin layer of hydrophilic cotton and moistened after which a pre-cut circle of a broad bean leave (Phaseolus vulgaris L.) was placed on top of the cotton. Thirty-three plant protection products (twenty-seven insecticides, 1 biological insecticide, 2 oils, 1 surfactant, 1 protectant and 1 herbicide) were applied as formulated product at registered dose rates (in Belgium) using calibrated spraying equipment with one central nozzle. All compounds were diluted in deionised water for application at a water volume rate of 400 L/ha to avoid pooling on the leave. To obtain the same amount deposit as in a field application the dose rates were corrected. A control item (deionised water) was included to assess the natural mortality of the test insects. Per test compound 10 female and 10 male earwigs were chosen randomly and exposed to dried fresh (1-2 hours) residue. The earwigs were added individually to a treated test unit with a darkened lid, no food or water was provided. All replicates were kept under controlled conditions (temperature 16°C, humidity 60% RH, photoperiod 12/12 h light/dark) after 5 days the earwigs were transferred to a rearing unit (petri-dish with plaster and a black lid) together with food and water (freshened weekly). Lethal effects on the insects were assessed 5, 12, 20, 26, 34 days after treatment by recording data on their condition as alive (showing normal behaviour), affected (upright and attempting to walk but with reduced co-ordination or spastic behaviour), moribund (on their back, twitching, unable to right themselves) or dead (verified by touching). We used the common practice of pooling dead and moribund individuals as dead and alive and affected individuals as alive to calculate the % reduction compared to the water-treated control according to Abbott (Abbott, 1925) We have to point out that this is a rather simple approach for a univoltine species, as some „affected“ earwigs show such severe sub-lethal symptoms that they are not likely to survive under natural conditions. We however adopt this approach until the impact of these behavioural changes is confirmed in further trials. Side effects were categorized according to the IOBC-classification for lab trials: 1 = harmless (<30% effect), 2 = slightly harmful (30-79% effect), 3 = moderately harmful (80- 99% effect), 4 = harmful (>99% effect).

Results and discussion

The side effect of the tested pesticides (classified in MoA-classes (IRAC, 2007)) on F. auricularia is given in Table 1. Mortality in the control group was low at the beginning but increased during the observation period: 0% at 0 DAT (days after treatment), 2.5% 12 DAT, 5% 20 and 26 DAT, 7.5% 34 DAT. Nine of the 17 represented classes of insecticides proved to be harmless to adults (carbamates, juvenile hormone mimics, feeding blockers, mite growth inhibitors, microbial disruptors, organotin acaricides, benzoylurea, diacylhydrazines, tetronic acid derivatives) as well as the biological insecticide, the paraffin oils, the surfactant, the protectant and the herbicide. Seven classes were slightly harmful to adults (organophosphates, pyrethroids, pyrethrins, neonicotinoids, avermectines, METI-acaricides, oxadizines) and 1 class moderately harmful (spinosyns). Side effects, slightly or moderately, didn’t appear shortly after the application but occurred only after a longer period (min. 12 days). Remarkably were the paralysing effects (60%-90%) of some products within the first days of observation causing up to 50% mortality after 34 days. Our results show lower toxicity to adults for some classes as known for juvenile stages (Sauphanor and Stäubli, 1994; Sauphanor et al., 1993; Sterk et al., 1999). We aim to extend our screening to include juvenile stages. Of course, due to recent regulatory restrictions, the 42

Table 1. Results of extended lab test: IOBC classes of side effect of pesticides (classified according to IRAC) on F. auricularia L. after exposure of adult earwigs to dried residue on leaves.

MOA-classes # Tested 5 12 20 26 34 products DAT DAT DAT DAT DAT Carbamates 1 1 1 1 1 1 Organophosphates 1 1 1 1 2 2 Pyrethroids 4 1 2 2 2 2 Pyrethrins 1 1 2 2 2 2 Neonicotinoids 3 1 1 1 2 1 Spinosyns 1 1 2 2 3 3 Avermectins 1 1 1 1 2 2 Juvenile hormone mimics 1 1 1 1 1 1 Feeding blockers 1 1 1 1 1 1 Mite growth inhibitors 2 1 1 1 1 1 Microbial disruptors 1 1 1 1 1 1 Organotin acaricides 2 1 1 1 1 1 Benzoylurea 1 1 1 1 1 1 Diacylhydrazines 2 1 1 1 1 1 METI-acaricides 3 1 1 1 2 2 Oxadizines 1 1 1 2 2 2 Tetronic acid derivatives 1 1 1 1 1 1 Biological insecticide 1 1 1 1 1 1 Paraffin oil 2 1 1 1 1 1 Surfactant 1 1 1 1 1 1 Protectant 1 1 1 1 1 1 Herbicide 1 1 1 1 1 1

test compounds of each group are milder than some of the test compounds used in these early studies. Also, as we disregard “affected” individuals in our Abbott calculation, we might understimate the effects for adults. Affected earwigs probably are more vulnerable to predation and dehydration and thus have less chance to survive. A simple inclusion of the category “affected” in mortality would give a worst-case scenario that needs to be compared with more realistic field exposure. We have to bear in mind that univoltine species as the earwig have a different vulnerability and recovery time than multi-generation species. Repeated application of plant protection products belonging to even “slightly harmful” classes may have a large impact on earwigs. Therefore it is crucial to avoid sensitive periods in earwig phenology (see Gobin et al. 2008, for details), i.e. products affecting juveniles pose more risk in late spring.

Acknowledgements

The Institute for the Promotion of Innovation by Science and Technology of Flanders (IWT) supported this work financially by an agricultural research grant 040667.

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References

Abbott, W.S. 1925: A method for computing the effectiveness of an insecticide. Journal of Economic Entomology 18: 265-267. Candolfi, M.P., S. Blümel, R. Forster, F.M. Bakker, C. Grimm, S.A. Hassan, U. Heimbach, M.A. Mead-Briggs, B. Reber, R. Schmuck, and Vogt, H. 2000: Guidelines to evaluate side-effects of plant protection products to non-target arthropods. IOBC/WPRS, Gent. Carroll, D.P. and Hoyt, S.C. 1984. Augmentation of European earwigs (Dermaptera: Forficulidae) for biological control of apple aphid (Homoptera: Aphididae) in an apple orchard. Journal of Economic Entomology 77: 738-740. Glenn, D.M. 1977: Predation of codling moth eggs, Cydia pomonella, the predators responsible and their alternative prey. Journal of Applied Ecology 14:445-456. Gobin, B., Moerkens, R., Herwig Leirs, H. & Peusens, G. 2008: Earwigs in fruit orchards: phenology predicts predation effect and vulnerability to side-effects of orchard management. IOBC/wprs Bull. 35: 35-39. Hassan, S.A., F. Bigler, P. Blaisinger, H. Bogenschütz, J. Brun, P. Chiverton, E. Dickler, M.A. Eaterbrook, P.J. Edwards, W.D. Englert, S.I. Firth, P. Hunag, C. Inglesfield, F. Klingauf, C. Kühner, M.S. Lediueu, E. Naton, P.A. Oomen, W.P.J. Overmeer, P. Plevoets, J.N. Reboulet, W. Rieckmann, L. Samsoe-Peterson, S.W. Shires, A. Stäubli, J. Stevenson, J.J. Tuset, G. Vanwetswinkel, and Van Zon, A.Q. 1985: Standard methods to test the side-effects of pesticides on natural enemies of insects and mites developed by the IOBC/WPRS Working Group "Pesticides and Beneficial Organisms". EPPO Bulletin. 15: 214-255. IRAC 2007: Mode of Action Classification Scheme v5.3, July 2007. http://www.irac-online.org/ documents/IRAC%20MoA%20Classification%20v5_3.pdf. Karsemeijer, M.M.D. 1973: Observations on the enemies of the oyster shell scale, Lepidosaphes ulmi, on apple in The Netherlands. Journal of Plant Pathology 79: 122-124. McLeod, J.H., and Chant, D.A. 1952: Notes on the parasitism and food habits of the European earwig, Forficula auricularia L. (Dermaptera : Forficulidae). Canadian Entomologist 84: 343-345. Mueller, T.F., L.H.M. Blommers, and Mols, P.J.M. 1988: Earwig (Forficula auricularia) pre- dation on the woolly apple aphid, Eriosoma lanigerum. Entomologia Experimentalis et Applicata 47: 145-152. Nicholas, A.H., R.N. Spooner-Hart, and Vickers, R.A. 2003: Control of woolly aphid, Eriosoma lanigerum (Hausmann) (Hemiptera: Pemphigidae) on mature apple trees using insecticide soil-root drenches. Australian Journal of Entomology 42: 6-11. Phillips, M.L. 1981: The ecology of the common earwig Forficula auricularia in apple orchards. Thesis. University of Bristol. Sauphanor, B. and Stäubli, A. 1994: Evaluation au champ des effets secondaires des pesticides sur Forficula auricularia et Anthocoris nemoralis: validation des resultats de laboratoire. IOBC/WPRS Bulletin 17 (10): 83-88. Sauphanor, B., L. Chabrol, F. Faivre d'Arcier, F. Sureau and Lenfant, C. 1993: Side effects of diflubenzuron on a pear psylla predator: Forficula auricularia. Entomophaga 38: 163-174. Sauphanor, B., C. Lenfant, E. Brunet, F.F. D'Arcier, A. Lyoussoufi, and Rieux, R. 1994: Régula- tion des populations de psylle du poirier, Cacopsylla pyri (L.) par un predateur generaliste, Forficula auricularia. IOBC/WPRS Bulletin 17 (2): 125-131. Sterk, G., S.A. Hassan, M. Baillod, F. Bakker, F. Bigler, S. Blümel, H. Bogenschütz, E. Boller, B. Bromand, J. Brun, J.N.M. Calis, J. Coremans-Pelseneer, C. Duso, A. Garrido, A. Grove, U. Heimbach, H. Hokkanen, J. Jacas, G.B. Lewis, L. Moreth, L. Polgar, L. Roversti, L. Samsoe- Petersen, B. Sauphanor, L. Schaub, A. Stäubli, J.J. Tuset, A. Vainio, M. van de Veire, G. Viggiani, E. Vinuela and Vogt, H. 1999: Results of the seventh joint pesticide testing programme of the IOBC-WPRS-working group "Pesticides and Beneficial Organisms". BioControl 44: 99-117. Pesticides and Beneficial Organisms IOBC/wprs Bulletin Vol. 35, 2008 pp. 44-50

About the presence and abundance of beneficials in overwintering sites of Anarsia lineatella (Lepidoptera: Gelechiidae) in peach orchards of northern Greece

Petros Damos and Matilda Savopoulou-Soultani Aristotle University of Thessaloniki, Faculty of Agriculture, Laboratory of Applied Zoology and Parasitology, 54 124, Thessaloniki, Greece, e-mail: [email protected]

Abstract: A report is given about the presence and abundance of beneficials in overwintering sites of Anarsia lineatella Zeller (Lepidoptera, Gelechiidae). The study was conducted in two important regions of peach production in Northern Greece (Veria 40.32oN and Velvendo 40.16oN). For 3 years (2005-2007) hibernacula of overwintering larvae were collected from conventional and IPM peach orchards and transferred to the laboratory in order to ascertain the level and type of beneficial activity. The presence of two Braconid parasitoids was high, causing a significant high larval mortality. In some cases almost 57% of inspected samples were parasitized. In addition, a comprehensive number of beneficial mites were also observed inside the hibernacula. Despite the fact that some of them are not directly linked to the predation of A. lineatella, they had a high presence during the years. Moreover, most of the observed species belonged to the families Phytoseiidae, Pyemotidae and Tydeidae. The observations attest the fact that the overwintering sites of A. lineatella constitute an important microenviroment of beneficial activity. Considering the increasing interest in biological control and that all the above-mentioned beneficials are subjected to mortality induced by pesticides, the different strategies for the control of A. lineatella in Northern Greece peach orchards are discussed.

Key words: Anarsia lineatella, parasitoids, hibernaculum, Braconidae

Introduction

Sustaining biological diversity has become one of the principal goals of conservation and gradually the goals have moved from concern for specifically threatened species to the broader desire to protect different types of ecosystems (Lewis and Whitfield 1999). In particular for agroecosystems, the identification and conservation of native species potentially important as bio-control agents, along with the development of protocols to test side effects of pesticides on non target and beneficial organisms, are considered as a corner stone when enhancing biological control in integrated pest management (IPM) (Carl 1996, Norris et al. 2003). The peach twig borer Anarsia lineatella Zeller (Lepidoptera: Gelechiidae) is one of the major economic pests of stone fruits in the Old and New World (Balachowski and Mesnil 1935, Jones 1935, Summers 1955). In Greece, A. lineatella has 3-4 generations per year (Damos and Savopoulou-Soultani 2007). The species overwinters in bark crevices as 2nd or 3rd instar forming hibernacula. These tiny instars become active in spring and they are able to cause early season injury burrowing into new twigs. Later during summer, new hatched larvae originating from next generations, feed mainly on fruits causing significant injury on yield (Balachowski 1935, Jones 1935, Bailey 1948, Summers 1955, Balachowski 1966).

44 45

In Northern Greece peach production is considered to be essential for economy and during the past few years efforts are made to improve pest control using IPM in order to qualify high standards of products. Moreover, A. lineatella has been increasingly damaging to some peach varieties, and along with the oriental peach moth Grapholitha molesta (Lepidoptera: Tortricidae), they are key pests for implementation of effective control strategies in terms of IPM (Kyparissoudas 1989, Damos and Savopoulou Soultani 2007, Damos and Savopoulou-Soultani 2008). This study reports the presence and abundance of a number of beneficial species, inside of hibernacula of A. lineatella in IPM and conventional orchards. In addition, the substantial mortality caused by the parasitoids on the overwintering larvae is also recorded. Finally the effects on the abundance and diversity of the observed beneficials are outlined in respect to IPM.

Materials and methods

The faunistic survey was conducted in two important regions of peach production in Northern Greece (Veria 40.32oN and Velvendo 40.16 oN). For 3 years (2005-2007) hibernacula of overwintering larvae were collected randomly from conventional and IPM peach orchards. Commercial orchards (4 plots of 1.5 acre each) were treated according to local pest manage- ment guidelines (all chemical treatments conducted using broad spectrum conventional pesticides), while IPM peach orchards (3 plots of 1.5 acre each) followed pest management practices under the guidelines of public IPM companies’ specialists. Decision making for pesticide application in IPM orchards was based on male moth flight monitoring with pheromone traps (Pherocon®) and degree-days heat accumulation. Environmentally sound pesticides, mostly Bacillus thuringiensis (B.t.) and insect growth regulators (IGR’s), were used for the IPM orchards. Finally, peach orchards (4 plots of 1 acre each) where no chemical treatment was applied were used as control. Each hibernaculum was considered as a sampling unit and the day of sampling all the collected material was transferred to the laboratory and examined. Each sampling unit was split open under a stereoscope and inspected in order to ascertain the level and type of beneficial activity. Larval mortality caused by parasitoids or predators was also recorded. Data analysis Analysis of variance (ANOVA) was carried out to test the effects of year and management practices on the different dependent variables of abundance. Frequency data were transformed to meet assumptions of normality and variance homogeneity if necessary. Means were compared using the Tukey multiple range test (a = 0.05) (Sokal and Rohlf 1995). Statistics were performed using SPSS 1.14. software (SPSS 2006).

Results

Two parasitoid species were reared from the overwintering larvae of A. lineatella. These species belong to the Braconidae family (Hymenoptera) and caused, in some cases, a significant mortality of A. lineatella larvae.

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Conventional IPM Control

2005

25% 36% 38% 41% 58% 40% 17% 21% 7% 14% 3%

2006 40% 45% 29% 34% 63% 40% 8% 18% 3% 18% 2% 0%

2007 14% 46% 40% 24% 40% 40% 57% 5% 12% 7% 13% 2%

Braconidae Tydeidae

Phytoseiidae Pyemotidae

Fig 1. Relative abundance (proportion) of beneficials inside of hibernacula of A. lineatella in relation to orchard management in Northern Greece (2005, 2006 and 2007).

In addition, the overwintering larval parasitization was especially high during the winter of the year 2006-2007. Moreover, the mortality of A. lineatella, which was due to Braconidae parasitization was significantly higher in the peach orchards, where no chemical treatment was applied (control). Parasitization level was also higher in most of the IPM orchards (10- 23%), when compared with the conventional orchards, where the usual conventional plant protection guidelines were applied (~ 10%) (Table 1). A comprehensive number of predatory mites was recorded inside of the hibernacula. The mite species belong to the Tydeidae, Pyemotidae and Phytoseiidae families. In addition, the mean number of individuals belonging to the Tydeidae family was higher when compared to those of the families Phytoseiidae and Pyemotidae (Table 1).

47

Table 1. Hibernacula (n) of overwintering larvae of A. lineatella and corresponding number of beneficials from Conventional, IPM and Control peach Orchards

Beneficial Activity (mean number of individuals) Larval

Orchard Parasiti- Family Manage zation ment

Year (n) df Tydeidae Pyemotidae Phytoseiidae Braconidae (% )

2005 143 3 4 0 2 8 11 a1 Conven- tional 2006 187 3 7 0 0.3 7.4 10 a 2007 141 3 3 1 4 9 21 ab 2005 140 2 5 0 2 7 16 ab IPM 2006 190 2 16.8 0.3 0.6 9 10 ab 2007 140 2 11 0.5 3 10.3 23 b 2005 143 3 12 1 4 12 28 bc Control 2006 145 3 11 2 3 2 57 c 2007 142 3 11 2 3 12 28 bc

1 Percentages followed by the same letter within a column are not significantly different (p<0.05, Tukey HSD test)

The type of orchard management had no effect on the relative diversity of the beneficials that were recorded. During all years and under all different management strategies, beneficials belonging to all the above four families were collected (Fig.1).

Discussion

The lower number of beneficials recorded in the conventional peach orchards contrasted with higher populations of some IPM orchards and indicate that the present orchard pest management practices may interfere with the establishment and diversity of beneficials, although differences were not significant in all cases. A current theory suggests that the highest species diversity should be found in relatively undisturbed to moderate disturbed habitats (Petraitis et al. 1989), but if the disturbance is too severe or too frequent, species may be lost from the community (Lewis and Witfield 1999). Daane et al. (1993), demonstrated that dormant-season sprays with an organophosphate insecticide (diazinon) for the control of A. lineatella leads to a significant decrease on the related parasitoid microfauna. Consequently, the status of a pest in relation to its natural enemies within an agroecosystem is not fixed and it depends on a number of factors, among them type, time and frequency of pesticide treatments (Easterbrook et al. 1985). Braconidae, in general, constitute one of the most species-rich families of insects, estimated by the taxonomists to have 40-50.000 species worldwide. The vast majority of Braconids are primary parasitoids of other insects, especially of larval stages of Coleoptera, Heteroptera, Diptera and Lepidoptera (Askew 1971). Their natural role as potential biological agents is indisputable because of their ability to keep insect and other arthropod populations 48

under control through predation or parasitism (Hanson 1995, Hanson and Gauld 1995, LaSalle and Gauld 1993). Balachowski (1966) referred to a number of Hymenoptera parasitoids of A. lineatella. The species Apanteles emarginatus Nees, A. xanthostigmus Hal. var: anarsiae, Paralitomastix (Encyrtus) variicornis Nees, Paralitomastix (Copidosoma) pyralidis Ashm and Euderus (Secodella) cushmani Crowford, were the most common. In previous studies, Daane et al. (1993) reported the presence of one Braconidae species (unidentified) and also the species Macrocentrus ancylivorus Rowher, Spilochalcis n. sp. aff tovrina (Cresson), Paralitomastix pyralidis (Ashmead), Erynnia totricis (Coquillett) and Euderus cushmani Crawford parasitizing A. lineatella, while Molinari et al. (2005) report three parasitoid species from overwintering A. lineatella larvae, the Ichneumonidae Aethecerus discolor Wesmel, the Braconidae Baeognatha armeniaca Talenga and the Encyrtidae Paralitomastix variicornis Nees, which caused a significant larval mortality. The role of mites (acari) for pest control is discussed in detail by Gerson et al. (2003). On the other hand, a recent survey of the mite fauna in a large number of peach orchards in northern Greece revealed Euseius finlandicus (Acari: Phytoseiidae) as the dominant predatory species. Moreover, the overwintering Phytoseiidae females were mostly found in bark crevices of the hibernation cocoons of the peach moth Adoxophyes orana (Lepidoptera: Tortricidae) (Broufas et al. 2002). This study reveals that mite fauna expansion occurs also inside the hibernacula of A. lineatella. Furthemore, in some cases a limited number of Pyemotidae individuals was also recorded. The species most commonly referred to in bibliography is Pyemotes tritici which parasitizes about 150 insect species and P. ventricosus (Cross et al. 1975). Finally, Tydeids are very common and fast moving inhabitants of plant and soil, where they subsist on many plant and animal foods. Some species like Proctotydeus spp. live on insects (Kaźmierski 1998) and some other proved to be important in suppressing Eriophyiidae species (Hessein and Pering 1986). However, the nature of the relationship of these species with plant or insect hosts is still unclear (Gerson 2003). In northern Greece, the implementation of IPM in peach orchards has made a number of advances. Automatic data and monitoring systems have been installed in most representative regions. Phenological models are already developed and applied for the control of A. orana (Milonas and Savopoulou-Soultani 2000, Milonas and Savopoulou-Soultani 2006). Moreover, reliable intervention thresholds are established for A. lineatella and species can be successfully managed in terms of IPM using both monitoring and degree-days as a decision tool for targeting a particular developmental stage (Damos and Savopoulou-Soultani 2007). If proper time application is achieved, environmentally sound insecticides such as diflubezuron and Bacilus thuringiensis are applied, with high success and resulting in a significant decrease in the total number of sprays. In contrast, pest control in conventional peach orchards depends on the phenological stage of the peach-host. As a result, in most cases, preventive chemical treatments are applied without evident necessity. However, peach growers face difficulties in practice when trying to apply alternatives. Firstly, because of the near absence of registered narrow-spectrum substances in the market. Thus, most of the pesticides used are toxic or very persistent and do not fulfil the requirements in order to be used in integrated fruit production (Cross and Dickler 1994, Malavolta et al. 2003, Milonas and Savopoulou-Soultani 2000, Milonas and Savopoulou- Soultani 2006). Secondly, specific environmental conditions in Greece (e.g. prolonged high mean temperatures and relative humidity during the spring and summer season) as well as pesticide resistance, can cause pest population outbreaks. Thus, pest populations outbreaks are very difficult to be controlled without the use of conventional broad-spectrum pesticides (Cravedi and Jörg 1995, Cravedi 2000). As a result, the natural enemies action can be 49

disrupted extensively also in IPM orchards as well and this trend could explain the low number of beneficials that were observed during the field trial in some cases of IPM orchards. Moreover, the peach production landscape constitutes a mosaic of IPM and conventional orchards, where it is difficult to establish long scale IPM strategies. Finally, it is noticeable that in some cases evaluating the action of natural enemies is difficult, uncertain and demonstrating little practical relevance (Cravedi 2000). However, it is part of IPM aims to shift the system progressively from reliance on chemical control to reliance mostly on biological control. Additionally, species that appear as well established indigenous predators in the fruit growing areas of northern Greece should be brought in as potential biocontrol agents.

References

Askew, R. R. 1971: Parasitic Insects. New York: Elselvier. Balachowski, A. & Mesnil, L. 1935: Les insects nuisibles aux plantes cultivées. Traité d’Entomologie agricole concernant la France, la Corse, l’Afrique du Nord et les Régions limitrophes. Paris: 442-449. Balachowski, A. S. 1966: Entomologie appliqué a l’agriculture. Traité. Tome II. Lepidoptères. Masson et Cie éditeurs, Saint Germain Paris: 442. Bailey, S. F. 1948: The peach twig borer. Calif. Agric. Exp. Stn. Bull. 708: 3-56. Broufas, G. D., Koveos D. S. & Georgatsis, D. I. 2002: Overwintering sites and winter mortality of Euseius finlandicus (Acari: Phytoseiidae) in peach orchards in northern Greece. Experimental and Applied Acarology 26: 1-12. Carl, K. 1996: Ecological studies and prospects for classical biological control of apple pests in Europe and elsewhere. IOBC/wprs Bulletin 19(4): 67-74. Cravedi, P. & Jörk E. 1996: Special challenges for IFP in stone and soft fruit. International conference on intergrated fruit production. IOBC/wprs Bulletin 19(4): 48-56. Cravedi, P. 2000: Integrated peach production in Italy: Objectives and criteria. Pflanzen- schutz-Nachrichten Bayer 53: 177-197. Cross, J. & Dickler, E. (Eds.) 1994: Guidelines for integrated production of pome fruits in Europe. Technical Guideline III, 2nd Edition. IOBC/wprs Bulletin 17(9). Cross, W.H., McGovern, W. L. & Cross, E. A. 1975: Insect host of the parasitic mites called Pyemotes ventricosus (Newport). J. Geor. Entomol. Soc. 10:1-8. Daane, K. M., Yokota G. Y. & Dlott J. 1993: Dormant-season sprays affect the mortality of peach twig borer (Lepidoptera : Gelechiidae) and its parasitoids. J. Econ. Ent. 86: 1679- 1685. Damos, P. & Savopoulou-Soultani, M. 2008: Préférence de Ponte par Anarsia lineatella (Lepidoptera: Gelechiidae) sur le pêcher et des autres hôtes en laboratoire. In: Proceedings of IOBC Workshop on Integrated Stone Fruit Production, October 2006, Balandran, France. IOBC/wprs Bulletin. In press. Damos, P. & Savopoulou-Soultani, M. 2007: Flight patterns of Anarsia lineatella (Lepidoptera: Gelechiidae) in relation to degree – days heat accumulation in northern Greece . Comm. Appl. Biol. Sci. Ghent University. 72: 465-468. Easterbrook, M. A., Solomon, M. G., Cranham, J. E. & Souter, E. F. 1985: Trials of an intergrated pest management programme based on selective pesticides in English apple orchards. Crop. Prot. 4: 215-230. Gerson, U., Smiley, L. R. L. & Ochoa, R. 2003: Mites (Acari) for Pest Control. U.K., Blackwell Science: 539 pp. 50

Hanson, P. E. 1995: Economic importance of Hymenoptera. In: The Hymenoptera of Costa Rica. Hanson P. E. and Gaud I. D., Eds, Oxford University Press, New York: 89-101. Hanson, P. E. & Gauld, I.D. 1995: The biology of Hymenoptera. In: The Hymenoptera of Costa Rica. Hanson P. E. and Gaud I. D., Eds, Oxford University Press: 20-88. Hessein, N. A. & Pering, T. M. 1986: The importance of alternate foods for the mite Homeopronematus anconai (Acari: Tydeidae). Ann. Entomol. Soc. Am. 81: 488-492. Jones, L. S. 1935: Observations of the habits and seasonal life history of Anarsia lineatella in California. J. Econ. Ent. 28: 1002-1011. Kaźmierski, A. 1998: A review of the genus Proctotydeus (Actinedida: Tydeidae: Pronemat- inae). Acarologia 39: 33-47. Kyparissoudas, D. S. 1989: Simoultaneous control of Cydia molesta and Anarsia lineatella in peach orchards of Northern Greece by combining mating disruption and insecticide treatments. Entomologia Hellenica 7: 13-16. LaSalle, J. & Gauld, I. D. 1993: Hymenoptera: their diversity, and their impact on the diversity of other organisms. In: Hymenoptera and Biodiversity. Eds. J. LaSalle, ID Gauld. Wallingford, UK: CAB Int.: 1-26. Lewis, C. N. & Witfield, J. B. 1999: Braconid wasp (Hymenoptera: Braconidae) diversity in forest plots under different silvicultural methods. Environ. Entomol. 28: 986-997. Malavolta, C., Cross, J.V., Cravedi, P. & Jörg, E. 2003. Guidelines for integrated production of stone fruits (2nd edition). IOBC/WPRS Bulletin 26 (7). Milonas, P. G. & Savopoulou-Soultani, M. 2000: Development, survivorship, and reproduc- tion of Adoxophyes orana (Lepidoptera: Totricidae) at constant temperatures. Ann. Entomol. Soc. Am. 93: 96-102. Milonas, P. & Savopoulou-Soultani, M. 2006. Seasonal abundance and population dynamics of Adoxophyes orana (Lepidoptera: Totricidae) in northern Greece. International Journal of Pest Management 52 (1): 45-51. Molinari, F., Chiapini, E. & Sambado, P. 2005: Preliminary investigation on the natural enemies of the peach twig borer Anarsia lineatella in Northern Italy. IOBC/wprs Bulletin 28(7): 135-138. Norris, R. F., Caswell-Chen, E. P. & Cogan, M. 2003: Concepts in IPM. Ecosystem bio- diversity and IPM. Prentice Hall. Petraitis, P. S., Latham, R. E. & Niesenbaum, R. A. 1989: The maintenance of species diversity by disturbance. Quart. Rev. Biol. 64: 393-418. Summers, F. M. 1955. The peach twig borer. Calif Agric. Exp. Stn. Circ. 449. Sokal, R. R. & Rohlf, F. J. 1995: Biometry 3rd ed. Freeman, New York. SPSS. 2006. I, VERSION 1.14. SPSS, Chicago IL.

Pesticides and Beneficial Organisms IOBC/wprs Bulletin Vol. 35, 2008 pp. 51-59

Is the use of some selected insecticides compatible with two noctuid endoparasitoids: Hyposoter didymator and Chelonus inanitus?

Pilar Medina1, José Javier Morales1, Manuel González-Núñez2, Elisa Viñuela1

1 Unidad de Protección de Cultivos. Escuela Técnica Superior de Ingenieros Agrónomos. Universidad Politécnica de Madrid (UPM). Ciudad Universitaria, s/n. 28040. Madrid. España. E-mail: [email protected] 2 Dpto. Protección Vegetal. Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA). Carretera de La Coruña Km 7,5. 28040-Madrid. Spain.

Abstract: Studies were conducted in the laboratory to evaluate the toxicity of three insecticides (imidacloprid, fipronil and natural pyrethrins+piperonyl butoxide) at field rates on pupae and adults of Hyposoter didymator and Chelonus inanitus, both of them solitary endoparasitoids of several noctuid larvae. Topical application on pupae and residual, topical and ingestion bioassays on adults of the two parasitoid species were used to assess percentages of adult emergence and life-span, in case of treated pupae and life-span for adults. Out of the three compounds tested, only fipronil significantly reduced the life-span of emerged adults, after topical treatment of H. didymator and C. inanitus pupae (90 and 75%, respectively). The life-span of treated adults was significantly reduced in both parasitoid species by all the insecticides tested irrespective of the uptake route, with the exception of C. inanitus adults treated with imidacloprid. Fipronil was clearly the most toxic insecticide.

Key words: Hyposoter didymator, Chelonus inanitus, fipronil, imidacloprid, natural pyrethrins +piperonyl butoxide.

Introduction

About 10% of the horticultural crop production in Spain is concentrated in the South-eastern region, in Almeria province, where the surface of protected crops yields about 37.500 ha (MAPA, 2007). Predominant species over a range of horticultural insect pests include the noctuids Autographa gamma (Linnaeus), Chrysodeixis chalcites (Esper), Helicoverpa armigera (Hübner), Spodoptera littoralis (Boisduval) and Spodoptera exigua (Hübner). The main strategy to control these pests has traditionally been the use of insecticides and this has resulted in the development of high levels of resistance, especially within the genus Spodoptera (Smagghe et al., 1999). An alternative control tactic that could mitigate the development of resistance is the use of natural enemies into an integrated pest management (IPM) program. Moreover, in the horticultural greenhouses, there is an important incidence of virus diseases transmitted by vectors as Bemisia tabaci Gennadius, Trialeurodes vaporari- orum (Westwood) or Frankliniella occidentalis (Pergande). The surface under biological control has sharply increased in the last year in Spain because, in the frame of the National Programme for Controlling Insect Vectors of Virosis, most of the measures aim to improve the biological control of vector insects by the use of plant protection products harmless to native as well as introduced beneficials (BOE, 2004). Hyposoter didymator (Thünberg) (Hym.; Ichneumonidae) and Chelonus inanitus L. (Hym.; Braconidae) are two solitary koinobiont endoparasitoids of noctuid larvae of a great

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variety of field and protected crops in many Spanish regions, the latter being more restricted to the genus Spodoptera (Jones, 1985). Both of them depend upon its host larva for food and shelter to complete its development from the embryo to the third instar and then, they emerge from the host and spin a silk cocoon (Grossniklaus-Bürgin et al., 1994). C. inanitus females lay eggs in a host egg mass whereas H. didymator parasitizes only larvae. Among the insecticides that can be applied in crops where noctuids and their parasitoids are also present, we have chosen those representatives of groups with different and, in two cases, relatively novel mode of action. Fipronil is a phenylpyrazole that blocks the GABA- regulated chloride channels of the central nervous system resulting in neural excitation and insect death (Cole et al., 1993). Imidacloprid is a chloronicotinyl insecticide that acts as nicotinic acetylcholine receptor agonist and is widely used to control sucking pests (Elbert et al., 1998). Natural pyrethrins, with excellent properties due to their knock-down effect on insects and low mammalian toxicity, are commercialized at present with addition of PBO (piperonyl butoxide), which has a synergic activity (Philogène et al., 2002). Monitoring natural enemy populations before and after application of insecticides in the field is the best method for evaluating the impact caused on insects, although it is either simple nor cost-effective. Techniques as topical application, exposure to fresh residues of insecticides or ingestion treatments can help to estimate hazard of these insecticides on natural enemies in the field (Tillman & Mulrooney, 2000). The goals of this paper were 1) to examine differential effects of several insecticides on pupae and adults of the parasitoids H. didymator and C. inanitus, because both of them are considered as important control agents of noctuid pests that cause severe losses in different horticultural crops; 2) to enhance our knowledge about the impact of different uptake routes of some currently used pesticides on the parasitoids; 3) to study different susceptibilities to the insecticides between the two parasitoids.

Materials and methods

Insect rearing A laboratory colony of H. didymator was established in 1998 from parasitized larvae of Heliothis peltigera (Denis & Schiffermüller) found in pine nurseries at Bilbao (north Spain). To avoid the bias towards males (Schneider & Viñuela, 2007), genetic variability was increased by regular infusion of individuals collected on cotton in Sevilla (south Spain) and a population from the Institut National de la Recherche Agronomique (I.N.R.A., Montpellier, France). C. inanitus are the progeny of a rearing kept in the Division of Developmental Biology (Berna University, Switzerland). Both adult wasps were kept separately in plastic cylinder cages (30 cm high, 20 cm diameter) with a cotton sleeve on both sides for ventilation, at a density of 100 males and 100 females each for H. didymator and just the half for C. inanitus. Wasps were fed with pure honey applied by a brushstroke on a plastic surface placed on the bottom of rearing cages. Water was provided in drinking troughs. H. didymator was routinely reared on third instar larvae of S. littoralis according to Schneider & Viñuela, (2007), as this instar was the best to obtain the higher numbers of females and diminish the risk of encapsulation. C. inanitus was offered single layers of host eggs patches for parasitization. S. littoralis larvae were mass-reared and fed on an artificial diet (modified from Poitout and Bues, 1974). The rearing was started with individuals kindly given by the Public University of Navarra, Spain; and it was renewed with individuals from University of Cordoba and University of Almeria, Spain.

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Insects were maintained in a growth chamber at standard conditions of 25 ± 2 ºC, 75 ± 5 RH and a 16:8 (L:D) h photoperiod. All the experiments were conducted at the same conditions. Insecticides The commercial products Regente® (80% fipronil, Basf), Confidor® (20% imidacloprid, Bayer), Pelitre Hort® (4% natural pyrethrins + 16% piperonyl butoxide, C.Q. Massó), were applied at the maximum recommended field rates in Spain. Fresh solution of every insecticide were prepared prior to the experiments. Water was replaced by acetone in topical treatments. A summary of the insecticides and concentrations used in the experiments is given in Table 1.

Table 1. Summary of the insecticides and concentration used in the experiments.

Insecticide Commercial Concentration Dose (mg a.i./l) (µg a.i./insect) Fipronil Regente® 80 WG 30 0.03 Imidacloprid Confidor® 20 SL 150 0.15 NP + PBO1 Pelitre Hort®4+16 SC 80+320 0.08+0.32 1Natural pyrethrins+piperonyl butoxide

Pupae bioassays A topical bioassay was conducted to determine the contact toxicity of the insecticides to parasitoid pupae. H. didymator and C. inanitus pupae (<48 h old) were collected from stock rearing, placed in ventilated plastic cages in groups of 5, and individually treated with 1 µl of the corresponding insecticide solutions using a Burkard hand microapplicator (Burkard, UK). Ventilated boxes consisted of a plastic jar (∅: 11 cm, h: 5 cm), kept in place with the lid which had a 5 cm diameter hole covered with a gauze to facilitate ventilation. Five replicates of five pupae per insecticide and control were used for the bioassay. Healthy pupae of H. didymator and C. inanitus need about 7 and 10 days in average, respectively, to become adults at the environmental conditions mentioned above. As such, more than a week after treatment, adult emergence) was scored and percentages calculated on basis of the total of treated individuals. Insects were maintained in the same cages described above and provided with food and water, added two days before probable emergence date, to assure adult survival. Furthermore, life-span (the mean time that all members of a replicate remain alive) was measured. Toxicity bioassays on adults Adults of both parasitoid species were exposed to the maximum field rate of insecticides using different uptake routes: residual, topical and ingestion applications. Groups of 10 males and 5 females less than 48 h-old per replicate, and 5 replicates per insecticide and control, were used in each type of treatment for H. didymator. Longevity of H. didymator males is lower than that of females (Schneider & Viñuela, 2007), so in experiments, a higher number of males was always used in order to have enough couples in case it could be necessary to study the influence of the insecticide on the parasitoid reproduction. Ten adults per replicate were used for C. inanitus. The evaluation of results on adult bioassays was based on life-span.

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Residual bioassay To evaluate residual contact activity, a group of adults per replicate was kept in glass dismountable cages consisting of two glass plates and a round glass frame (modified from Jacas & Viñuela, 1994). They were provided with honey and water. Plates were treated under a Potter precision spray Tower with a standard deposit of 1.45 ± 0.2 mg cm-2 for H. didymator and 1.55 ± 0.2 mg cm-2 for C. inanitus (1 ml; 55 kPa). As soon as the plates were dry, they were mounted with two bolts and adults were introduced into the cages. These cages were then moved to the climatic chamber and connected to forced ventilation. Adults of both parasitoid species were exposed to the dried residue until the death of the last insect. Topical bioassay Adults were topically treated on the pronotum with 1 µl of the insecticide solutions using a Burkard hand microapplicator (Burkard, UK). Controls were treated with acetone alone. Prior topical application, adults were anaesthetized with cold temperatures (some minutes at -5 ºC). After the treatment, insects were transferred to ventilated plastic round boxes (11 cm diameter by 5 cm height) as described above and provided with honey and water ad libitum. Ingestion bioassay Adults were fed with aqueous solutions of the different insecticides or water for control, offered continuously in the drinking troughs during their life-span, in the ventilated plastic boxes previously described. Aqueous solutions were replaced every 5 days because of evaporation. Statistical analysis Data (means values and standard errors (S.E.)) were subjected to one-way analysis of variance (ANOVA) and a LSD test was used to compare responses at field rates. Statistical tests were performed using Statgraphics (STSC, 1987). Non-transformed data are shown in tables. If any of the premises of analysis of variance was still violated after appropriate transformations, the non-parametric test Kruskal-Wallis was applied. Median values were considered significantly different if 95% confidence intervals of medians did not overlap.

Results

Topical treatment on pupae After topical application of the insecticidal solutions on H. didymator and C. inanitus pupae, the emergence of adults was not significantly different compared to controls (Table 2). Nevertheless, life-span of emerged adults was drastically reduced when pupae had been treated with fipronil (90 and 75% of reduction for H. didymator and C. inanitus, respectively). Adults emerged from pupae treated with fipronil died quickly, H. didymator being slightly more sensitive than C. inanitus. It was observed that pharate adults coming from treated cocoons with fipronil were dead inside the silk cocoon and, in some cases, they were able to open a hole in order to escape, without success. Bioassays on adults Residual bioassay Residual application of all insecticides affected the evaluated parameters in both parasitoid species by a drastic reduction of life-span compared to controls (Table 3). C. inanitus showed a slightly higher resistance to the insecticides than H. didymator, except for fipronil. Topical bioassay Topical application of natural pyrethrins+PBO and fipronil adversely affected both parasitoid species compared to controls. Adults only survived some hours, showing tremors starting just 1 hour after treatment. Toxicity of imidacloprid varied among the species. Imidacloprid was highly toxic to H. didymator, but harmless to C. inanitus (Table 3).

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Table 2. Effects of insecticides applied topically to H. didymator and C. inanitus pupae on several life parameters.

Compound Adult emergence IOBC Life-span IOBC IOBC (%) Class1 (days) Class Total2 Hyposoter didymator Control 100a - 20.0±2.2a - - Fipronil 72.0±10.2a 1 2.2±0.2b 3 3 Imidacloprid 84.0±9.8a 1 20.6±3.1a 1 1 NP+PBO3 88.0±4.9a 1 19.8±2.6a 1 1 F (df=3,16) 2.38 16.2 P 0.11 <0.0001 Chelonus inanitus Control 70.0±9.3a - 29.2±4.6a - - Fipronil 65.0±12.7a 1 7.3±0.2b 2 2 Imidacloprid 74.7±3.3a 1 26.7±2.2a 1 1 NP+PBO3 72.0±4.9a 1 23.8±2.9a 1 1 F (df=3,16) 0.23 11.25 P 0.87 0.0003 Means ± SE in the same column and insect species following by a different letter are significantly different at P<0.05 (ANOVA, LSD). 1 IOBC toxicity classes: 1=harmless (<30% reduction), 2=slightly harmful (30-70% reduction), 3=moderately harmful (80-99% reduction), 4= harmful (<99% reduction). 2 Highest value among the two IOBC classes calculated previously (adult emergence and life-span). 3 Natural pyrethrins + piperonyl butoxide.

Ingestion bioassay All the insecticides tested reduced life-span of H. didymator adults compared to controls. Life-span of H. didymator adults that ingested fipronil continuously did not exceed 2.1 days in mean. Life-span of adults treated with imidacloprid and natural pyrethrins+PBO was 5.9 and 8.1 days, respectively, whereas control life-span reached 20.3 days. Similar results as those obtained for H. didymator were found for C. inanitus, even though effects were not so drastic (Table 3). Imidacloprid was harmless for this species (IOBC class 1).

Discussion

Although pupae are one of the most protected stages, it is clear that they can easily be contaminated by direct contact with insecticide droplets during spraying. Pupae of H. didymator and C. inanitus topically treated to mimic the previous described effect in the field, continued their development to adult emergence in spite of the treatment. However, fipronil significantly reduced the adult parasitoids life-span. The successful adult emergence after topical treatment of a broad range of insecticides on pupae of H. didymator was also reported by Schneider et al. (2003a,b), and also these authors described a potent effect of neurotoxic insecticides as spinosad, and insect growth regulators as pyriproxyfen or azadirachtin on life- span of the parasitoid. Based on the rate of penetration through the silk cocoon, they concluded that, irrespective of the insecticide, most of them remained in the cocoon and do not affect the pupal body. So, the rate of penetration did not seem to explain the toxicity detected. It is likely that fipronil was ingested by adults when they made the hole with their

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jaws in the cocoon before emergence. It can be objected that high mortality should also be recorded when pupae were treated with NP+PBO or imidacloprid because these compounds have shown high toxicity on other development stages of this parasitoid species (Morales et al., unpublished data). Nevertheless, adults need among 7 and 10 days for emergence, enough time for these compounds to be mostly degraded whereas fipronil is reported as a highly persistent insecticide (Tingle et al., 2000).

Table 3. Effects of insecticides on H. didymator and C. inanitus adults with different uptake routes.

Compound Life IOBC Life IOBC Life IOBC span class1 span class span class Residual Topical Ingestion Hyposoter didymator Control 19.6±1.2a - 24.0±2.5a - 20.3±0.9a - Fipronil 1.0±0.0b 3 1.0±0.0b 3 2.1±0.1b 3 Imidacloprid 1.0±0.0b 3 1.0±0.0b 3 5.9±0.8b 2 NP +PBO2 1.0±0.0b 3 1.0±0.0b 3 8.1±0.6b 2 F(df) or K 1034.1 18.5 136.14 (3,16) P <0.0001 0.0003 <0.0001 Chelonus inanitus Control 24.1±0.7a - 31.7±1.8a - 25.9±2.6ab - Fipronil 1.0±0.0c 3 1.0±0.0b 3 4.5±0.4d 3 Imidacloprid 2.3±0.1b 3 29.3±3.5a 1 20.1±3.5bc 1 NP +PBO 2.0±0.0b 3 1.0±0.0b 3 14.6±1.8c 2 F(df) or K 17.88 77.88 (3,16) 28.7 (3,16) P 0.0004 <0.0001 <0.0001 Means ± SE in the same column following by a different letter are significantly different at P<0.05 (ANOVA, LSD; Kruskal-Wallis). 1IOBC classes: 1=harmless (<30% reduction), 2=slightly harmful (30-70% reduction), 3=moderately harmful (80-99% reduction), 4= harmful (<99% reduction). 2Natural pyrethrins + piperonyl butoxide.

Adults of endoparasitoids are the most exposed stage to insecticides for basically one reason: they have to move continuously to find food and a suitable host to oviposit their eggs, increasing the possibility to contact pesticides. Their effects on natural enemies will mainly depend, among other factors, on the application method, formulation and persistence (Elbert et al., 1998; Gels et al., 2002). Fipronil showed the highest toxicity compared to the other insecticides, reducing severely the life-span of adults, depending on the mode of exposure. The toxicity of fipronil for beneficial insects has been cited by several authors. Balanca & Visscher (1997a) showed that very low doses of fipronil (0.6-2 g a.i./ha) reduced the populations of species from families Scelionidae and Sphecidae drastically, causing an impact similar to classic organophosphates (Balanca & Visscher, 1997b). Elzen et al., (1999) on the ectoparasitoid Catolaccus grandis (L.) and Abdallahi et al., (2000) on the braconid Psyttalia concolor (Szèpligeti) found fipronil as the most toxic compound among the range of insecticides tested. Fipronil seems to be a very efficient compound to control a number of important pests, but its

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compatibility with natural enemies is rather arguable because its efficacy at low doses and high persistence do not permit population of beneficials to survive or at least to re-establish after treatment. Imidacloprid was as toxic as fipronil to H. didymator, except in the ingestion treatment. Adults of this parasitoid that had ingested imidacloprid had a mean life-span of about 6 days, 4 more than that measured after fipronil treatment. C. inanitus was extremely sensitive to this insecticide by residual treatment, whereas topical application was harmless, showing that the method of treatment might lead to completely different results. The strongly sclerotized thorax of C. inanitus might be a potent barrier to the penetration of a single droplet, whereas the parasitoid could contact more intensively the insecticide for days through legs and antenna when walking on the treated glass plates. The reduction on life-span after ingestion treatment was more severe for H. didymator than for C. inanitus, as it was also detected in topical treatment. The adverse effects of imidacloprid in adult survival of the order Hymenoptera has been reported by several authors as Stapel et al. (2000) and Tzeng & Kao (1999). Even though natural pyrethrins tend to be quickly degraded in contact to light (Soderlund & Bloomquist, 1989), a strong knock-down effect was detected together with the tremors commonly suffered by insects contaminated by neurotoxic insecticides. This effect was much more pronounced by contact (topical or residual treatment) than by ingestion. Again, C. inanitus was less sensitive than H. didymator. Tosacano et al., (1997) alerted about the negative effects of natural pyrethrins on beneficials. To conclude, fipronil, imidacloprid and natural pyrethrins+PBO are not compatible with both parasitoid species, H. didymator and C. inanitus, based on laboratory experiments. The application of these insecticides at maximum field rates currently used would imply the disappearance of beneficials. Besides, we cannot trust in a rapid recover of insects in the case of fipronil, because its high persistence is known, and also, because its degradation in the field leads to an even more toxic metabolite. Further studies in field should be performed to verify if such described toxicity still remains. Another interesting conclusion in this study is, that C. inanitus was found to be a more resistant insect than H. didymator, which makes it a better control agent in those cases where biological and chemical control coexist.

Acknowledgements

This work was supported by the Spanish Ministry of Education and Culture (project AGL2004-07516-C02-01/AGR) to E. Viñuela. We should like to express our thanks to Dr. Volkoff from INRA Montpellier (France) for having sent insects and kindly offered their wise advices concerning rearing and also to Dr. Lanzrein from University of Berna (Switzerland) for the rearing of C. inanitus.

References

Abdallahi, E., Adán, A. & Viñuela, E. 2000: Estudio de la actividad de piriproxifen y fipronil sobre Opius concolor Szépligeti (Hymenoptera: Braconidae) y su huésped de sustitución Ceratitis capitata (Wiedemann) (Diptera: Tephritidae). Bol. San. Veg. Plagas 26: 503- 511. Balança, G. & de Visscher, N. 1997a: Impacts on nontarget insects of a new insecticide compound used against the desert locust [Schistocerca gregaria (Forskal 1775)]. Arch. Environ. Contam. Toxicol. 32: 58-62.

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Balança, G. & de Visscher, N. 1997b: Effects of very low doses of fipronil on grasshoppers and non-target insects following field trials for grasshopper control. Crop Prot. 16 (6): 553-564. B.O.E. 2004: Real Decreto 1938/2004, de 27 de septiembre, por el que se establece el Programa Nacional de Control de los Insectos Vectores de los Virus de los Cultivos Hortícolas. Cole, L. M., Nicholson, R. A. & Casida, J. E. 1993: Action of phenylpyrazole insectides at the GABA-gate chloride channel. Pestic. Biochem. Physiol. 46: 47-54. Elbert, A., Nauen, R. Y. & Leicht, W. 1998: Imidacloprid, a novel chloronicotinyl insecticide: Biological activity and agricultural importance. In: Ishaaya, I., Degheele, D. (Eds.), Insecticides with novel models of action: mechanism and application, Springer-Verlag, Berlin: 50-73. Elzen, G. W., Maldonado, S. N. & Rojas, M. G. 1999: Toxicological responses of the boll weevil (Coleoptera: Curculionidae) ectoparasitoid Catolaccus grandis (Hymenoptera: Pteromalidae) to selected insecticides. J. Econ. Entomol. 92: 309-313. Gels, J. A., Held & D W., Potter, D. A. 2002: Hazards of insecticides to the bumblebees Bombus impatiens Cresson (Hymenoptera: Apidae) foraging on flowering white clover in turf. J. Econ. Entomol. 95(4): 722-728. Grossniklaus-Bürgin, C.,Wyler, T., Pfister-Wilhelm, R. & Lanzrein, B. 1994: Biology and morphology of the parasitoid Chelonus inanitus (Braconidae, Hymenoptera) and effects on the development of its host Spodoptera littoralis (Noctuidae, Lepidoptera). Invertebr. Reprod. Dev. 25(2): 143-158. Jacas, J. & Viñuela, E. 1994: Analysis of a method to test the effects of pesticides on adult females of Opius concolor Szepl. (Hym., Braconidae), a parasitoide of the olive fruit fly, Batrocera oleae (Gmelin) (Dipt., Tephritidae). Biocontrol Sci. Technol. 4: 147-174. Jones, D. 1985: Endocrine interaction between host (Lepidoptera) and parasite (Cheloninae, Hymenoptera): Is the host or the parasite in control? Ann Entomol. Soc. Am. 78: 141- 148. M.A.P.A. 2007. Anuario de Estadística Agroalimentaria 2006. URL: http://www.mapa.es/es/estadistica/pags/anuario/introduccion.htm (online: 19 November, 2007). Philogene, B.J.R., Regnault-Roger, C. & Vincent, C. 2002: Produits phytosanitaires insecticides d´origine végétale: promesses d´hier et d´aujourd´hui. In: Regnault-Roger, C., Philogène, B.J.R., Vincent, C. (Eds.), Biopesticides d´origine végétale. Tec&Doc Editions, London, UK: 2-17. Poitout, S. & Bues, R. 1974: Élevage de chenilles de vingt-huit espèces de lépidoptères Noctuidae et de deux especes d` Arctiidae sur milieu artificiel simple. Particularités de l` élevage selon espèces. Ann. Zool. Ecol. Anim. 6 (3): 431-441. Schneider, M. & Viñuela, E. 2007: Improvements in rearing method for H. didymator considering sex allocation and sex determination theories used for Hymenoptera. Biol. Control 43 (3): 271-277. Schneider, M. I., Smagghe, G. & Viñuela, E. 2003a: Susceptibility of Hyposoter didymator (Hymenoptera: Ichneumonidae) adults to several insect growth regulators and spinosad by different exposure methods. IOBC/wprs Bull. 26 (5): 111-122. Schneider, M., Smagghe, G., Gobbi, A. & Viñuela, E. 2003b: Toxicity and pharmacokinetics of insect growth regulators and other novel insecticides on pupae of H. didymator, a parasitoid of early larval instars of noctuid pests. J. Econ. Entomol. 96: 1054-1065. Smagghe, G., Carton, B., Wesemael, W., Ishaaya, I. & Tirry, L. 1999: Ecdysone agonist- mechanism of action and application on Spodoptera species. Pestic. Sci. 55: 343-389.

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Soderlund, D. M. & Bloomquist, J. R. 1989: Neurotoxic actions of pyrethroid insecticides. Ann. Rev. Entomol. 34: 77-96. Stapel, J. O., Cortesero, M. & Lewis, W.J. 2000: Disruptive sublethal effects of insecticides on biological control: altered foraging ability and life-span of a parasitoid alter feeding on extrafloral nectar of cotton treated with systemic insecticidas. Biol. Control 17: 243- 249. STSC 1987: User´s guide Statgraphics. Graphic Software System, STSC Inc., Rockville, M.D, USA. Tillman P. G. & Mulrooney J. E. 2000: Effect of selected insecticides on the natural enemies Coleomegilla maculata and Hippodamia convergens (Coleoptera: Coccinellidae), Geocoris punctipes (Hemiptera: Lygaeidae), and Bracon mellitor, Cardiochiles nigriceps and Cotesia marginiventris (Hymenoptera: Braconidae) in cotton. J. Econ. Entomol. 93 (6): 1638-1643. Tingle, C. C., Rother, J. A., Dewhurst, C. F., Lauer, S. & King, W. J. 2000: Health and environmental effects of fipronil. Pesticide Action Network UK. Briefing A/1. 30 pp. Tosacano, N. G., Yoshida, H. A., & Henneberry, T. J. 1997: Responses to azadirachtin and pyrethrum by two species of Bemisia (Homoptera: Aleyrodidae). J. Econ. Entomol. 90 (2): 583-589. Tzeng, C. C. & Kao, S. S. 1999: Toxicity of nine insecticides to Eretmocerus orientalis adults, a parasitoid of silverleaf whitefly (Bemisia argentifolii). Plant Prot. Bull. 41(1): 83-86.

Pesticides and Beneficial Organisms IOBC/wprs Bulletin Vol. 35, 2008 pp. 60-65

The extended laboratory test guideline for Aphidius rhopalosiphi: some areas of debate relating to the methodology

Mead-Briggs, Michael Mambo-Tox Ltd., 2 Venture Road, Chilworth Science Park, Southampton SO16 7NP, UK. [email protected]

Abstract: After several years of slow evolution, a final draft version of the extended laboratory test guideline was put forward by the Aphidius Ring-Test Group in 2006. However, upon wider circulation, some proposals made in the guideline were challenged. These related to a) why only female wasps were being evaluated; b) the relevance of the repellency assessments and what happens where settling rates on the treated plants were poor; c) which of the surviving wasps should be selected for the reproduction assessments; only those classed as ‘alive and unaffacted’, or also those appearing to be ‘affected’? It is hoped that by explaining the reasons behind certain decisions that were made and by presenting additional data, we can now move forward and finalise the long-overdue publication of the guideline.

Key words: Aphidius rhopalosiphi, parasitoid, extended laboratory test

Introduction

An extended laboratory test method for evaluating pesticide effects on the parasitoid Aphidius rhopalosiphi (DeStephani-Perez) (Hymenoptera, Braconidae) was developed by the Aphidius Ring Testing Group as a continuation of the work of the European ‘Joint Initiative’ (Candolfi et al., 2000). Although not formally published, this method has already been used for regulatory studies over a number of years and has resulted in reliable experiments. The test method involves the treatment of pots of seedling barley, over which adult female wasps are then confined using Perspex cylinders. Since the insects are capable of flying away from the treated plants and settling on the walls of the arena, observations of the numbers walking on the plants are typically taken during the initial 3 h to confirm that exposure to the fresh residues has occurred. Wasp mortality is assessed over 48 h. The surviving wasps (ideally 15 per treatment) are then individually confined over pots of untreated aphid-infested barley for a further 24 h and the numbers of aphids in which wasp pupae (or ‘mummies’) subsequently develop is recorded. Having circulated the finalised draft guideline in 2006 to interested parties outside of the Ring Testing Group, a number of questions were raised regarding the design of the test. The principal areas of contention can be summarised as: a] why are only female wasps evaluated? b] how important are the repellency assessments and what happens where settling rates on the treated plants are poor? c] which of the surviving wasps should be selected for the reproduction assessments; only those classed as ‘alive and unaffected’, or also those appearing to be ‘affected’? The aim of this paper is to discuss these three issues and, where necessary, make proposals so that the guideline can be put forward for formal publication.

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Discussion

The use of female wasps only This decision to only use female wasps in the extended laboratory test was originally based on the hypothesis that the females might spend more time in contact with the (treated) plants, since they would be actively searching for host aphids to parasitise. Although this hypothesis was not formally tested, there are other reasons why this approach is considered more practical. For instance, where up to half of the 30 test insects in a treatment die during the intial 48 h, there are still at least 15 females left for the reproduction assessments. This would not be the case if half of the test insects were male. The ideal of having a minimum of 15 wasps per treatment for the reproduction assessments is based on previous statitical analyses reported as part of the laboratory test guideline (Mead-Briggs et al., 2000). It is considered impractical to increase replication further in studies where there may already be up to seven treatments, each with six replicate units, or to increase the numbers of wasps in individual replicates, which would hamper observations during the bioassay. In addition, having just females present in the arenas makes it easier when, after the 48 h mortality assessment, wasps have to be collected for the reproduction assessments. More importantly, there is no evidence of a difference in the sensitivities of male and female A. rhopalosiphi, despite the fact that the male wasps are, on average, smaller than the females. A recent analysis of past regulatory studies carried out by a single laboratory has confirmed the absence of any difference in the sensitivities of the sexes (BART – pers. com.). For that analysis the results of 101 separate laboratory studies with A. rhopalosiphi (i.e. Tier 1 rate-response tests following the guideline of Mead-Briggs et al., 2000) were collated. In these, both male and female wasps had been used and the fate of the individual sexes had been recorded after 48 h. When the data for the individual test item treatment rates (440 in total) were presented as a scatter plot and a linear regression applied to the data points, the slope of the regression was 0.97, the intercept was 0.02 and the square of the correlation coefficient was 0.82 (Figure 1). This showed the comparable sensitivity of males and females and, therefore, it is considered acceptable to only use females in the extended laboratory test.

Behavioural data One constraint in using A. rhopalosiphi for bioassays is that it can fly away from a treated surface and settle on the walls of the test arena, thus avoiding the potential harmful effects of the test item. This may happen whether the bioassay involves either a three-dimensional treated plant (as here) or, to a lesser extent, a two-dimensional treated leaf surface lining the floor and ceiling of a shallow arena (Grimm et al. 2002). Thus it is important that sufficient exposure to the treated plants should be demonstrated. For this reason the guideline recommends that an assessment of the numbers of wasps settled on the plants (as opposed to the walls of the test arenas) is made on five separate occasions during the initial 3 h of the bioassay. The data are analysed statistically. It is also advisable that where repellent effects are apparent during the intial 3 h of the bioassay, additional assessments are made of the settling positions of the wasps after 24 and 48 h, to hopefully confirm that exposure of wasps to residues does occur during the course of the experiment. To find out how frequently wasps settle on the treated plants in extended laboratory tests, data from three independent contract research organisations was collated. Each laboratory provided the results from 10 impartially-selected studies (each with up to five test item treatment rates) and the data are presented in Figure 2.

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Figure 1. A linear regression applied to data for the mortality of male and female wasps in 101 laboratory tests with A. rhopalosiphi.

100

75

50

% wasps settling on plants settling % wasps 25

0 0 5 10 15 20 25 30 individual tests

control highest rate high rate medium rate low rate lowest rate

Figure 2. Trends in the initial settling behaviour of wasps in 30 extended laboratory tests carried out at three separate laboratories. The data presented are the percentage of observations where wasps were settled on the treated plants during the first 3 h of each bioassay. The results of individual tests with up to five test-item treatment rates and a water control are presented in vertical alignment. (Lab A = tests 1-10, Lab B = tests 11-20, Lab C = tests 21-30).

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Statistically, a significant reduction in settling rates on the freshly-treated plants was observed in approximately one third of these studies and it was often the case that lower settling activity was observed at higher test-item rates than in lower test-item treatment rates (Fig. 2). Naturally, in some instances reduced settling correlated with other treatment effects, such as significant mortality or reduced reproduction rates of the exposed wasps. However, the full relevance of this behavioral is difficult to make without further study. The intention of extended laboratory tests is to simulate the effects of fresh treatment residues under more realistic exposure conditions. This particular test does exactly this, allowing the wasps to move between treated and non-treated areas. However, since food for the wasps is only available on the plants (sugar solution is sprayed onto the seedlings before application of the test item – thus adding a potential oral exposure to the test design), some level of exposure is ensured. Nevertheless, the question for a minimal settling level has to be addressed to grant that the test results are reliable and the Aphidius Ring-Test Group continues to discuss and evaluate this study design detail with the ultimate aim of setting a maximum difference in settling rates that would be allowable between test item and control treatments.

Selection of wasps for the reproduction assessments The laboratory test guideline (Mead-Briggs et al., 2000) recommended that one should evaluate, where possible, 15 wasps from each treatment to determine any side-effects on the reproduction rates of exposed females. This number of replicates was based on an analysis of historical data that showed that the investigator could then determine statistically a 50% treatment effect with at least 80% confidence. Since the assessment is intended to be of the reproductive capacity of the wasps, not a further assessment of survivorship, one ideally wants the 15 replicate wasps to survive the entire 24 h oviposition period. In that way the parasitisation rates of individuals are compared over a similar time. Where individuals have

200

175 150

125

100 75

50

number of test item treatments 25

0 0% 1-10% 11-20% 21-30% 31-40% 41-50% 51-60% 61-70% 71-80% % of living wasps classed as 'affected' in individual tests

Figure 3. A summary of the percentage of the total living wasps that were classed as ‘affected’ at the time of the 48 h mortality assessment in 60 extended laboratory tests with Aphidius. There were in total 206 test item treatments included in these studies, 181 of which had no (0%) affected individuals.

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died during the oviposition period, this is noted and these replicates are normally eliminated from the statitical comparison of treatments. To minimise the reduction of replicates through mortality, it is common practice to select just healthy-looking live wasps for these assessments and not individuals already categorised as being ‘affected’. Consequently, it may be argued that affected wasps are not accounted for in either the mortality assessments or the reproduction assessments. In reality, the proportion of living wasps recorded as affected in extended laboratory studies is relatively small. The data from 60 studies with a rate-response design was collated from three independent contract research organisations. In these studies it was found that there were no wasps classed as affected in 181 out of 206 test item treatments (i.e. 88%) and in all but three of the remaining 25 treatments, less than 20% of the surviving wasps were recoded as being affected (Figure 3). To negate the potential influence of any ‘affected’ wasps on the overall reproductive capacity of the treatment group, a correction factor might be used when calculating a value for effects on reproduction. This proposal that has yet to be fully considered by the Aphidius Ring Testing Group, but might be based on the following equation:

Corrected mean reproduction = Mean number mummies per surviving female x 1 - Number affected wasps at 48 h determined using live (not affected) wasps Number live + affected wasps at 48 h

This effectively gives a zero reproduction score to the proportion of the living wasps recorded as being affected (a worst-case scenario). The benefit of a retrospective correction of any values mathematically, rather than actually assessing the affected wasps, is that we do not risk diminishing the statistical vigour of the reproduction assessments carried out using just live wasps.

Conclusions

The extended laboratory test guideline for Aphidius rhopalosiphi, the design of which was proposed some time ago by the Aphidius Ring-Test Group, has been used over a number of years for evaluating the potential side-effects of plant-protection products on this sensitive species of wasp. It has already proven to be a suitable method for determining reliable rate-response data. A recent analysis of historic data has shown that it is quite acceptable to use just female wasps in the bioassay, these being as sensitive as the male insects. Furthermore, recording the settling rate of wasps on the treated plants during the initial phase of the bioassay is still considered to be a worthwhile assessment, although further analysis and discussion is required before deciding how data are to be handled when significant repellency is observed.

Acknowledgements

I wish to thank BART (the Beneficial Arthropods Regulatory Testing Group) for proving the data relating to male:female wasp sensitivity. I also wish to thank the members of the Aphidius Ring-Testing Group, and specifically Dr Monika Moll (Ibacon GmbH) and Christian Warmers (Eurofins-GAB GmbH), for their help in collating data.

References

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Candolfi, M.P., Blümel, S., Forster, R., Bakker, F.M., Grimm, C., Hassan, S.A., Heimbach, U., Mead-Briggs, M.A., Reber, B., Schmuck, R. & Vogt, H. (Eds.) 2000: Guidelines to evaluate side-effects of plant protection products to non-target arthropods; IOBC, BART and EPPO Joint Initiative. Gent, IOBC/wprs. ISBN 92-9067-129-7. Grimm, C. Candolfi, M.P. & Fisch, R. 2002: A comparison of rate-response toxicity tests with Aphidius rhopalopsiphi (Hymenoptera: Aphidiidae) using glass, leaves and whole plants as substrate. Chemosphere 48: 581 – 589. Mead-Briggs, M.A., Brown, K., Candolfi, M.P., Coulson, M.J.M., Miles, M., Moll, M., Nienstedt, K.M., Schuld, M., Ufer, A. & McIndoe, E. 2000: A laboratory test for evaluating the effects of plant protection products on the parasitic wasp, Aphidius rhopalosiphi (DeStephani-Perez) (Hymenoptera, Braconidae). In: Guidelines to evaluate side-effects of plant protection products to non-target arthropods; IOBC, BART and EPPO Joint Initiative. Eds. Candolfi, M.P., Blümel, S., Forster, R., Bakker, F.M., Grimm, C., Hassan, S.A., Heimbach, U., Mead-Briggs, M.A., Reber, B., Schmuck, R. & Vogt, H.. Gent, IOBC/wprs. ISBN 92-9067-129-7. pp 13-25. Pesticides and Beneficial Organisms IOBC/wprs Bulletin Vol. 35, 2008 pp. 66-77

Pesticides selectivity list to beneficial arthropods in four field vegetable crops

Jean-Pierre Jansen1, Louis Hautier1, Nicolas Mabon2 and Bruno Schiffers2 1 Ecotoxicology Laboratory, Department of Biological control and Plant genetic resources, Walloon Centre of Agricultural Research, Chemin de Liroux, 2, 5030 Gembloux, Belgium 2 Phytopharmacy Laboratory, Analytical Chemistry Unit, Faculté Universitaire des Sciences Agronomiques, Passage des déportés, 2, 5030 Gembloux, Belgium

Abstract: Selectivity of pesticides to beneficial arthropods is a key data for the implementation of IPM program. In the context of field vegetable crops, a set of 16 fungicides, 16 herbicides and 13 insecticides commonly used in Belgium were tested on 5 indicator species: the parasitic hymenoptera Aphidius rhopalosiphi (De Stefani-Perez) (Hym., Aphidiidae), the aphid foliage dwelling predators Adalia bipunctata (L.) (Col., Coccinellidae) and Episyrphus balteatus (Dipt., Syrphidae) and the ground-dwelling predators Aleochara bilineata (Col., Staphylinidae) and Bembidion lampros (Col., Carabidae). Pesticides were tested according to a testing scheme including a first assessment on inert substrate and, for products that were toxic, a second assessment on natural substrate. The effects of the product were assessed on basis of onion fly pupae parasitism reduction for A. bilineata and on basis of corrected mortality for the 4 remaining species. According to the final results obtained at the end of this testing scheme, the products were listed in toxicity classes: green list if effect ≤30%, yellow list 30% < effect ≤ 60% and orange list 60% < effect ≤ 80%. Products with toxicity higher than 80% on plants or on soils, or that reduce parasitism more than 80% on soil were put in the red list and are not recommended for IPM. Results showed that all fungicides and herbicides were included in the green list except tebuconazole and boscalid + pyraclostrobin that were labeled as yellow for A. bipunctata. In opposite, no foliar insecticide was totally selective for all beneficial tested. However some products are in green list for one or several species. Soil insecticides were all very toxic for ground dwelling arthropods and classed in red list. In conclusion, fungicides and herbicides tested are compatible with IPM programs. For foliar insecticides, some treatments can be used carefully according to the selectivity. But for soil insecticide treatments, their toxicity raise the question of their use in IPM programs in vegetables and the need of new compounds or development of alternative pest control programs.

Key words: Adalia bipunctata, Aleochara bilineata, Aphidius rhopalosiphi, Bembidion lampros, Episyrphus balteatus, selectivity list, vegetable

Introduction

In vegetable production in open field, as in other crops, beneficial arthropods are a key factor in the biological control of several pests (Hughes & Salter, 1959; Read, 1962; Coaker & William, 1963; Lövei & Sunderland, 1996). In the context of a sustainable agriculture and IPM implementation, these beneficial arthropods must be preserved from adverse effects, especially from non-selective pesticides. By eliminating pest natural enemies, non selective insecticides can enhance pest outbreak, with population levels that even reach higher levels than those observed without any insecticide treatment (Ripper, 1956; Pimentel, 1961; Besemer, 1964; Vickerman & Sunderland, 1977; Croft & Slone, 1998). Resurgence of pests of

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secondary importance, simply because their natural enemies have been eliminated is also a consequence of the use of non-selective compounds, even with a simple fungicide or herbicide application (Nanne & Radcliffe, 1971; Sotherton & Moreby, 1988; Lagnaoui & Radcliffe, 1998). Both pest outbreak and pest resurgence multiply pest problems and insecticide use, increase cost production and negative impact of pest control on human health and environment. This situation could be avoided at the beginning, if selective pesticide were available and used instead of non-selective compounds. Actually, there is a trend to try to use selective products, as claimed by certification standard guidelines as EUREPGAP and Chart PERFECT. However, clear information that can directly be used by producers and pesticide users are missing. In the context of IPM implementation and pesticide users information, the selectivity of pesticides used in Belgium in carrot, onion, pea and bean has been determined according to the methodology previously used for building the selectivity list in potato (Hautier et al., 2006). 16 fungicides, 16 herbicides and 13 insecticides have been tested on 2 to 5 beneficial arthropods species selected as indicator species for these crops. According to the results obtained, products were rated in different toxicity classes and selectivity lists were established.

Material and methods

Products that were taken into consideration are listed in table 1. A first bibliographic survey for products that were well documented was carried out and data were retained when the methods used fulfill the IOBC standard (Hassan, 1994) and were similar or close to those used to build the selectivity list (residual contact toxicity test with inert and/or natural substrate, susceptible life stage, product application and tested rate, exposure time, .....). Only clear results, products that were undoubtedly harmless or harmful at the recommended field rate for Belgium, were retained. All the other products were assessed for toxicity. Toxicity tests were realized on 2 to 5 different species, according to the use of the product (crop, timing of application and beneficial exposition risk). The tested species were: adult of the parasitic wasp Aphidius rhopalosiphi De Stefani-Perez (Hym.; Aphidiidae), larvae of the ladybird Adalia bipunctata (L.) (Col.; Coccinellidae) and the hoverfly Episyrphus balteatus (De Geer.) (Dipt.; Syrphidae), adults of the carabid beetle Bembidion lampros (Herbst.) (Col.; Carabidae) and the rove beetle Aleochara bilineata Gyll. (Col.; Staphylinidae). Toxicity tests fulfilled the SETAC recommendations (Barrett et al., 1994). Details of testing methods for A. rhopalosiphi, A. bipunctata and E. balteatus are available from previous work (Copin et al., 2001; Hautier et al., 2006). Methods used for B. lampros were similar as those used for Poecilus cupreus (Heimbach et al., 2000), except for insect origin (field collected beetles instead of laboratory rearing) and feeding (Ephestia kuehniella sterilized eggs instead of fly pupae, no assessment of feeding capacity). For A. bilineata, methods followed were those described by Grimm et al. (2000). Both methods for rove and carabid beetles were validated and used in the context of pesticide registration at European level. Pesticides were tested at the maximum recommended field rate for one application, on basis of available commercial formulations. Herbicides were only tested on the carabid and rove beetle, as the exposure risk for plant dwelling predator and parasitoid was negligible or inexistent. Insecticides and fungicides were tested on the 5 species. Most products were applied as spray mixtures in water, with rates of 200 l x ha-1 ± 10% on glass plates and 400 l x 68

ha-1 ± 10% on sand and soil. Thiram was applied as dusting powder and, when required, soil insecticides were incorporated into the soil as granule. For all products, tests followed a classical IOBC testing scheme (fig 1.) Pesticides were first tested at their maximum field rate on an inert substrate (glass plates or sand). Products that lead to effects (mortality or reduction in parasitism rate with Aleochara) higher than 30% were further tested on natural substrate (plants or soils). For the ground-dwelling beneficials, two different soils, one sandy-loamy soil (Soil “A”) and one loamy-clayed soil (Soil “B”) were used. Soil characteristics are given in table 2. According the results of the tests, products were labeled as harmless and included in a green list (class 1), slightly harmful – yellow list (class 2), moderately harmful – orange list (class 3) and harmful – red list (class 4). The green list includes all selective compounds and the red list all harmful products that must be avoided if possible. Yellow and orange list comprise intermediate products, that have to be used carefully, when no equivalent in the green list exists or for very specific uses, as pest resistance management.

1. Test on inert substrate (glass, sand)

≤ 30% Effects Green list

> 30%

2. Test on natural substrate (plant, soil)

≤ 30% Effects Green list 30% - 60 %

60% - 80% > 80%

Yellow list Orange list Red list

Figure 1. Sequential testing scheme of pesticide selectivity assessment

Results and discussion

Parasitic hymenoptera: Aphidius rhopalosiphi Results of tests carried out with A. rhopalosiphi are listed in table 3. All results were provided by toxicity tests (no bibliographic data). As there was no risk of exposure, the herbicides applied in field vegetables crops were not assessed on this species.

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Table 1: List of tested products. Commercial name, formulation type, active(s) ingredient(s) and tested dose. (* sowing line applied insecticide= product /m of sowing line)

Tested Formu- Active(s) ingredient(s) Commercial name dose / ha lation

Alpha-cypermethrin EC Fastac 0.25 l Bifenthrin SC Talstar 8 0.5 l Carbofuran GR Curater 1.25 g* Carbosulfan GR Sheriff 1 Gr 6.25 g* Chlorpyriphos-ethyl GR Dursban 5G 34 g/m² Deltamethrin WG Décis Micro 200 g Diazinon SC Disonal 8.5 l Dimethoate EC Hermootrox 0.5 l

Insecticides Lambdacyhalothrin CS Karate Zeon 0.1 l Methiocarb SC Mesurol 500 1.5 l Pirimicarb WG Pirimor 250 g Pirimicarb + lambdacyhalothrin EC Okapi 1.5 l Pyrethrins (plant extract) EC Bio-pyretrex 5 l Azoxystrobin SC Ortiva 1 l Boscalid + pyraclostrobin WG Signum 750 g Chlorothalonil SC Bravo 4.5 l Difenoconazole EC Geyser 0.5 l Dimethomorph + mancozeb WG Acrobat extra 2500 g Dithianon WG Ditho 1800 g Fluazinam SC Shirlan 0.5 l Iprodione WG Rovral 1000 g Mancozeb WG Dequiman 3200 g

Fungicides Maneb WG Tricarbamix extra 6300 g Myclobutanil EC Systhane 24 0.25 l Procymidone SC Sumisclex 1 l Sulfur WG Hermovit 5000 g Tebuconazole EW Horizon 1 l Thiram DP Luxan thiram 100 20000 g Vinclozolin SC Ronilan 1.5 l Bentazone SG Basagram 800 g Chlorpropham EC Chloor IPC 6 l Clomazone CS Centium 360 0.25 l Cycloxydim EC Focus Plus 6 l Fluazifop-P-butyl EC Fusilade 2 l Glufosinate-ammonium SL Basta S 3 l Glyphosate SG Roundup energy 3700 g Isoxaben SC AZ 500 0.2 l Linuron SC Linuron 500 1 l

Herbicides Metoxuron WP Dosanex 4500 g Paraquat SL Gramoxone 5 l Paraquat + diquat SL Priglone 5 l Pendimethalin SC Stomp 400 2.5 l Propachlor SC Ramrod 10 l Quizalofop-ethyl D EC Targa Prestige 1.5 l Tepraloxydim EC Aramo 2 l 70

Table 2: Textural and physicochemical characteristics of different substrates used.

Texture (%) Chemical characteristics

Sand Loam Clay organic C (g/kg) Humus (%) CEC (meq/100g)

Sand (inert subtstrate ) 100 0 0 0 0 0

Sandy loam (soil A) 72.7 18.9 8.3 14.2 2.8 8.22

Loamy-clayed (soil B) 6.6 76.2 17.2 8.9 1.8 13.54

Table 3: Result of toxicity test with A. rhopalosiphi, corrected mortality (A=glass plate, B=extended lab) and selectivity class: 1–harmless, 2-slightly harmful, 3-moderately harmful and 4-harmful.

A B Class Alpha-cypermethrin 100% 38% 2 Bifenthrin 100% 83% 4 Delthamethrin 100% 75% 3 Dimethoate 100% 100% 4 Lambda-cyalothrin 100% 1% 1 Methiocarb 100% 100% 4

Insecticides Insecticides Pirimicarb 100% 12% 1 Pirimicarb + lambdacyalothrin 100% 3% 1 Pyrethrins (plant extract) 100% 97% 4 Azoxystrobin 63% 7% 1 Boscalid + pyraclostrobin 4% - 1 Chlorothalonil 10% - 1 Difenoconazole 0% - 1 Dimethomorph + mancozeb 2% - 1 Dithianon 35% 24% 1 Fluazinam 8% - 1 Iprodione 6% - 1 Mancozeb 6% - 1 Maneb 0% - 1 Fungicides Myclobutanil 4% - 1 Procymidone 11% - 1 Sulfur 17% - 1 Tebuconazole 92% 5% 1 Thiram 98% 0% 1 Vinclozolin 0% - 1

Ground dwelling predators: Aleochara bilineata and Bembidion lampros On basis of bibliographic analysis, the insecticides carbofuran, chlorpyriphos-ethyl, diazinon and dimethoate were rated as harmful for carabid and rove beetles according to their high toxicity for these insects (Mowat & Coaker, 1967; Hassan, 1969; Edwards & Thompson, 1975; Finlayson, 1979; Finlayson et al., 1980; Kirknel, 1978; Cockfield & Potter, 1983; Vickerman et al., 1987; Floate et al., 1989; Kegel, 1989; Casteels and De Clerq, 1990; Bale et 71

al., 1992; Samsøe-Petersen, 1993; Sivasubramanian & Wratten, 1995). On the opposite, pirimicarb was included in the green list according to its selectivity for these ground dwelling beneficial insects (Unal & Jepson, 1992; Samsøe-Petersen, 1993).

Table 4. Result of toxicity test with B. lampros and A. bilineata, corrected mortality on sand, and soil A and B. Selectivity class: 1–harmless, 2-slightly harmful, 3-moderately harmful and 4-harmful.

A. bilineata test B. lampos test

Sand Soil A Soil B Class Sand Soil A Soil B Class Carbosulfan 100% 100% 100% 4 100% 97% 100% 4 Deltamethrin 100% 55% 30% 1-2 72% 13% 30% 1 Lambdacyhalothrin 100% 100% 84% 4 100% 10% 10% 1 Methiocarb 100% 99% 100% 4 100% 100% 100% 4 Pirimicarb + lambdacyhal. 100% 99% 100% 4 96% 20% 17% 1 Insecticides Pyrethrins (plant extract) 100% 0% 0% 1 100% 80% 43% 2-3 Azoxystrobin 1% - - 1 4% - - 1 Boscalid + pyraclostrobin 0% - - 1 0% - - 1 Difenoconazole 0% - - 1 20% - - 1 Dimethomorph +mancozeb 0% - - 1 0% - - 1 Dithianon 0% - - 1 0% - - 1 Fluazinam 5% - - 1 0% - - 1 Iprodione 0% - - 1 0% - - 1 Mancozeb 24% - - 1 0% - - 1 Fungicides Maneb 0% - - 1 0% - - 1 Myclobutanil 0% - - 1 4% - - 1 Sulfur 0% - - 1 0% - - 1 Tebuconazole - - - 1 0% - - 1 Vinclozolin 0% - - 1 0% - - 1 Bentazone 0% - - 1 4% - - 1 Chlorpropham 100% 7% 0% 1 100% 0% 10% 1 Clomazone 0% - - 1 14% - - 1 Cycloxydim - - - 1 0% - - 1 Fluazifop-p-butyl - - - 1 4% - - 1 Glufosinate-ammonium - - - 1 7% - - 1 Glyphosate - - - 1 0% - - 1 Isoxaben 2% - - 1 0% - - 1 Linuron 16% - - 1 10% - - 1

Herbicides Metoxuron 2% - - 1 3% - - 1 Paraquat 1% - - 1 3% - - 1 Paraquat + diquat 18% - - 1 3% - - 1 Pendimethalin 29% - - 1 0% - - 1 Propachlore 0% - - 1 19% - - 1 Quizalofop-ethyl D 2% - - 1 30% - - 1 Tepraloxydim 0% - - 1 0% - - 1

With herbicides and fungicides, Samsøe-Petersen (1995a,b) has concluded that cycloxydim and tebuconazole were not toxic for A. bilineata. Fluazifop-p-butyl and 72

glyphosate were also harmless for this rove beetle (Naton, 1989), as glufosinate was (EFSA Scientific Report, 2005). For the other products, no pertinent information was found in the literature and they were tested according to the sequential testing scheme described. All the results are listed in table 4.

Foliage dwelling predators: Adalia bipunctata and Episyrphus balteatus Results of tests carried out with A. bipunctata and E. balteatus larvae are listed in table 5. As for A. rhopalosiphi, no herbicides were assessed on these insects due to no or little exposure risk.

Table 5. Result of toxicity test with A. bipunctata and E. balteatus, corrected mortality (A=glass plate, B=extended lab) and selectivity class: 1–harmless, 2-slightly harmful, 3- moderately harmful and 4-harmful.

A. bipunctata test E. balteatus test

A B Class A B Class Alpha-cypermethrin 100% 100% 4 16% - 1 Bifenthrin 100% 100% 4 68% 16% 1 Delthamethrin 100% 100% 4 75% 77% 3 Dimethoate 100% 100% 4 100% 100% 4 Lambda-cyalothrin 100% 100% 4 0% - 1 Methiocarb 100% 100% 4 100% 100% 4 Insecticides Pirimicarb 21% - 1 80% 94% 4 Pirimicarb + lambdacyalothrin 100% 100% 4 100% 100% 4 Pyrethrin (plant extract) 100% 100% 4 100% 70% 3 Azoxystrobin 21% - 1 14% - 1 Boscalid + pyraclostrobin 80% 60% 2 0% - 1 Chlorothalonil 0% - 1 21% - 1 Difenoconazole 3% - 1 21% - 1 Dimethomorph + mancozeb 0% - 1 17% - 1 Dithianon 17% - 1 0% - 1 Fluazinam 100% 20% 1 0% - 1 Iprodione 30% - 1 10% - 1 Mancozeb 3% - 1 0% - 1

Fungicide Maneb 0% - 1 16% - 1 Myclobutanil 0% - 1 0% - 1 Procymidone 53% 13% 1 0% - 1 Sulfur 45% 11% 1 7% - 1 Tebuconazole 96% 32% 2 10% - 1 Thiram 61% 7% 1 10% - 1 Vinclozolin 13% - 1 10% - 1

Discussion

Results obtained from this study (table 6), including bibliographic analysis, showed that most herbicides and fungicides actually registered can be considered as safe for the 5 beneficial species tested. Among 101 tests with herbicides and fungicides, only 2 combinations (boscalid + pyraclostrobin and tebuconazole) were slightly toxic for ladybirds, all the other being finally 73

harmless for all species, even if some of them were first harmful on glass plates. According to the slight effect level observed for the two combinations under extended laboratory conditions, no adverse effects are expected in the field. This very low toxicity is probably the direct consequence of the 91/414 EEC application, harmful fungicides or herbicides being more difficult to register than in the past, when no specific requirements for beneficial arthropods were available. With insecticides, the situation is not so simple. If some products were harmless for one or more species, no insecticide was harmless for the 5 species tested. The most severe problem occurred for soil applied insecticides and soil dwelling predators. All the products used in this context (carbofuran, carbosulfan, chlorpyriphos-ethyl and diazinon) were harmful to both rove beetle and carabid beetle. These both insects are the only tested that can play a significant role in the control of soil insect pests as the carrot fly, the main pest problem in carrots in Belgium. Consequently, the use of chemical control for this pest together with biological or integrated control are completely impossible. Thus, for these pest, new insecticides that can be used in combination with beneficial insects or alternative control methods are urgently needed to limit pesticide use. This point has been strengthened as these toxic products were not included in the Annex I of the European pesticide list and thus cannot be used in the future. With foliar insecticides, the situation can be totally different between products. While insecticides as dimethoate and methiocarb are harmful (class 4) for all beneficial species, other products can be harmless or harmless/slightly harmful for 3 or 4 in 5 species tested. For example, pirimicarb was only toxic for E. balteatus and lambdacyhalothrin was toxic for A. bipunctata and A. bilineata but safe for the three remaining species. Thus, with the availability of several foliar insecticides, the preservation of natural enemies of insect pests and effective chemical control could be compatible by appropriate selection of the product and treatment timing. However, number of selective products is actually quite limited and new products are welcome especially in vegetables. New selective insecticides as pymetrozine or flonicamide used in other crop could be a solution for aphid control, but products with a similar ecotoxicological profile are required for other pests. Results obtained with natural pyrethrin extracts (Bio-Pyretrex®), an insecticide registered in organic farming, showed that this insecticide has the same impact on natural enemies than synthetic pyrethroids as deltamethrin or bifenthrin. The use of such products in organic farming can be criticized in term of their compatibility with biological control, even if this product is short-lived. A comparison of the sensitivity of the different species to the same products is indicating that at ground level, A. bilineata is probably a little more sensitive than B. lampros. For the other beneficials, final toxicity classes were indicating that A. rhopalosiphi is not a sensitive species under extended laboratory conditions, with several products more toxic on plants for A. bipunctata or E. balteatus than for the parasitic wasp. Thus, if this species can be a highly sensitive species on glass plates and therefore selected as an indicative species for beneficial in the context of pesticide registration at European level, the question of the use of extended laboratory study data with this species as an indication of toxicity for larger group species than parasitic hymenoptera can be criticized. 74

Table 6: Selectivity list of products used in carrots, beans, peas and onions. Class: 1– harmless, 2-slightly harmful, 3-moderately harmful and 4-harmful. Under bracket= bibliographic data, others= toxicity test on inert and on natural substrates in the laboratory.

Rove beetle Carabid Parasitoid Hoverfly Ladybird Active(s) ingredient(s) beetle A. bilineata B. lampros A. rhopalosiphi E. balteatus A. bipunctata Alpha-cypermethrin - - 2 1 4 Bifenthrin - - 4 1 4 Carbofuran (4) (4) Carbosulfan 4 4 no risk of exposure (soil insecticide) Chlorpyriphos-ethyl (4) (4) Deltamethrin 2 1 3 3 4 Diazinon (4) (4) no risk of exposure (soil insecticide) Dimethoate (4) (4) 4 4 4 Insecticides Lambda-cyhalothrin 4 1 1 1 4 Methiocarb 4 4 4 4 4 Pirimicarb (1) (1) 1 4 1 Pirimicarb + l-cyhalothrin 4 1 1 4 4 Pyrethrins (plant extract) 1 3 4 3 4 Azoxystrobin 1 1 1 1 1 Boscalid + pyraclostrobin 1 1 1 1 2 Chlorothalonil - - 1 1 1 Difenoconazole 1 1 1 1 1 Dimethomorph + mancozeb 1 1 1 1 1 Dithianon 1 1 1 1 1 Fluazinam 1 1 1 1 1 Iprodione 1 1 1 1 1 Mancozeb 1 1 1 1 1

Fungicides Maneb 1 1 1 1 1 Myclobutanil 1 1 1 1 1 Procymidone - - 1 1 1 Sulfur 1 1 1 1 1 Tebuconazole 1 1 1 1 2 Thiram - - 1 1 1 Vinclozolin 1 1 1 1 1 Bentazone 1 1 Chlorpropham 1 1 Clomazone 1 1 Cycloxydim 1 1 Fluazifop-P-butyl 1 1 Glufosinate-ammonium 1 1 Glyphosate 1 1 Isoxaben 1 1 no or low exposure risk Linuron 1 1

Herbicides Metoxuron 1 1 Paraquat 1 1 Paraquat + diquat 1 1 Pendimethalin 1 1 Propachlor 1 1 Quizalofop-ethyl D 1 1 Tepraloxydim 1 1 75

Acknowledgements

This research program was funded by the Federal Public Service Health, Food, Chain Security and Environment – Directorate Animals Plants and Food and by the Ministry of the Walloon Region – Agriculture General Directorate. The authors would like to thank the technical staff that has carried out the test, as Brigitte Jonard, Thibault Defrance and Anne Michele Warnier.

References

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Grimm, C., Reber, B., Barth, M., Candolfi, M.P., Drexler, A., Maus, C., Moreth, L., Ufer, A. & Waltersdorfer, A. 2000: A test for evaluating the chronic effects of plant protection products on the rove beetle Aleochara bilineata Gyll. (Col. Staphylinidae) under laboratory and extended laboratory conditions. In: Guidelines to evaluate side-effects of plant protection products to non-target arthropods, an IOBC, BART and EPPO Joint Initiative”, eds. Candolfi, M.P., Blümel, S., Forster, R., Bakker, F.M., Grimm, C., Hassan, S.A., Heimbach, U., Mead-Briggs, M.A., Reber, B., Schmuck, R. & Vogt, H.: IOBC/ WPRS Gent: 1-12 Hassan, S.A. 1969: Observations on the effect of insecticides on coleopterous predators of Erioischia brassicae (Diptera: Anthomyiidae). Entomol. Exp. Appl. 12: 157-168. Hassan, S.A., Bigler, F., Bogenschütz, H, Boller, E., Brun, J., Calis, J.N., Chiverton, P., Coremans-Pelseneer, J., Duso, C., Lewis, G.B., Mansour, F., Moreth, L., Oomen, P.A., Overmeer, W.P., Polgar, L., Rieckmann, W., Samsøe-Petersen, L., Stäubli, A., Sterk, G., Tavares, K., Tuset, J.J. & Viggiani, G. 1991: Results of the fifth joint pesticide testing programme carried out by the IOBC/WPRS-Working Group "Pesticides and Beneficial Arthropods". Entomophaga 36: 55-67. Hassan, S.A. 1994: Activities of the IOBC/WPRS working group “Pesticides and beneficial organisms”. IOBC/wprs Bulletin 17(10): 1-5. Hautier, L., Jansen, J-P., Mabon, N., Schiffers, B., Deleu, R. & Moreira, C. 2006: Building selectivity list of plant protection products on beneficial arthropods in open field: a concrete example with potato crop. IOBC/wprs Bulletin 29(10): 21-32. Hautier, L., Jansen, J-P., Mabon, N., Schiffers, B. 2007: Influence of organic matter on bio- availability of carbosulfan and its toxicity on a carabid beetle. Communications in Agricultural and Applied Biological Sciences, in press. Heimbach, U., Dohmen, P., Barret, K.L., Brown, K., Kennedy, P.J., Kleiner, R., Römbke, J., Schmitzer, S., Schmuck, R. & Wilhelmy, H. 2000: A method for testing effects of plant protection products on the carabid beetle Poecilus cupreus (Coleoptera, Carabidae) under laboratory and semi-field conditions. In: Guidelines to evaluate side-effects of plant protection products to non-target arthropods, an IOBC, BART and EPPO Joint Initiative”, eds. Candolfi, M.P., Blümel, S., Forster, R., Bakker, F.M., Grimm, C., Hassan, S.A., Heimbach, U., Mead-Briggs, M.A., Reber, B., Schmuck, R., and Vogt, H. IOBC/WPRS Gent: 87-106. Hughes, R.D. & Salter, D.D. 1959: Natural mortality of Erioschia brassicae (Bouché) (Dipt. Anthomyiidae) during the immature stages of the first generation. J. Anim. Ecol. 28: 343- 357. Kegel, B. 1989: Laboratory experiments on the side effects of selected herbicides and insecticides on the larvae of three sympatric Poecilus-species (Col., Carabidae). J. Appl. Entomol. 108: 144-155. Kirknel, E. 1978: Influence of Diazinon, Trichloronate, Carbofuran and Chlorfenvinphos on the parasitization capacity of the rove-beetle Aleochara bilineata (Gyll.). Tidsskr. Planteavl. 82: 117-129. Lagnaoui, A. & Radcliffe, E.B. 1998: Potato fungicides interfere with entomopathogenic fungi impacting population dynamics of green peach aphid. Am. Potato J. 75: 19-25. Lövei, G.L. & Sunderland, K.D. 1996: Ecology and behaviour of ground beetles (Coleoptera: Carabidae). Annu. Rev. Entomol. 41: 231-256. Mowat, D.J. & Coaker, T.H. 1967: The toxicity of some soil insecticides to carabid predators of the cabbage root fly. Ann. Appl. Biol. 59: 349-354. Nanne, H.W. & Radcliffe, E.B. 1971: Green peach aphid populations on potatoes enhanced by fungicides. J. Econ. Entomol. 64: 1569-1570. 77

Naton, E. 1989: Die Prüfung der Nebenwirkung von Pflanzenschutzmitteln auf Aleochara bilineata Gyll. (Col., Staphylinidae). Anz. Schädlingskd. Pfl. 62: 1-6. Pimentel, D. 1961: An ecological approach to the insecticide problem. J. Econ. Entomol. 54: 108-114. Read, D.C. 1962: Notes on life history of Aleochara bilineata (Gyll.) (Coleoptera: Staphylin- idae) and on its potential value as a control agent for cabbage maggot, Hylemya brassicae (Bouche) (Dipt.: Anthomyiidae). Can. Entomol. 94: 417-424. Ripper, W.E. 1956: Effect of pesticides on balance of arthropod populations. Annu. Rev. Entomol. 1: 403-438. Samsøe-Petersen, L. 1993: Effects of 45 insecticides, acaricides and molluscicides on the rove beetle Aleochara bilineata (Col.: Staphylinidae) in the laboratory. Entomophaga 38: 371- 382. Samsøe-Petersen, L. 1995a: Effects of 37 fungicides on the rove beetle Aleochara bilineata (Col.: Staphylinidae) in the laboratory. Entomophaga 40: 145-152. Samsøe-Petersen, L. 1995b: Effects of 67 herbicides and plant growth regulators on the rove beetle Aleochara bilineata (Col. Staphylinidae) in the laboratory. Entomophaga 40: 95- 104. Sivasubramaniam, W. & Wratten, S.D. 1995: Effects of insecticides on the abundance of arthropod predators in carrots in Canterbury; Proceedings of the 48th New Zealand Plant Protection Conference 48: 302-308. Sotherthon, N.W. & Moreby, S.J. 1988: The effects of foliar fungicides on beneficial arthropods in wheat fields. Entomophaga 33: 87-99. Unal, G. & Jepson, P.C. 1991: The toxicity of aphicide residues to beneficial invertebrates in cereal crops. Ann. Appl. Biol. 118: 493-502. Vickerman, G.P. & Sunderland, K.D. 1977: Some effects of Dimethoate on arthropods in winter wheat. J. Appl. Ecol. 14: 767-777. Vickerman, G.P., Coombes, D.S., Turner, G., Mead-Briggs, M.A. & Edwards, J. 1987: The effects of Pirimicarb, Dimethoate and Deltamethrin on Carabidae and Staphylinidae in winter wheat. Med. Fac. Landbouwkundige Wetenschappen Rijksuniversiteit Gent 52: 213-223.

Pesticides and Beneficial Organisms IOBC/wprs Bulletin Vol. 35, 2008 pp. 78-84

Concerns and solutions in non-target arthropod regulatory risk assessment of plant protection products

Pierre-François Chaton, Christine Vergnet & Anne Alix Afssa (Agence Française de Sécurité Sanitaire des Aliments), Direction du Végétal et de l’Environnement, 10 rue Pierre Curie, 94704 Maisons-Alfort Cedex, France

Abstract: According to the Directive 91/414/EC, the risks of plant protection products to non-target arthropods have to be assessed in the field and outside the field (off-field area). For the products that require a refined risk assessment, an evaluation of all risk types relies on many extrapolations from available experimental data, especially for the off-field assessment. Due to these extrapolations, some concerns could occur in the assessment, like the questionable relevance of the tested species. The introduction of off-field specific topics in the existing tests and the use of model could be helpful to solve these concerns.

Key words: non-target arthropod, risk assessment, plant protection product

Principles of risk assessment to non-target arthropods for plant protection products

Criteria for the regulatory risk assessment to non-target organisms for plant protection product (PPP) in the European Union (EU) are established by the Directive 91/414/EC. Data requirements for active substances and formulated products are listed in Annexes II and III respectively, and decision making criteria are proposed in Annex VI. According to Annex VI, “where there is a possibility of beneficial arthropods other than honeybees being exposed, no authorization shall be granted if more than 30 % of the test organisms are affected in lethal or sublethal laboratory tests conducted at the maximum proposed application rate, unless it is clearly established through an appropriate risk assessment that under field conditions there is no unacceptable impact on those organisms after use of the plant protection product according to the proposed conditions of use.” This is a stepwise approach since it evolves from laboratory to field studies (the latter corresponding to “the unless clause”). The 30% effect criteria was linked with the former Escort guidance document (Barrett et al., 1994), which brought technical indications on how to conduct laboratory and field testing and on the species to use in tests. This guidance document has further been revised in order to introduce useful recommendations about the risk assessment scheme, particularly with regard to considerations at the population level, including recovery issue (Candolfi et al., 2001). Criteria for effects of 50%, measured under extended laboratory conditions or field studies, were proposed instead of the 30% trigger mentioned in Annex VI. Finally, the Escort 2 document (Candolfi et al., 2001) introduced the distinction between in-field and off-field risk assessments. The in-field risk assessment refers to the field area, which is treated, while the off-field area refers to boundaries, bushes, etc… which receive only a fraction of the application rate of PPP corresponding mostly to the spray drift. As far as protection aims are concerned, the in- field risk assessment focuses on acceptable effect on the NTA of the field and, in the case of impacts at the intended application rate, a possible recovery from the observed effects should

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usually be demonstrated within one year (Candolfi et al., 2001). The off-field risk assessment focuses on acceptable effect on populations being exposed to the drift-rate and, in the case of impacts at the expected drift rate, a possible recovery from the observed effects has to be demonstrated within an ecologically relevant time (Candolfi et al., 2001). The in- and off-field risk assessments are summarized the Figures 1 and 2. The Tier 1 risk assessment is based on the estimation of a Hazard Quotient (HQ), which is the comparison of the estimated exposure and the Lethal Rate 50% (LR50) for the most sensitive indicator species. HQ values are calculated for each standard species for the in-field risk assessment and for the off-field, all of which are compared to the threshold value of 2. If the resulting quotient is greater than or equal to 2 for at least one species, a potential hazard to non-target in-field or off-field arthropods is concluded. Based on Escort 2 discussions, this Tier 1 assessment is expected to be conservative since the laboratory tests are performed with two sensitive species (Vogt, 2000; Candolfi et al., 1999) of which the exposure is maximized on a glass plate. If a refined assessment is still indicating a possible risk for NTA (i.e. one HQ above 2), additional species, at least one for the in-field assessment and two for the off-field assessment, have to be tested. The increased number of test species aims at reducing the uncertainty about the sensitivity of NTA populations. The number of additional species required is higher for the off-field risk assessment, in order to take into account the possible higher species diversity in this area in comparison to the field. In a first step of refinement, some laboratory tests are conducted on artificial and/or natural substrates for the additional species and on natural substrate for the sensitive indicator species affected in Tier 1 testing. The exposure of the tested arthropods to the PPP on natural substrate in extended laboratory studies is more

Figure 1. General scheme of the in-field risk assessment to non-target arthropods

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Figure 2. General scheme of the off-field risk assessment to non-target arthropods. (i): the vegetation distribution factor includes the dilution of the exposure in a vegetated area (3 dimensions). (ii): the correction factor represents the uncertainty with the extrapolation from T. pyri and A. rhopalosiphi as sensitive indicator species to all off-field non-target arthropods.

relevant of the exposure in the environment and could be necessary for substances that induced important effects. In the refined risk assessment, sublethal effects on reproduction or predation are also considered and the 50% trigger applied for both lethal and sublethal effects. In the case where the exposure of NTA under more realistic conditions (i.e. extended laboratory tests) does not demonstrate an acceptable impact, a further refinement is possible by considering the product degradation in aged-residue studies. The aim of these studies is to assess the duration of effects of a PPP to NTA. As an example, potted plants are treated and placed outdoor for residue ageing, and the residual toxicity on arthropods is tested indoor. Residual toxicity may also be investigated under semi-field tests. In these tests, the product is applied on crops outdoor and the toxicity is also tested outdoor. The aged-residue and semi- field tests are generally performed with the arthropod species for which the highest sensitivities had been observed under laboratory and extended laboratory tests. Aged-residue and semi-field tests may also be performed directly after a Tier 1 risk assessment. The highest Tier risk assessment corresponds to field tests. These tests may help in investigating the level of effects of the PPP on field NTA populations under the intended use of the product and thus may be directly used to assess the risks. In field tests, the product should then be applied according to the intended use with regard to application rate, stage of the crop and number of applications. A reduced rate is often applied in order to assess the effect of a drift rate.

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Current limits in the refined risk assessment for NTA

A refined risk assessment is frequently required in the European evaluation of PPP. An analysis was performed for 100 active substance dossiers selected randomly among insecticides, herbicides and fungicides. Out of these dossiers, 63% contained Tier 2 (extended laboratory studies) or higher Tier data being triggered by the results of a Tier 1 risk assessment (Table 1). 98% of these data were used in order to refine the in-field risk assessment and 52% were used to refine the off-field risk assessment. Less substances required a refinement for off-field risks than for in-field risks, mainly because the exposure is reduced off-field in comparison to in-field, and because some intended uses would induce no off-field exposure (e.g. soil treatments with granules …).

Table 1. Analysis of data for 100 active substance dossiers selected randomly among insecticides, herbicides and fungicides.

Number of active substances 100 Number of active substances with higher Tier data 63 Number of active substances with higher Tier risk 62 assessment for in-field Number of active substances with higher Tier risk 33 assessment for off-field

Then, according to European evaluation criteria, around on third of the assessments triggered higher Tier assessments for the off-field area in addition to the in-field area. Problems then arise when the same higher Tier data set is used to solve different risk assessment purposes. For the in-field risk assessment purpose, all higher Tier data generated should in principle be representative of species and exposure conditions (Table 2). The relevance of the species used in the higher Tier studies is more appropriate for the in-field than for the off-field scenario as the protection is mainly focused on beneficial species (i.e.: parasitoids) and tests are performed on beneficial species. The relative sensitivity of the two standard species (A. rhopalosiphi and T. pyri) to PPP has been widely investigated and is proved to be sufficient to cover the risk for the typical in-field populations. The exposure mode is also representative of expected exposure of in-field NTA with regard to the rate of product applied and the application mode (spray direction and further 3- dimensional dilution on foliage), so that the uncertainty is quite low with regard to these aspects. The recovery potential, since it is deduced from field studies on the relevant crops, may not be challenged for in-field risk assessment purposes. Nevertheless a condition for that is that the size of the treated areas involved in the tests are comparable to the common cropped areas, so that the results achieved in plots should in principle be extrapolated to real crop size situations, particularly when the recovery observed relies on recolonization. Data are currently missing to discuss recovery potential for species as a function of the distance of the field to refuges and the relative size of the field compared to refuge areas. It is however expected that the recovery would not occur equally and would be lower in intensive agricultural lands with poor or affected refuge area. 82

Table 2. Relevance of the data from higher Tier studies to address in-and off-field issues (X: yes; ?: questionable).

In-field Off-field Extended laboratory tests Exposure mode X X Tested species X ? Aged residues tests Exposure mode X X Tested species X ? Tested rates X ? Semi-field and field tests Exposure mode X ? Tested species X ? Tested rates X X

Another aspect that is difficult to deal with is the consequence of using beneficial species in the risk assessment when the PPP exerts a direct action on prey/host species. Indeed, any indirect effects on a predator due to low prey populations (food shortage) is generally not well assessed or not considered in the field studies, so that recovery potential may be incompletely assessed. For the off-field risk assessment purpose, the relevance of the species used in the higher Tier studies is more questionable as the protection is focused on all arthropods species and the species diversity and ecological traits encountered is probably higher in off-field areas. The expected differences among communities between the in- and off-field areas lead to uncertainties with regard to the relative sensitivity to PPP of these communities and to the potential for species to recover from effects. Data are currently missing to compare the sensitivity of the two standard species A. rhopalosiphi and T. pyri, and of the other beneficial species on which laboratory tests are performed, to the sensitivity of typical off-field areas species. With regard to ecological aspects, in-field communities are made of species that may adapt to changing habitat due to crop constraints. R-strategy species may be more adapted to these changing environmental conditions, while off-field communities may contain both species with a high reproduction rate (r-strategy) and species with lower reproduction rate (K-strategy) so that again extrapolation of a recovery observed in field tests may hardly be extrapolated to off-field situations. In consequence, further research is needed on the ecology of off-field non-target arthropods especially with regard to the relative species distribution between in-field and off- field habitats and on the relative sensitivity of off-field species to pesticides compared to standard test species. With regard to the exposure route, due to the difference of habitats and condition of exposure, it is difficult to extrapolate the drift rate-related effects observed in the semi-field or field studies to off-field situations. Tests investigating off-field impacts are conducted with lower rates being uniformly applied on a crop. The relevance of uniform applications of the PPP on a crop is questionable for off-field where the vegetation cover is most of the time 83

different, except maybe in the case of an adjacent untreated crop or of an untreated zone in the crop, and where the exposure is represented by an heterogeneous deposit of droplets. The representativeness of the rate tested in the aged residue tests is also questionable for the off-field issue since generally only the rate according to the intended use is tested. Therefore, a recovery/re-colonisation potential at lower rates or drift rates can hardly be extrapolated. Finally, refined off-field risk assessment may conclude that acceptable level of effects can only be achieved through the implementation of mitigation measures. In nearly all the cases, mitigation measures correspond to buffer zones that are deduced from higher Tier studies where the impacts of application rates that correspond to drift rates are acceptable. This approach proposed by Escort 2 is further included in Directive 2003/82/EC through specific safety precaution phrases (Spe3: To protect non-target arthropods/insects respect an unsprayed buffer zone of (distance to be specified) to non-agricultural land). Such measures appear to be easy to include into the risk assessment, since a simple drift value is applied to the application rate to deduce an exposure rate, but remain difficult to implement in agricultural practice by risk manager. Buffer zones are in fact not well described: if they should correspond to a pathway, hedge, grassbuffer strip, zone in the field limits, is not defined. In addition, the characterisation and effectiveness of different kind of “buffer zones” to reduce the risks to NTA would need to be supported by field data. The fact that buffer zone are in most cases deduced from field studies performed at drift rates and thus based on effects on field population does not help in attempts to compare their efficacy as mitigation measures with measured data.

Towards solutions proposal

Once the limits of the current risk assessment for NTA are clearly described, the opportunities to improve the relevance can be proposed for the specific sensitivity, ecology and recovery- related issues. As far as the sensitivity issue is concerned, it is generally approved that the two standard species A. rhopalosiphi and T. pyri are among the most sensitive species (Vogt, 2000; Candolfi et al., 1999) for beneficials arthropods, while their relative sensitivity at a wider range of NTA, including off field species, is not well demonstrated. Further investigation to address the relative sensitivity of off-field species to pesticides compared to standard test species are needed. If such a comparability of sensitivities would be demonstrated, this would make further extrapolation from higher Tier studies to off-field issue easier, particularly in the case where the results of extended laboratory tests or aged residues tests conducted with drift rates are sufficient to demonstrate an acceptable impact. As far as the community issue is concerned, drift rate tested in the field studies could be relevant only if the in-field community would be representative of the off-field community, which is currently not demonstrated. Thus field studies could be simplified by removing the modalities that involve off-field rates, since reduced application rates are in fact not useful for in-field risk assessment and not appropriate, for the reasons mentioned above, for off-field risk assessment. One possibility to cover this would preferably be to introduce samplings in the off-field area of a field study, in order to describe the community that is present and to directly appreciate the impacts of the drift rate on this community. Another possibility could be to use models in order to simulate effects on the non-tested species. This possibility requires adapted models and basic data describing ecological traits of typical off-field species that would be candidate to represent off-field arthropods. Further research in both entomology and modeling (like ALMaSS; Jepsen et al., 2005) is needed to support such an approach. 84

As far as recovery issue is concerned, the extrapolation of the recovery observed for a species to other species that have not been tested in the laboratory or not investigated under field studies is often challenged. Extrapolation is even worse for off-field situations. In this case, the use of model (as an example ALMaSS; Jepsen et al., 2005) could be helpful to solve this question.

Conclusion

From the experience gained over several years of use of current PPP guidelines and data bases, it is concluded that the risk in-field can be adequately assessed with the available data package. The risk off-field is currently more challenged, since many extrapolations are needed and some precisions have to be made on buffer zones (i.e.: implementation, mitigation, …). Nevertheless, an increase of the number of studies is not felt necessary and an upgrade of existing guidelines with off-field specific topics could be more appropriate. In parallel, more knowledge in population ecology of off-field areas and comparative ecotoxicity of PPP to beneficials and off-field/non beneficials species would help to reduce many uncertainties. Finally, the development of models would bring additional tools to reduce uncertainties that can not be covered by experiments, e.g. for species that can not be tested or for long-term, i.e. several years investigation.

References

Barrett, K.L., Grandy, N., Harrison, E.G., Hassan, S. and Oomen, P. (eds.) 1994: Guidance document on regulatory testing procedures for pesticides with non-target arthropods. SETAC Europe, Brussels. ISBN 0 9522535 2 6. Candolfi, M.P., Barrett, K.L., Campbell, P.J., Forster, R., Grandy, N., Huet, M.C., Lewis, G., Oomen, P.A., Schmuck, R. and Vogt, H. (eds.) 2001: Guidance document on regulatory testing and risk assessment procedures for plant protection products with non-target arthropods. From the ESCORT 2 workshop. SETAC, Pensacola, 46 p. Candolfi, M., Bakker, F., Cañez, V., Miles, M., Neumann, C., Pilling, E., Priminani, M., Romijn, K., Schmuck, R., Storck-Weyhermüller, S., Ufer, A. and Waltersdorfer, A. 1999: Sensitivity of non-target arthropods to plant protection products: could Typhlodromus pyri and Aphidius spp. be used as indicator species? Chemosphere 39: 1357-1370. Commission Directive 2003/82/EC of 11 September 2003 amending Council Directive 91/414/EEC as regards standard phrases for special risks and safety precautions for plant protection products. Official Journal L228: (12 September) (2003). Directive 91/414/EEC, Council Directive of 15 July 1991, concerning the placing of plant protection products on the market (91/414/EEC). Official Journal L230: (19 August) (1991). Jepsen, J.U., Topping, C.J., Odderskær, P. and Andersen, P.N. 2005: Evaluating consequences of land-use strategies on wildlife populations using multiple-species predictive scenarios Agr. Ecosyst. Environ. 105: 581-594. Vogt, H. 2000: Sensitivity of non-target arthropods species to plant protection products according to laboratory results of the IOBC WG “Pesticides and Beneficial Organisms”. IOBC/wprs Bulletin 23 (9): 3-15. Pesticides and Beneficial Organisms IOBC/wprs Bulletin Vol. 35, 2008 pp. 85-91

Toxicity of certain pesticides to the predatory mite Euseius finlandicus (Acari: Phytoseiidae)

Georgios D. Broufas1, Maria L. Pappas2, George Vassiliou1, Dimitrios S. Koveos2 1 Department of Agricultural Development, Democritus University of Thrace, Pantazidou 193, 68 200 Orestiada Greece (e-mail: [email protected]) 2 Laboratory of Applied Zoology and Parasitology, School of Agriculture, Aristotle University of Thessaloniki, 54 124 Thessaloniki, Greece

Abstract: The acute and residual toxicity of certain widely used pesticides in plum orchards in Greece to the predatory mite Euseius finlandicus were determined with laboratory and semi-field experiments. The acute toxicity of the tested products was evaluated under laboratory conditions using detached bean leaf disks which were sprayed with a Potter spraying tower calibrated to approximately 1.5 mg wet deposit per cm2. Protonymphs of E. finlandicus were transferred on the sprayed leaf disks and subsequently pre-imaginal survival, adult survival and fecundity were determined according to the IOBC protocols. Based on mortality and fecundity, the pesticides carbaryl, cypermethrin, acetamiprid, methomyl and deltamethrin were considered as harmful, diflubenzuron slightly harmful and Bacillus thuringiensis as harmless to E. finlandicus. The residual toxicity of the tested pesticides to E. finlandicus was evaluated using 3 year old potted plum trees (cv. Vanilia) which were sprayed till run- off with a hand sprayer and maintained in the field. At regular time intervals of 3, 7, 10, 15, 20 and 25 days after spraying, leaves were detached from the plants and protonymphs of E. finlandicus were transferred on them. Based on the mortality percentages, the pesticides carbaryl, cypermethrin, acetamiprid and methomyl were highly toxic to the predator for more than two weeks, whereas diazinon for 7 to 10 days. These results could be useful for the selection of suitable pesticides for use in integrated pest management programs in orchards in northern Greece.

Keywords: Toxicity, residual toxicity, Euseius finlandicus, Phytoseiidae, carbaryl, cypermethrin, acetamiprid, methomyl, deltamethrin, diflubenzuron, Bacillus thuringiensis

Introduction

The predatory mite Euseius finlandicus Oudemans is a widespread arboreal phytoseiid species in Europe, and an important natural enemy of eriophyid and tetranychid mites such as the European red spider mite Panonychus ulmi Koch (Van de Vrie, 1975; Gruys, 1982; Kropczyńska & Petanovic, 1987; Kropczyńska & Tuovinen, 1988; Duso 1992; McMurtry and Croft, 1997; Broufas & Koveos, 2000). In Northern Greece, E. finlandicus is the dominant phytoseiid species in commercial stone fruit orchards comprising an important and potentially an efficient indigenous biocontrol agent of phytophagous mites (Broufas & Koveos, 2000). However, the use of highly toxic, broad spectrum insecticides during growing season for the control of the key insect pests (mainly Lepidopteran ones) may adversely affect the populations of this indigenous phytoseiid predator and disrupt biological control of phytophagous mites. Conservation of indigenous predatory insects and mites as integral part of an Intergrated Pest Management (IPM) program could be accomplished to some extent by using pesticides with lower toxicity and persistence to beneficial organisms. In the last decades a huge amount of data on the side-effects of several pesticides on certain phytoseiid species has been published

85 86

(Hassan et al., 1988; 1994; Blümel & Gross 2001; Pozzebon et al., 2002; IOBC, 2005; Castagnoli et al., 2005; Barbar et al., 2007; Bonafos et al., 2007). However, published data on the effects of pesticides on E. finlandicus are rather limited (Hassan et al., 1988). The objective of this study was to evaluate the acute and residual toxicity of the most commonly used insecticides in plum orchards in Greece to E. finlandicus. These results are a part of an ongoing project concerning the evaluation of the toxicity of the most commonly used pesticides in stone fruit orchards on indigenous phytoseiid species. These results could be useful for the selection and use in IPM projects of those pesticides with the lowest toxicity to predatory mites.

Materials and methods

Mite populations Two months before starting the bioassays, a laboratory stock colony of E. finlandicus was established with approximately 350 mites collected from a commercial plum orchard from the area of Alexandria, Northern Greece. The colony was maintained on detached bean leaves (Phaseolus vulgaris L.) on wet cotton wool inside plastic cups at 25°C and a photoperiod of 16:8 LD, as described by Broufas and Koveos (2001). Adequate quantity of Typha sp. pollen was provided daily as food for the mites.

Pesticides The commercial formulations of the selected pesticides and their respective maximum recommended rate for field application used in the bioassays are shown in table 1. Plants sprayed with dionized water were used as the control group.

Table 1. Commercial names and active ingredients of the pesticides used in the experiments.

Commercial Tested rate Formulation Active ingredient name (g or ml a.i./hL) Profil SG 20 acetamiprid 5 Sevin WP 85 carbaryl 64 Ale EC 10 cypermethrin 4 Decis EC 2.5 deltamethrin 0.75 Diziktol EC 60 diazinon 60 Dedevap SL 50 dichlorvos 100 Dimilin WP 25 diflubenzuron 37.5 Makhteshim SL 20 methomyl 36 Agree WP 3.8 Bacillus thuringiensis subsp. kurstaki 1.9

Toxicological tests Laboratory bioassays Toxicological tests were carried out using a modification of the detached leaf method, according to Oomen (1988). Each test unit consisted of a detached bean leaf disc (4cm in diameter) placed upside down on wet cotton wool inside a plastic Petri dish (5cm in diameter). The leaf disks were sprayed with a calibrated Potter Precision Tower (Burkard Manufacurer®, Rickmansworth, UK) producing a wet deposit of 2mg/cm2. Following 87

pesticide application, spray residues on the leaf disks were allowed to dry for 1 hour and afterwards a sufficient quantity of Typha sp. pollen was added on the leaf surface as food for the mites. Pollen grains were gently sprinkled with a thin camel brush over the leaf disc surface in such a way that no refuges for the mites were created. On each leaf disc 15 protonymphs were transferred on the leaf surface of the experimental units. Subsequently, the experimental units with the mites were transferred and maintained in a climatic room at 25oC and a photoperiod of 16:8 LD. For each treatment 10 replicates of 15 predator protonymphs each were used. Every day the experimental units were inspected under a stereomicroscope and mortality percentages of the predatory mites were recorded. Dead individuals were removed from the experimental units. Cumulative mortality was assessed after exposure for 7 days to the spray residues. Cumulative mortality percentages were calculated by adding the number of predatory mites which had escaped from the leaf bean disks to those which were dead and compared with the number of mites transferred on the leaf disks at the start of the experiments. Mortality percentages were adjusted for the control mortality using Abbott’s formula (Abbott, 1925). Fecundity of the surviving females was assessed from 7th to 14th day following the spray application and mean cumulative number of eggs per female was calculated as described by Blümel et al. (2002). Additionally, the total effect values (E) were calculated, according to Overmeer & Van Zon (1982).

Extended laboratory bioassays (duration of harmful activity) In a second group of experiments the persistence of the pesticides was assessed. Three years old plum potted trees (cv Vanilia), were sprayed till run off with a hand sprayer and subsequently maintained in the field. For each of the selected pesticides the concentration of the spray solution was adjusted to the maximum recommended rate for field application, as shown in Table 1. Each treatment included five trees. Control trees were sprayed with dionized water. At certain time intervals (i.e. 3, 7, 10, 15, 20 and 25 days) following spray application, leaves were cut from the trees and transferred to the laboratory. The leaves were placed upside down in contact with wet cotton wool inside plastic Petri dishes (5cm in diameter). A wet cotton barrier at the edges of each leaf was used to prevent escape of the mites. Each leaf was an experimental unit. On each leaf a sufficient quantity of Typha sp. pollen was added as food for the mites and 15 protonymphs of the predatory mite were transferred with the help of a fine camel hair brush. For each treatment (i.e. pesticide and time interval following the spray application) 10 replicates with 15 predator protonymphs each were used. Subsequently, the leaves with the mites were transferred and maintained in a climatic room at 25oC and a photoperiod of 16:8 LD. Mortality and egg production of the surviving mites were scored, as described above for the laboratory bioassays.

Classification of effect The effect of the different pesticides were categorised according to the IOBC/WPRS (International Organization for Biological and Integrated Control of Noxious Animal and Plants) classification (table 2) (Sterk et al., 1999).

Results and discussion

The effect of the pesticides on E. finlandicus tested under laboratory and extended laboratory conditions are summarized in tables 3 & 4.

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Table 2. IOBC/WPRS classification of pesticides, according to their toxicity and persistence.

Toxicity Persistence Classification % Effect observed Classification Time until loss of toxicity Class 1 Harmless <30% Class A Short Lived < 5 days Class 2 Slightly 30 - 79% Class B Slightly 5 - 15 days Harmful Persistent Class 3 Moderately 80 - 99% Class C Moderately 16 - 30 days Harmful Persistent Class 4 Harmful >99% Class D Persistent > 30 days

Table 3. Toxicity of certain selected pesticides to the predatory mite E. finlandicus (laboratory tests).

Toxicity Pesticide Mortality (%) *E (%) Class** acetamiprid 91.3 34.0 2 carbaryl 90.0 85.3 3 cypermethrin 100 - 4 deltamethrin 100 - 4 diazinon 90.4 44.1 2 dichlorvos 90.0 81.1 3 diflubenzuron 25.1 25.1 1 methomyl 100 - 4 B. thuringiensis subsp. kurstaki 5.6 9.5 1

Note: * E= overall effect, ** According to Table 2

Only diflubenzuron and B. thuringiensis were classified as harmless and short lived under field conditions (tables 3 & 4). Similar results have been reported for other phytoseiid species such as Typhlodromus pyri and Amlyseius andersoni (Sterk et al., 1999; Hassan et al., 1988). Among the rest of the tested insecticides acetamiprid and diazinon were classified as slightly harmful with slightly persistent toxic effect under field conditions (tables 3 & 4). According to an earlier report refering to the arboreal predatory species T. pyri, acetamiprid and diazinon were classified as harmless and harmful, respectively (IOBC, 2005). This difference could be related among other reasons to the innate intraspecific differences between the two populations or to some extent to previous exposure of the two populations to different pesticides. In either case, such results may suggest that extrapolation of toxicity side- effect data from one species to another could be rather complicated. Methomyl and the two pyrethroids cypermethrin and delatmethrin at the maximum field recommended rate for application were harmful to E. finlandicus. Furthermore, the toxic 89

effect of the latter insecticides under field conditions was extended to approximately 25 to 30 days. Similar moderate persistence in the toxic effect under field conditions has been recorded in the case of carbaryl and dichlorvos which are classified as moderately harmful (tables 3 & 4).

Table 4. Toxicity of certain selected pesticides to the predatory mite E. finlandicus (extended laboratory tests).

*E (%) after Persistence Pesticide 0 3 7 10 15 20 25 30 Class** days following application acetamiprid 49.3 38.8 30.1 28.7 27.1 11.0 - - B carbaryl 89.1 80.1 75.1 52.7 35.6 21.6 19.6 - C cypermethrin M M M 88.1 66.8 48.5 38.7 22.1 C deltamethrin M M M 83.1 81.5 42.1 28.8 10.0 C diazinon 47.2 38.0 33.6 26.2 21.1 14.3 - - B dichlorvos 88.6 65.5 44.9 36.5 31.2 19.9 - - C diflubenzuron 28.5 20.1 ------A methomyl M 86.2 64.0 67.7 40.9 35.2 23.6 8.1 C B. thuringiensis 7.9 ------A subsp. kurstaki Note: M= 100% mortality, * E= overall effect, ** According to Table 2

According to our results among the tested insecticides methomyl, cypermethrin, delatmethrin, carbaryl and dichlorvos should not be recommended for use in an IPM program in plum fruit production in Northern Greece, due to their high toxicity and persistence under field conditions. In a second group of tested pesticides, acetamiprid and diazinon were slightly harmful to E. finlandicus, whereas persistence of their toxic effect under field conditions was approximately 1 week. These two insecticides could be used early in March before the predatory mites leave their hibernation sites (Broufas et al. 2002) and therefore will be protected from exposure to toxic residues or even throughout the growing season without dramatically affecting field populations of E. finlandicus. However, further field experiments are required in order to prove such hypothesis. From the tested pesticides, only diflubenzuron and B. thuringiensis subsp. krustaki were harmless and thus could be incorporated in IPM programs without interfering with the indigenous phytoseiid predator E. finlandicus.

Acknowledgements

This study is a part of a project ‘Pythagoras I’ founded by European Union – European Social Fund & National Resources EPEAEK. 90

References

Abbott, W.S. 1925. A method of computing the effectiveness of an insecticide. J. Econ. Entomol. 18: 265-267. Barbar, Z., Tixier, M.-S. & Kreiter, S. 2007. Assessment of pesticide susceptibility for Typhlodromus exhilaratus and Typhlodromus phialatus strains (Acari: Phytoseiidae) from vineyards in the south of France. Exp. Appl. Acarol. 42: 95-105. Blümel, S. & Gross, M. 2001. Effect of pesticide mixtures on the predatory mite Phytoseiulus persimilis A.H. (Acarina, Phytoseiidae) in the laboratory. J. Appl. Entom. 125:201-205. Blümel, S., Bakker, F.M., Baier, B., Brown, K., Candolfi, M.P., Goßmann A., Grimm, C., Jäckel, B., Nienstedt, K., Shirra, K.J., Ufer, A. & Waltersdorfer, A. 2002. Laboratory residual contact test with the predatory mite Typhlodromus pyri Scheuten (Acari: Phytoseiidae) for regulatory testing of plant protection products. In: Candolfi, M.P. et al. (eds.): "Guidelines to evaluate side-effects of plant protection products to non-target arthropods" IOBC/WPRS; Gent, Belgium: 158 pp. Bonafos, R., Serrano, E., Auger, P. & Kreiter, S. 2007. Resistance to deltamethrin, lambda- cyhalothrin and chlorpyriphos-ethyl in some populations of Typhlodromus pyri Scheuten and Amblyseius andersoni (Chant) (Acari: Phytoseiidae) from vineyards in the south- west of France. Crop Protection 26: 169-172. Broufas, G.D. & Koveos, D.S. 2000. Functional response of Euseius finlandicus and Amblyseius andersoni to Panonychus ulmi on apple and peach leaves in the laboratory. Exp. Appl. Acarol. 24: 247-256. Broufas, G.D. & Koveos, D.S. 2001. Development, survivorship and reproduction of Euseius finlandicus (Acari: Phytoseiidae) at different constant temperatures. Exp. Appl. Acarol. 25: 441-460. Broufas, G.D., Koveos, D.S. & Georgatsis, D.I. 2002. Overwintering sites and winter mortality of Euseius finlandicus (Acari: Phytoseiidae) in a peach orchard in northern Greece. Exp. Appl. Acarol. 26: 1-12. Castagnoli, M., Liguori, M., Simoni, S. & Duso, C. 2005. Toxicity of some insecticides to Tetranychus urticae, Neoseiulus californicus and Tydeus californicus. BioControl 50: 611-622. Duso, D. 1992. Biological control of the tetranychid mites in peach orchards of northern Italy: Role of Amblyseius andersoni (Chant) and Amblyseius finlandicus (Oud.) (Acari: Phytoseiidae). Acta Phytophathol. Entomol. Hungarica 27: 211-217. Gruys, P. 1982. Hits and misses: The ecological approach to pest control in orchards. Ent. Exp. Appl. 31: 70-87. Hassan, S.A., Bigler, F., Bogenschütz, H., Boller, E., Brun, J., Chiverton, P., Edwards, P., Mansour, F., Naton, E., Oomen, P.A., Overmeer, W.P.J., Polgar, L., Rieckmann, W., Samsøe-Petersen, L., Stäubli, A., Sterk, G., Tavares, K., Tuset, J.J., Viggiani, G. & Vivas, A.G. 1988. Results of the fourth joint pesticide testing programme carried out by the IOBC/WPRS-Working Group “Pesticides and Beneficial Organisms”. J. Appl. Ent. 105: 321-329. Hassan, S.A., Bigler, F., Bogenschütz, H., Boller, E., Brun, J., Calis, J.N.M., Coremans- Pelseneer, J., Duso, C., Grove, A., Heimbach, U., Helyer, N., Hokkanen, H., Lewis, G.B., Mansour, F., Moreth, L., Polgar, L., Samsøe-Petersen, L., Sauphanor, B., Stäubli, A., Sterk, G., Vainio, A., van de Veire, M., Viggiani, G. & Vogt, H. 1994. Results of the sixth joint pesticide testing programme of the IOBC/WPRS-Working Group “Pesticides and Beneficial Organisms”. Entomophaga 39: 107-119. 91

IOBC 2005. IOBC Database on Selectivity of Pesticides. http://www.iobc.ch/2005/IOBC_Pesticide%20Database_Toolbox.pdf. Kropczyńska, D. & Petanovic, R. 1987. Contribution to the knowledge of the predacious mites (Acari, Phytoseiidae) of Yugoslavia. Biosistematika 13: 81-86. Kropczyńska, D. & Tuovinen, T. 1988. Occurrence of phytoseiid mites (Acari: Phytoseiidae) on apple trees in Finland. Annal. Agric. Fenniae 27: 305-314. McMurtry, J.A. & Croft, B.A. 1997. Life-styles of phytoseiid mites and their roles in bio- logical control. Ann. Rev. Entomol. 42: 291-321. Oomen, P.A. 1988. Guideline for the evaluation of side-effects of pesticides on Phytoseiulus persimilis. IOBC/WPRS Bulletin 11 (4): 51-64. Overmeer, W.P.J. & Van Zon, A.Q. 1982. A standardized method for testing the side effects of pesticides on the predacious mite Amblyseius potentillae (Acari: Phytoseiidae). Entomophaga 27: 357-364. Pozzebon, A., Duso, C. & Pavanetto, E. 2002. Side effects of some fungicides on phytoseiid mites (Acari, Phytoseiidae) in north-Italian vineyards. Anzeiger für Schädlingskunde 75: 132-136. Sterk, G., Hassan, S.A., Baillod, M., Bakker, F., Bigler, F., Blümel, S., Bogenschütz, H., Boller, E., Bromand, B., Brun, J., Calis, J.N.M., Coremans-Pelseneer, J., Duso, C., Garrido, A., Grove, A., Heimbach, U., Hokkanen, H., Jacas, J., Lewis, G., Moreth, L., Polgar, L., Roversti, L., Samsøe-Petersen, L., Sauphanor, B., Schaub, L., Stäubli, A., Tuset, J.J., Vainio, A., Van de Veire, M., Viggiani, G., Viñuela, E. & Vogt, H. 1999. Results of the seventh joint pesticide testing programme carried out by the IOBC/WPRS- Working Group “Pesticides and Beneficial Organisms”. BioControl 44: 99-117. Van de Vrie, M. 1975. Some studies on the predator prey relationship in Amblyseius potentillae Garmans, A. finlandicus Oud. and Panonychus ulmi (Koch) on apple. Parasitica 31: 43-44.

Pesticides and Beneficial Organisms IOBC/wprs Bulletin Vol. 35, 2008 pp. 92-95

Side effects of pesticides used in vineyards in the Aegean region on the predatory mite Typhlodromus perbibus Wainstein&Arutunjan (Acari: Phytoseiidae) under laboratory conditions

M. Ali Göven, Bilgin Güven Bornova Plant Protection Research Institute, Gençlik Street 6, 35040, Izmir, Turkey, e-mail: [email protected]

Abstract: In this study the side-effects of pesticides on predatory mite Typhlodromus perbibus Wainstein et Arutunjan were tested under laboratory conditions during the period 2002-2004. The susceptible life stages of T. perbibus were exposed to fresh residues on glass of five commonly used pesticides in vineyards. The tests were conducted according to the standard laboratory test method of the IOBC/WPRS working group “Pesticides and Beneficial Organisms”(Blümel et al., 2000). In laboratory tests, Thiovit (a.i. sulphur) was the only compound demonstrating moderately toxic effect. Ekalux (a.i. quinalphos), Folidol M (a.i. parathion-methyl), Korvin (a.i. carbaryl) and Antracol (a.i. propineb) showed harmless effect.

Key words: Typhlodromus perbibus, vineyard, pesticides, side-effects, laboratory

Introduction

Typhlodromus perbibus Wainstein et Arutunjan (Acari: Phytoseiidae) was found as a wide- spread predator of spider mites and was recorded as a new species of Turkish fauna in vineyards of Aegean region (Göven et al, 1999). In vineyards of Aegean region under IPM program 10-11 pesticides applications are applied, but in conventional vineyards number of pesticides applications is about 19 and in this situation, predatory mite populations have been seriously reduced (Göven et al, 2002). The purpose of our study was to detect the side-effects of some pesticides used in vineyards on the predatory mite T. perbibus.

Material and methods

The tests were conducted according to the standard laboratory test method of the IOBC/ WPRS working group “Pesticides and Beneficial Organisms”(Blümel et al., 2000). The IOBC classification is based on the mortality values of Boller et al. (2006). Insect rearing T. perbibus was reared in the laboratory at 25 ± 2°C, 70% rel. humidity and 16 h day light conditions. Broad bean (Vicia faba L.) pollen and two spotted spider mite Tetranychus urticae Koch were used as food of T. perbibus. T. urticae was reared on potted bean (Phaseolus vulgaris L.) plants at the same conditions. Typhlodromus perbibus rearing unit A rearing unit consists of a plastic water container, water saturated foam within the container, black plexiglass plate with 2 holes situated on top of the foam and cotton tapers put in the holes for the water supply.

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Test units and number of replications One test unit consists of a glass plate (50 cm²) put on water-saturated foam within the container with the pesticide sprayed on its upper surface. The test arena on a test unit was 12 cm² with 1 hole in the middle and cotton taper put in the hole for the water supply. In order to keep protonymphs within the test unit after the application of the PPP the test arena were bordered as a square by a barrier of non-drying glue gel (Tangle-Trap). The experimental design included 3 treatments: the test substance, a reference substance (Dimethoate (Poligor) 400g a.i./l EC) and a water treated control. Each treatment consisted of 5 units (= 5 replicates) with 20 (24 h old) protonymph larvae of T. perbibus per unit. Application of pesticides The glass plates were sprayed by using a little hand sprayer with adjustable angle full cone spray type (0.2 mm nozzle orifice, at the pressure of 1.5 atm.) at the highest recommended per hectar rate of the test substance in a water volume of 200 l/ha. In order to ensure standard pesticide amount of 2 mg fluid/cm² glass plates were weighed before and after application. Glass plates with dried pesticide film were put on the foam and 20 protonymphs were transferred with a fine brush on to the plates. The pesticides were tested with the rates as indicated in Table 1.

Table 1. Pesticides tested on T. perbibus under laboratory conditions.

Pesticide amount Active ingredient Trade name per ha (formulated Mode of action product) sulphur, 80% Thiovit 4000 g Multi site activity parathion-methyl, 360 g/l Folidol M EC 360 1000 ml Cholinesterase inhibitor quinalphos, 250 g/l Ekalux 1250 ml Cholinesterase inhibitor carbaryl, 85 % Korvin 85% 1200 g Cholinesterase inhibitor propineb, 70 % Antracol WP 70 1000 g Multi site activity dimethoate, 400 g/l (reference item) Poligor 1000 ml Cholinesterase inhibitor

Validity criteria Control Mortality rate: Maximum acceptable mean mortality in the control should not exceed 20 % on day 7 after treatment application. Reproduction: The cumulative mean number of eggs per female in the control from day 7 to day 14 should be ≥4 eggs /female. Reference item The cumulative mean mortality (control corrected) of protonypmhs on day 7 should range between 50% and 100%.

Observations Mortality assessment Mortality on day 7 after treatment was assessed by adding the number of predatory mites which had escaped to the number of those which had died. Finally, mortality due to the residual action of the pesticide was obtained by correcting the values observed in the control series according to Abbott (1925). 94

Reproduction assessment The mean cumulative number of eggs per female was determined by counting the number of females and eggs on 3 assessment days (day 7, 10 and 14). Reproduction performance was expressed as percent reduction obtained from the expression given below.

R = (1–Rt/Rc) x 100%

Rt = Reproduction in the treatment groups Rc = Reproduction in the control groups

Results

Mortality Classification of tested pesticides was done according to the corrected mortality values (Table 2). As to validity criteria, maximum acceptable cumulative mortality rate and the cumulative mean number of eggs per female were met in the control. Futhermore, the level of mortality in the reference item treatment was above 50%. For that reason, the effects were considered as treatment related.

Table 2. Effects of pesticides to T. perbibus.

Treatments Control Dimethoate Test Item IOBC class* Active ingredient Mortality (reference Corrected (%) item) Mortality Mortality(%) (Abbott%) sulphur, 80% 10 88.9 36.0 2 (M) parathion–methyl, 360 g/l 20 75.0 27.0 1 (N) quinalphos, 250 g/l 20 81.2 21.0 1 (N) carbaryl, 85 % 15 70.1 28.0 1 (N) propineb, 70 % 20 75.0 3.0 1 (N) * Laboratory (Boller et al. 2006): 1 = harmless, not toxic (< 30 %), 2 = slightly to moderately harmful (30-79 %), 3 +4= harmful, toxic (>80 %)

Table 3. Effects of pesticides on reproduction of T. perbibus.

Active ingredient Fecundity Eggs/female/7day Reduction% IOBC class sulphur, 80 % 5.9 0 1 parathion–methyl, 360 g/l 6.5 27 1 quinalphos, 250 g/l 4.6 27 1 carbaryl, 85 % 5.5 11 1 propineb, 70% 4.3 46 2 95

Reproduction Sulphur showed no effect on reproduction. Parathion-methyl, quinalphos and carbaryl were harmless, and propineb showed slight effects (Table 3).

Conclusion

In this study it should be taken into consideration that organophosphate and carbamate pesticides had no adverse effect on T. perbibus. Duso et al. (1992) found a definite resistance in Italian T. pyri and A. andersoni strains to parathion, azinphos-methyl and carbaryl. Bonafos et al. (2007) confirmed in their studies resistance in the two predatory mites, T. pyri and A. andersoni to the organophosphate chlorpyriphos-ethyl. There is no available data with regard to resistance in T. perbibus; therefore these results are not sufficient to say that this predatory mite has definitely shown resistance to organophosphates and carbamates. For this reason it is concluded that studies to identify the resistance of T. perbibus are required.

References

Abbott, W.S. 1925: A method of computing the effectiveness of an insecticide. J. Econ. Entomology. 18: 265-267. Boller, E.F., Vogt, H., Ternes, P. and Malavolta, C. 2006: IOBC Database on Selectivity of Pesticides. Tool Box for Organisations seeking IOBC Endorsement, 5: http://www.iobc.ch/toolbox.html. Bonafos, R., Serrano, E., Auger, P. & Kreiter, S. 2007: Resistance to deltamethrin, lambda- cyhalothrin and chlorpyrifos-ethyl in some populations of Typhlodromus pyri Scheuten and Amblyseius andersoni (Chant) (Acari: Phytoseiidae) from vineyards in the south- west of France. Crop Protection 26: 169-172. Blümel, S., Bakker, F.M., Baier, B., Brown, K., Candolfi, M.P., Gobman, A., Grimm, C., Jäckel, B., Nienstedt, K., Schirra, K.J., Ufer, A. and Waltersdorfer, A. 2000: Laboratory residual contact test with the predatory mite Typhlodromus pyri Scheuten (Acari, Phytoseiidae) for regulatory testing of plant protection products. In: Candolfi et al. (Eds): Guidelines to evaluate side-effects of plant protection products to non-target arthropods. IOBC, BART and EPPO Joint Initiative, IOBC Gent, 158 p.. Duso, C., Camporese, P. and van der Geest, L.P.S. 1992: Toxicity of a number of pesticides to strains of Typhlodromus pyri and Amblyseius andersoni (Acari: Phytoseiidae) BioControl 37: 363-372. Göven, M.A., Çobanoğlu, S., Güven, B. and ve Topuz, M. 1999: Ege Bölgesi Bağ Alanlarındaki Faydalı Akar Faunası Üzerinde Araştırmalar. Türkiye 4 Biyolojik Mücadele Kongresi, Adana, 491-501. Göven, M.A., Güven, B. and ve Çobanoğlu, S. 2002: İzmir (Menemen) ve Manisa (Saruhanlı) illerinde entegre ve geleneksel mücadele programı uygulanan bağların phytoseid (Acarina: Phytoseidae) populasyonları yönünden değerlendirilmesi. Türkiye 5. Biyolojik Mücadele Kongresi, Erzurum.

Pesticides and Beneficial Organisms IOBC/wprs Bulletin Vol. 35, 2008 pp. 96-100

Effects of ten pesticides to Anystis baccarum (Acari: Anystidae)

Noubar J. Bostanian, Marie-Claude Laurin Horticulture Research and Development Center, Agriculture & Agri-Food Canada, 430 Gouin Blvd., St. Jean-sur-Richelieu, Quebec, Canada, J3B 3E6; Institut des Sciences de l’Environnement, Université du Québec à Montréal, C.P. 8888, Succursale Centre-ville, Montréal, Québec, Canada, H3C 3P8

Abstract: Anystis baccarum (L.) (= Anystis agilis (Banks)) is a common predatory mite recently identified in apple orchards and in vineyards of Quebec, Canada. Studies of its susceptibility to pesticides used in these crops need to be carried out to encourage integrated pest management programs in these crops. A laboratory evaluation of the following insecticides: methoxyfenozide (Interprid® 2F), acetamiprid (Assail® 70WP), thiamethoxam (Actara® 25WG), imidacloprid (Admire® 24%), spinosad (Tracer® 44.1%), λ-cyhalothrin (Warrior® T), and carbaryl (Sevin® XLR) showed that residues of λ-cyhalothrin, and carbaryl were highly toxic to Anystis baccarum in 48 h Petri dish bioassays. The label rate of λ-cyhalothrin is 9.9 g A.I /ha applied in 540 liters of water (0.0184 g A.I./L) which would be 26 fold the estimated LC50 (0.0007 g A.I./liter) for this predator. The field rate for carbaryl is 1.06 kg A.I / ha again applied in 540 liters of water (1.960 g A.I./liter) and it would be 784 fold the estimated LC50 (0.0025 g A.I./liter). The other five insecticides, evaluated were non-toxic. Among the three fungicides evaluated mancozeb (Dithane® M-45) was slightly to moderately toxic. Whereas kresoxim-methyl, (Sovran® 50WG) and sulphur, (Microscopic sulphur® 92WP) were non toxic.

Key words: Anystis baccarum, neonicotinoids, carbamates, pyrethroids, spinosad, methoxyfenoxide, fungicides

Introduction

Predacious mites are vital for the success of integrated pest management programs for phytophagous mites in apple orchards and vineyards. Most of these programs are either based on conservation and augmentation or the rearing and massive releases of different species of phytoseiids. Anystis baccarum (L.) = Anystis agilis (Banks)) (Acari: Anystidae) is a voracious generalist, feeding on any prey that it can overpower. It is a relatively large fast-moving, orange-red mite (Smith Meyer and Ueckermann, 1987). It is found on agricultural crops grown from temperate to sub-tropical regions. In Canada, Anystis sp. was first reported feeding on Panonychus ulmi (Koch) (Acari: Tetranychidae) on peach trees in southern Ontario (Putman and Herne, 1966). Around Moscow, Russia, A. baccarum has been the most common predacious mite feeding on phytophagous mites on blackcurrants (Lange et al., 1974). Cuthbertson et al., (2003a, 2003b) reported it as a key predator of Aculus schlechtendali (Nalepa) (Acari: Eriophyidae) and Rhopalosiphum insertum (Walker) (Hemiptera: Aphididae) in Northern Ireland. Extensive toxicological tests have been carried out to elucidate the toxicity of pesticides to phytoseiids, but few to A. baccarum. Bushkovskaya (1974) carried out laboratory studies and reported carbaryl and dimethoate to be very toxic to A. baccarum. In the same study, she showed that field treatments of these

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insecticides with zineb, copper sulphate and colloidal sulfur fungicides completely wiped out the predator population. A similar devastating impact on A. baccarum was noted when multiple applications of azinphosmethyl, phosmet, chlorfenvinphos or a single application of cypermethrin was applied to kiwi trees in New Zealand (Ferguson et al. 1978). Field studies have also demonstrated that multiple applications of mancozeb and captan/penconazole used to control scab in apples had a detrimental impact on A. baccarum (Cuthbertson and Murchie 2003). In this study we report the residual toxicity of fresh residues of the insecticides imidacloprid (Admire® 24%), acetamiprid (Assail® 70WP), thiamethoxam (Actara® 25WG), methoxyfenozide (Intrepid® 2F), spinosad (Tracer® 44.1), carbaryl (Sevin® XLR) and λ- cyhalothrin (Warrior® T), and the fungicides sulphur (Microscopic Sulphur® 92WP), kresoxim-methyl (Sovran® 50WG), and mancozeb (Dithane® M-45) to A. baccarum adults under laboratory conditions.

Materials and methods

Field collection Specimens were collected from the AAFC experimental farm at Frelighsburg, Quebec. Acaricides and insecticides were not applied in this block for two seasons prior to this study. The field collection was made by tapping branches with A. baccarum into a 5-liter bucket and then transferring individual mites into a 30 ml Solo® plastic cup (Urbana, Illinois, USA). Each cup contained a single specimen because of their cannibalistic behaviour. The cups were placed in a cooler and brought to the laboratory within the hour following the termination of the collection. Residual toxicity tests As a pilot experiment with all the compounds showed no difference in mortality between the naked plastic floor and a leaf disc placed in the petri dish, we used plastic petri dishes (50 mm in diameter) as cages for the treatment of the mites, in order to simplify the system. The cover of each cage had a 30 mm wide circular window to help prevent condensation of water in the Petri dish. Each window was covered with a 40 micron mesh Pecap® polyester screen (Tetko Inc, New York, New York, USA). A thin-layer chromatography sprayer set at 10.3 kPa was used to apply the pesticides. The label rates g AI/ha for orchards were converted to concentrations of g AI/L on the premise that in eastern Canada concentrated sprays are applied in orchards at the rate of 540 or 600 liters of sprayable material per hectare and for dilute sprays 1000 L/ha are applied. The pesticides were first evaluated at their recommended converted label concentrations (g A.I./liter) and whenever LC50 values were to be estimated, they were evaluated at over and/or below these field concentrations. The concentrations for spinosad and acetamiprid were based on dilute applications of 1000 L/ha, whereas the concentrations of the remaining compounds were based on concentrated applications of 540 L/hectare. Mites were released into the treated petri dishes after drying of the residues. The treated cages with the A. baccarum were stored in a growth chamber set at 21°C, 80% RH and 16:8 light:dark photoperiod. As survival of A. baccarum without any prey was excellent for 96 h, mortality counts were made at 48h. Only one specimen was introduced in each treated cage to eliminate cannibalism. Treated specimens were considered dead when they were unable to move about 1mm when the cage was shaken. The toxic pesticides were replicated three times with 30 mites per replicate. The non-toxic pesticides were replicated two times with 30 mites per replicate. Probit analyses were carried out on the mortality data with Polo PC (LeOra Software, 1994) for the toxic pesticides. Mortality was corrected according to Abbott (1925).

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Results and discussion

Even when applied at several fold the recommended field rates, imidacloprid, acetamiprid and thiamethoxam as well as, methoxyfenozide and spinosad caused no residual toxicity to adult A. baccarum (Table 1). Imidacloprid, acetamiprid and thiamethoxam are neonicotinoids also known as chloronicotinyl insecticides. They manifest their toxic action as powerful agonists of the nicotinic acetylcholine receptors (Nauen et al., 1999).

Table 1. Residual toxicity of ten pesticides to Anystis baccarum adults following 48hrs of exposure.

Active ingredient Pesticide Field rate LC50 Times (A.I.) (g A.I. / liter) Methoxyfenozide Insecticide 0.2260 - - Spinosad Insecticide 0.0804 - - Acetamiprid Insecticide 0.1543 - - Imidacloprid Insecticide 0.1689 - - Thiamethoxam Insecticide 0.1778 - - Sulphur Fungicide 4.140 - - Kresoxim-methyl Fungicide 0.300 - - Mancozeb Fungicide 1.600 1.8768 0.9 X λ-cyhalothrin Insecticide 0.0184 0.0007 26 X Carbaryl Insecticide 1.960 0.0025 784 X

In an earlier study, Agistemus fleschneri Summers adults collected from the same plot showed only 16.7% mortality when treated by the slide dip method with imidacloprid at four times the recommended field rate (Bostanian and Larocque, 2001). Unacceptable toxicity by neonicotinoids to predacious mites has also been reported. Acetamiprid and imidacloprid were reported to be moderately toxic to Neoseiulus fallacis (Garman) in North Carolina USA, whereas thiamethoxam was non-toxic (Villanueva and Walgenbach 2005). Methoxifenozide is a diacylhydrazine insecticide that mimics the naturally-occurring insect growth regulator hormone. Once ingested, it causes premature molting and death of the larvae. Even at 32 fold the label rate, methoxyfenozide was non-toxic to A. baccarum (Table 1). Spinosad is a biological insecticide produced in the aerobic fermentation of Saccharopolyspora spinosa, a soil bacterium. It causes depolarization of the neurons at the nicotinic acetylcholine receptor. This depolarization causes prostration with tremors and paralysis of the poisoned insect. It is non-toxic to A. baccarum at the field rate of 0.0804 g A.I./liter (Table 1). Spinosad has been reported to cause the highest mortality of N. fallacis among eight reduced–risk insecticides (Villanueva and Walgenbach, 2005). Lambda-cyhalothrin, and carbaryl both caused 100% mortality to A. baccarum when applied at the recommended field concentrations. Lambda-cyhalothrin is a pyrethroid and it interferes with normal axonic transmission by delaying the closing of a small percentage of the sodium channels. This delay causes postsynaptic hyper stimulation leading to paralysis and death (Stenersen, 2004). Carbaryl is a carbamate and it bonds covalently with acetylcholinesterase. After the passage of an impulse, this bonding prevents the hydrolysis of acetylcholine to acetic acid and choline by the enzyme and it results in the accumulation of acetylcholine. This causes repetitive firing, blockage of nerve transmission and death.

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In contrast to the findings of Bushkovskaya (1974) sulphur was non-toxic. In this respect Hoy and Standow (1982) reported Galendromus (Metaseiulus) occidentalis, Phytoseiidae, to have developed resistance to sulphur in California vineyards. It is highly possibly that resistance may have also developed in A. baccarum in Quebec and it should be addressed in a further studies. In addtion to sulphur, kresoxim-methyl was also non-toxic.Whereas, Mancozeb was slightly to moderately toxic at the recommended field concentration (Table 1). In Northern Ireland mancozeb was also reported to be toxic to A. baccarum (Cuthbertson and Murchie, 2003). The LC50 values to A. baccarum along with their appropriate parameters are reported on Table 2. The recommended field concentrations of λ-cyhalothrin, carbaryl and mancozeb are 26, and 784 and 0.9 fold the LC50 estimates respectively (Table 2). Imidacloprid, acetamiprid, thiamethoxam, methoxyfenozide spinosad and the two fungicides sulphur and kresoxim-methyl are harmless to A. baccarum under laboratory conditions. The pronounced toxicity of λ-cyhalothrin, and carbaryl, makes even field testing of these insecticides questionable, in view of developing an IPM program based on A. baccarum.

Table 2. Estimates of LC50 values for three pesticides to Anystis baccarum adults collected in 2005-2006 at Frelighsburg, Quebec, Canada.

2 Insecticides n Slope (±SE) LC50 (CI 95%) df χ g (g A.I./liter) λ-cyhalothrin 90 2.708 (±0.267) 0.0007 13 10.1722 0.0374 (0.0006-0.0008) Carbaryl 90 3.431 (±0.443) 0.0025 13 29.1700 0.1750 (0.0019-0.0032) Mancozeb 60 0.681 (±0.266) 1.8768 8 7.7278 0.4124 (0.1761-3.8179)

Finally these findings are only part of the toxicity attributes of these compounds and effect on fecundity, egg hatch and repellence should be carried out to complete our understanding of the effects of these pesticides.

Acknowledgments

Gaétan Racette is thanked for helping out in the field collections, reviewing and formatting the manuscript. We would like to thanks summer students and trainees for field collection and laboratory works: Myriam Paquette, Maxime Lefebvre (Régime coopératif, Université de Sherbrooke, Sherbrooke, QC, Canada); Mélanie Locu, Vincent Huneau: (ÉSITPA, École d’Ingénieurs en Agriculture, Val de Reuil, France); Frédérique Sevel (ENSHAP, Institut National d’Horticulture, Angers, France); Laurent White, Dany Roy, Judith Paré, Viviane Goyette, Keaven Normadin Racine: (Federal Public Sector Youth Internship Program, Human Resources and Skills Development Canada, YMCA).

References

Abbott, W.S. 1925: A method of computing the effectiveness of an insecticide. J. Econ. Entomol. 18: 265-267.

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Bostanian, N.J. & Larocque, N. 2001: Laboratory tests to determine the intrinsic toxicity of four fungicides and two insecticides to the predacious mite Agistemus fleschneri. Phytoparasitica 29: 215-222. Bushkovskaya, L.M. 1974: The effect of chemicals on the mite Anystis. Zashch. Rast. (Moscow) 10: 53 (in Russian). Cuthbertson, A.G.S.& Murchie, A.K. 2003: The impact of fungicides to control apple scab (Venturia inaequalis) on the predatory mite Anystis baccarum and its prey Aculus schlechtendali (apple rust mite) in Northern Ireland Bramley orchards. Crop Prot. 22: 1125-1130. Cuthbertson, A.G.S., Bell, A.C. & Murchie, A.K. 2003a: Impact of the predatory mite Anystis baccarum (Prostigmata: Anystidae) on apple rust mite Aculus schlechtendali (Prostig- mata: Eriophyidae) populations in Northern Ireland ‘Bramley’ orchards. Ann. Appl. Biol. 142: 107-114. Cuthbertson, A.G.S., Fleming, C.C. & Murchie, A.K. 2003b: Detection of Rhopalosiphum insertum (apple-grass aphid) predation by the predatory mite Anystis baccarum using molecular gut analysis. Agric. For. Entomol.5: 219-225. Ferguson, A.M., Stratton, A.E. & Hartley, M.J. 1978: Insect control on kiwifruit. In: Proceedings of the 31st New Zealand Weed and Pest Control Conference. The New Zealand Weed and Pest Control Society Inc., August 8-10, 1978, Palmerston North, New Zealand. M.J. Hartley (ed.), Part 1: 135-139. Hoy, M.A.& Standow, K.A. 1982: Inheritance of resistance to sulfur in the spider mite predator Metaseiulus occidentalis. Entomol. Exp. Appl.31: 316-323. Lange, A.B., Drozdovskii, E.M & Bushkovskaya, L.M. 1974: The effectiveness of Anystis baccarum in the control of small predatory phytophages. Zashch. Rast. (Moscow) 1: 26- 28 (in Russian). LeOra Software. 1994: Polo-PC, Probit and logit analyses, Berkley, California. Nauen, R., Ebbinghaus U., and Tietjen, K. 1999. Ligands of nicotinic acetylcholine receptor as insecticides. Pestic. Sci. 55: 608-610. Putman, W.L. & Herne, D.H.C. 1966: The role of predators and other biotic agents in regulating the population density of phytophagous mites in Ontario peach orchards. Can. Entomol. 98: 808-820. Smith Meyer, M.K.P. & Ueckermann, E.A. 1987: A taxonomic study of some Anystidae (Acari:Prostigmata). Republic of South Africa Department of Agriculture and Water Supply. Entomology Memoir 68: 1-37. Stenersen, J. 2004: Chemical pesticides; mode of action and toxicology, CRC Press, London, New York. Villanueva, R.T. &. Walgenbach, J.F. 2005: Development, oviposition, and mortality of Neoseiulus fallacis (Acari: Phytoseiidae) in response to reduced-risk insecticides. J. Econ. Entomol. 98: 2114-2120.

Pesticides and Beneficial Organisms IOBC/wprs Bulletin Vol. 35, 2008 pp. 101-108

Influence of some insecticides and acaricides on beneficial mites and on Coccinella septempunctata (Coleoptera; Coccinellidae) larvae

Remigiusz W. Olszak, Małgorzata Sekrecka Research Institute of Pomology and Floriculture, Department of Plant Protection, Pomologiczna Str. 18, 96-100 Skierniewice, Poland

Abstract: During the period 2004-2007 several experiments have been conducted under laboratory and field conditions to assess the influence of a broad range of insecticides and acaricides on different beneficial mites and on Coccinella septempunctata larvae. In the laboratory the toxicity of the insecticides spinosad, methoxyfenozide and triazamate were investigated on the predatory mite Typhlodromus pyri (Phytoseiidae) . In other laboratory experiments the influence of the pesticides spirodiclofen, thiamethoxam, spinosad, propargite and novaluron were investigated on Coccinella septempunctata larvae. In the field, the side-effects of triazamate, thiacloprid, pirimicarb, novaluron, spinosad, indoxa- carb and fenithrotion were studied on the population of Typhlodromus pyri. Additionally, in the field, the toxicity of the acaricides hexythiazox and fenpyroximate were investigated on the predatory mite Zetzellia mali (Stigmaeidae). In a third experiment, carried out in an abandoned orchard, the influence of propargite, pyridaben, cyhexatin, and fenithrotion were studied on the population of beneficial mites belonging to the families Phytoseiidae, Tydeidae, Stigmaeidae and Tarsonemidae. The results of investigations indicate that most of used insecticides were harmless to the predatory mite Typhlodromus pyri and to the larvae of Coccinella septempunctata, with the exception of spinosad being harmful to phytoseiid mites. Similar data were received when the acaricide fenpyroximate was used on population of Zetzellia mali. In the field experiment, carried out in an abandoned apple orchard, all the used chemicals were toxic to beneficial mites.

Key words: beneficial mites, Coccinella septempunctata, insecticide, acaricide, Stigmaeidae, Phyto- seiidae, Tydeidae, Tarsonemidae, Typhlodromus pyri, Zetzellia mali

Introduction

Pesticides enjoy popularity in plant protection due to their fast activity. Up to date, their frequent use may be associated with negative effects, such as the appearance of resistant pest races (Helle and van de Vrie, 1974; Hurkova, 1980), the reduction or total destruction of the population of beneficials (Croft, 1976; Sterk, 1993) and pollution of the natural environment with agrochemicals. That is why studies aiming at popularization of non-chemical methods of controlling agricultural pests (based among other on the use of their natural enemies) are undertaken. However, the use of plant protection products is indispensable. Then it is important to apply products which are safe (selective or partly selective) for beneficials. The aim of our study was to define the influence that selected plant protection products exert on beneficial mites belonging to the families Phytoseiidae, Stigmaeidae, Tydeidae and Tarsonemidae. Laboratory tests also aimed at grasping the effect that some pesticides may exert on survival of second and fourth larval stages of the ladybird species Coccinella septempunctata. The presented results are continuation of studies conducted formerly in our Institute (Niemczyk, 1997; Niemczyk et al., 1998; Olszak, 1982; Olszak, 1999; Olszak et al., 1994).

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Material and methods

Pesticides A total of 16 plant protection products (comprised in the Table 1) was investigated in labora- tory and field tests.

Table 1. List of tested products.

Commercial name Active ingredient Rate kg or l/ha c.p. SpinTor 480 SC spinosad 0.3 l Runner 240 SC methoxyfenozide 0.4 l Actara 25 WG thiamethoxam 0.2 kg Aztec 140 EW triazamate 0.7 l Rimon 100 EC novaluron 0.75 l Calypso 480 SC thiacloprid 0.2 l

Insecticides Insecticides Pirimix 100 PC pirimicarb 1.5 l Steward 30 WG indoxacarb 0.3 kg Owadofos 540 EC fenithrotion 2.25 l

Envidor 240 SC spirodiclofen 0.4 l Omite 570 EW propargite 2 l Nissorun 050 EC hexythiazox 0.9 l Nissorun 10 WP hexythiazox 0.5 kg Ortus 05 SC fenpyroximate 1.l ; 1.5 l Aacaricides Aacaricides Sanmite 20 WP pyridaben 0.75 kg

Pennstyl 600 SC cyhexatin 0.6 l

Laboratory tests on the predatory mite Typhlodromus pyri (Phytoseiidae) In the laboratory tests with T. pyri we used direct overspray of adult females on apple leaf discs. For this purpose, discs cut from apple leaves and with 10 females of T. pyri each were placed at the bottom of a Petri dish on moistened pieces of cotton. Every disc with the predators on it was considered a replication. Five replications were used. The discs with the mites were sprayed in a Potter Tower using 2 ml of water solution of the insecticides spinosad (0.03%), methoxyfenozide (0.04%) and triazamate (0.07%). This corresponds to 27% of the per ha field rate and is within the range of deposits achieved on leaf surfaces in apple orchards. The reason is, that due to the three dimensional structure of orchards the amount applied per ha cannot be directly transfered to the leaves. According to deposit measures in apple orchards, between 10 and 30 % of the per ha rate can be found on leaves (Koch & Weisser, 2001). Control discs were sprayed with water. Treated predatory mites were fed with the spider mite Tetranychus urticae and with the tulip pollen. Both, the predatory and the spider mites came from a laboratory rearing. To evaluate survival and fecundity of the females, the numbers of living females, eggs laid as well as hatched larvae and nymphs were counted during 14 days. The tests were conducted in a growth chamber with following conditions: temperature 250C, relative humidity 75%, photoperiod 16L:8D. 103

The results obtained from the tests were statistically elaborated using the one-sided Walsh test (Siegel, 1956) for checking the significance of the differences between daily means.

Laboratory tests on the ladybird Coccinella septempunctata (L2 and L4) We tested the effect of direct spraying of the coccinellid larvae (L2 and L4). The test unit consisted of filter paper discs put inside circular plastic boxes of 77 mm in diameter. Five ladybird larvae coming from a laboratory mass rearing were then put into each box. One box with the predators placed inside constituted a single replicate (5 replicates per product). The boxes with ladybird larvae inside were then sprayed with a Potter Tower with 2 ml of water solution of the pesticides spirodiclofen (0.04%), thiamethoxam (0.02%), spinosad (0.04%), propargite (0.2%) and novaluron (0.075%). Control boxes were sprayed with water. Survival of the larvae was checked during 7 subsequent days. The test units were placed in a rearing chamber with following conditions: temperature 250C, relative humidity 70% and photo- period 16L:8D. Ladybird larvae were fed on pea aphids (Acyrthosiphon pisum) during the test period. The results of experiments were submitted to statistical analysis by means of ANOVA and Newman-Keuls test. Percentage of ladybird’s larvae mortality was corrected according to Abbott (1925).

Field tests on the beneficial mites The tests were conducted in three apple orchards localized in Central Poland. Experimental plots were fixed for each tested product and for the control. Every plot (replicate) had 3 to 6 trees growing in one row (depending on orchard). Thirty leaves were randomly sampled from each plot and than anlysed in the laboratory by Henderson-McBurnie method (1943) by counting the numbers of mites present on them. Every tested product was applied in 4-5 replicates, depending on the experiment. Control trees were treated with water. The treatments were accomplished using a knapsack turbine-motor sprayer and a spray liquid dose equivalent to 750 l per ha. In one orchard, seven plant protection products: triazamate, thiacloprid, pirimicarb, novaluron, spinosad, indoxacarb, fenithrotion were tested on the mite Typhlodromus pyri. Mortalities at 1, 3 and 5 weeks after treatment were scored. In another orchard, three products: hexythiazox (in two formulations) and fenpyroximate were assayed on the mite Zetzellia mali. Mortality at 2, 4, 6, and 8 weeks after treatment was scored. For both orchards, the products were classified according to IOBC toxicity classes (Hassan et al., 1985). In a third orchard, an abandoned one, the pesticides fenithrotion, propargite, fenpyroximate, pyridaben and cyhexatin were tested on the beneficial mites belonging to the families Phytoseiidae, Stigmaeidae, Tydeidae and Tarsonemidae. Based on pre-treatment samples, trees with similar number of mites were selected for the test. Numbers of living mites at 1, 2 and 3 weeks after treatment were counted. The results of the experiments were submitted to statistical analysis by means of ANOVA and Newman-Keuls test. The percentage of mites mortality was was calculated from results of the treatments and then corrected according to Abbott (1925).

Results and discussion

Laboratory tests on the predatory mite Typhlodromus pyri (Phytoseiidae) In laboratory tests the highest mortality of T. pyri was found on discs treated with spinosad. The insecticide triazamate decreased the survival and fecundity of the females of T. pyri to a 104

moderate degree. No significant differences in mean numbers of the predators have been found between discs treated with methoxyfenozide and untreated ones (Table 2, 3).

Table 2. Effect of direct overspray on adult females of T. pyri ( Phytoseiidae) – laboratory test, Skierniewice 2004.

% mortality Product Concentra- IOBC (active ingredient) tion toxicity class 1 DAA* 3 DAA 7 DAA 14 DAA SpinTor 480 SC 0.03 % 50 84 94 93 3 - 4 (spinosad) Runner 240 SC 0.04 % 0 0 0 3 1 (methoxyfenozide) Aztec 140 EW 0.07 % 2 0 6 7 1 (triazamate) * DAA – day after application

Table 3. Effect of direct overspray on reproduction of adult females of T. pyri ( Phytoseiidae) – laboratory test, Skierniewice 2004.

Mean number of Level of Pesticides Concentra- Mean number of eggs and immature significance (active ingredient) tion living females* (Walsh test) stages* compared to control Control - 8.7 25.2 - SpinTor 480 SC significant 0.03% 1.1 1.3 (spinosad) p=0.005 Runner 240 SC 0.04% 8.6 23.4 insignificant (methoxyfenozide) Aztec 140 EW significant 0.07% 8.2 19.3 (triazamate) p=0.005 * mean of 14 days and 5 replicates

Laboratory tests on the ladybird Coccinella septempunctata (L2 and L4) The laboratory tests showed selectivity of spirodiclofen, thiamethoxam and spinosad to L2 larvae as well as of spirodiclofen, propargite and novaluron to L4 larval stages of the ladybird C. septempunctata (Table 4). 105

Table 4. Effect of some pesticides to larvae of Coccinella septempunctata (Coccinellidae) – laboratory test, Skierniewice 2007

% mortality Product Concentration IOBC toxicity Species 7 days after (active ingredient) (%) class application Coccinella Envidor 240 SC 0.04 9 1 7-punctata (spirodiclofen) larvae L2 Actara 25 WG 0.02 29 1 (thiamethoxam) SpinTor 480 SC 0.04 13 1 (spinosad) Coccinella Envidor 240 SC 0.04 5 1 7-punctata (spirodiclofen) larvae L4 Omite 570 EW 0.2 43 1 (propargite) Rimon 100 EC 0.075 24 1 (novaluron)

Field tests on the beneficial mites In the field experiment almost all of the products revealed low toxicity to predatory mite Typhlodromus pyri. The pesticides triazamate, thiacloprid, pirimicarb, novaluron, indoxacarb and fenithrotion slightly diminished the numbers of predatory mites on leaves of apple trees treated with them, in comparison with these on control trees. The results of the experiment with spinosad were approximately similar to the effects from the laboratory test. For T. pyri the product was scored as belonging to the 3-rd or 4-th class of toxicity according to the IOBC classification. (Table 5). The influence of three acaricides on Zetzellia mali (Stigmaeidae) was examined in a different field experiment. The highest mortality of the predator was found on trees treated with fenpyroximate. The acaricide hexythiazox reduced of Z. mali in comparison with non- treated trees, but not so significantly as fenpyroximate (Table 6). The third field experiment was performed in an apple orchard where no plant protection products had been applied so far. The acaricides propargite, fenpyroximate, pyridaben as well as cyhexatin and fenithrotion strongly reduced the numbers of beneficial mites belonging to the families Phytoseiidae, Stigmaeidae, Tydeidae and Tarsonemidae. The family Stigmaeidae was the most sensitive to the tested acaricides and the insecticide fenithrotion. 106

Table 5. Effect of some pesticides to the predatory mite Typhlodromus pyri (Phytoseiidae) – field test, 2004 (orchard 1).

Mortality Product Rate per ha IOBC (active ingredient) (c.p.) Week(s) after toxicity class % treatment Aztec 140 EW 0.7 l 1 40 2 (triazamate) 3 50 2 5 50 2 Calypso 480 SC 0.2 l 1 0 1 (thiacloprid) 3 50 2 5 17 1 Pirimix 100 PC 1.5 l 1 20 1 (pirimicarb) 3 0 1 5 17 1 Rimon 100 EC 0.75 l 1 40 2 (novaluron) 3 33 2 5 33 2 SpinTor 480 SC 0.3 l 1 80 4 (spinosad) 3 67 3 5 67 3 Steward 30 WG 0.3 kg 1 40 2 (indoxacarb) 3 0 1 5 33 2 Owadofos 540 EC 2.25 l 1 20 1 (fenithrotion) 3 17 1 5 33 2

Table 6. Effect of some pesticides to the predatory mite Zetzellia mali (Stigmaeidae) – field test, 2005 (orchard 2).

Mortality Product Rate per ha IOBC (active ingredient) c.p. Weeks after toxicity class % treatment 2 24 1 Nissorun 050 EC 0.9 l 4 55 3 (hexythiazox) 6 16 1 8 39 2 2 24 1 Nissorun 10 WP 0.5 kg 4 36 2 (hexythiazox) 6 70 3 8 61 3 2 54 3 Ortus 05 SC 1 l 4 98 4 (fenpyroximate) 6 70 3 8 66 3 107

Phytoseiidae Tydeidae

2,5 just before 2,5 just before treatment 2 2 treatment 1,5 1,5

1 1

0,5 0,5

0 0 17.07 24.07 31.07 07.08 17.07 24.07 31.07 07.08

Stigmaeidae Tarsonemidae 2,5 2,5 number of mites / leaf 2 just before 2 just before treatment treatment 1,5 1,5

1 1

0,5 0,5

0 0 17.07 24.07 31.07 07.08 17.07 24.07 31.07 07.08 Date of sampling

Control fenithrotion propargite Control fenithrotion propargite fenpyroximate pyridaben cyhexatin fenpyroximate pyridaben cyhexatin

Figure 1. Mean number of beneficial mites collected in an abandoned apple orchard after treatment with selected pesticides (orchard 3).

References

Abbott, W.S. 1925: A method of computing the effectiveness of an insecticide. J. Econ. Entomol. 18, 265-267. Carnevale, R.A., Johnson, W.A., Reed, C.A. & Past, A. 1991: Agricultural chemical residues in food; evaluating the risks. Agriculture and the Environment. The 1991 Yearbook of Agriculture. US Government Printing Office, Washington D.C.: 227-233. Croft, B.A. 1976: Establishing insecticide-resistant phytoseiid mite predators in deciduous tree fruit orchards. Entomophaga 21(4): 383-399. Hassan, S.A. 1985: Standard methods to test side-effects of pesticides on natural enemies of insects and mites developed by the IOBC/WPRS Working Group “Pesticides and Beneficial Organisms”. – Bulletin OEPP/EPPO 15: 214-255. 108

Helle, W. & van de Vrie, M. 1974: Problems with spider mites. Outlook on Agric. 8(3): 199- 225. Henderson, C.F. & McBurnie, H.V. 1943: Sampling technique for determining populations of the citrus red mites and its predators. U.S. Dept. Agric. Circ. 671. Hurkova, J. 1980: Insecticide resistant Panonychus ulmi in apple orchards in Bohemia (Acari, Tetranychidae). Acta Entomol. Bohemoslovaca 77: 82-88. Koch, H. & Weisser, P. 2001: Spray deposits of crop protection products on plants – the potential exposure of non-target arthropods. Chemosphere 44:307-312. Niemczyk, E. 1997: The occurrence of different groups of phytophagous and predatory mites on apple plots sprayed according to different programs. Zahradnictvi Hort. Sci. Prague, 24(2): 45-52. Niemczyk, E., Olszak, R.W. & Sekrecki, R. 1998: Wpływ selektywnych i nieselektywnych insektycydów na występowanie pożytecznych i szkodliwych stawonogów w sadzie jabłoniowym. Rocz. AR Poznan CCCIV, Ogrod. 27: 183-194. Olszak, R.W. 1982: Impact of different pesticides on ladybird beetles (Coccinellidae, Col.). Rocz. Nauk. Roln. s.E.12: 141-149. Olszak, R.W. 1999: Influence of some pesticides on mortality and fecundity of the aphido- phagous coccinellid Adalia bipunctata L. (Col., Coccinellidae). J. Appl. Ent. 123: 41-45. Olszak, R.W., Pawlik, B. & Zając, R.Z. 1994: The influence of some insect growth regulations on mortality and fecundity of the aphidophagous coccinellids Adalia bipunctata L. and Coccinella septempunctata L. (Col., Coccinellidae). J. Appl. Ent. 117: 58-63. Siegel, S. 1956: Nonparametric Statistics for the Behavioral Sciences. McGraw-Hill Book Company, NewYork: 83-87. Smith, J.A.R. 1991: Food Safety in Agriculture and the Environment. Washington D.C., pp. 22-236 Sterk, G. 1993: Studies on the effect of pesticides on beneficial arthropods. Acta Hort. 347: 233-245.

Pesticides and Beneficial Organisms IOBC/wprs Bulletin Vol. 35, 2008 pp. 109-112

Effect of the entomopathogenic fungus Lecanicillium muscarium on the predatory mite Phytoseiulus persimilis as a non-target organism

András Donka, Helga Sermann, Carmen Büttner Humboldt University of Berlin, Institute of Horticulture Sciences, Department of Phytomedicine, Lentzeallee 55-57, D-14195 Berlin, e-mail: [email protected] berlin.de

Abstract: To combine different beneficial organisms in biological control systems with entomo- pathogenic fungi, it is necessary to examine their compatibility. Regarding the entomopathogenic fungus L. muscarium , the objective of the study was to determine the potential risk of our strain V 24 for the most important antagonist Phytoseiulus persimilis. In standardised trials in petri dishes and on potted plants, the effect of the fungus on predatory mites at different spore densities (2x106 and 2x107 sp./ml) was assessed. We could show that predatory mites can indeed pick up spores from the leaf surface. At spore densities of 2x106 and 2x107 sp./ml however, only few predatory mites died on plants (4,2 respectively 12,7%). There were no differences to the control regarding the development of the mite population on the plant.

Key words: biological control, entomopathogenic fungi, Lecanicillium muscarium, Verticillium lecanii, predatory mite, Phytoseiulus persimilis, mortality and side effect

Introduction

Entomopathogenic fungi are able to infect and kill different arthropods. They are not strongly host specific. In many experiments, our strain V 24 of Lecanicillium muscarium (former Verticillium lecanii) demonstrated a high efficacy infecting different sucking insects as white fly, aphids and thrips. This strain has therefore a high potential for biological control, also in combination with other control methods. In case of application in combination with beneficial arthropods it is possible that individuals come in contact with spores. Therefore it is necessary to examine the susceptibility of non-target organisms to the microbial. This risk was investigated for the predatory mite Phytoseiulus persimilis because of its high importance in biological control. The objective of the study was to determine the susceptibility of the predatory mite as regards mortality and effects on the population development after a random (indirect) contact with spores in relation to spore density. Biotests were run at optimal conditions in petri dishes and at nearby practical conditions on potted plants. The results of the experiments should give the following information: Is the fungus able to infect the predatory mite Phytoseiulus persimilis? If yes, under which conditions and at which grade of efficacy the infection takes place?

109 110

Material and methods

Fungus and mites A conidial spore suspension of Lecanicillium muscarium strain V 24 from our strain collection was used in all experiments. The suspension came from a laboratory production emers at Maltextractagar. Spore densities were chosen from 2x106 to 2x107 per ml. P. persimilis was obtained from Katz Biotech AG. Tetranychus urticae on Phaseolus vulgaris was used as prey and was harvested from our mass breeding. Biotest in petri dish This biotest procedure in petri dish has been standardised over a long time, using detached bean leaves. 20 to 40 mites of T. urticae serving as food were released on the leaves. Then, for each density, 1 ml spore suspension per leaf was applied with a hand sprayer. One prepared bean leaf per dish was laid on a wet filter paper. After drying of the suspension, 2 or 3 adults of P. persimilis were put per leaf. Petri dishes were closed and incubated in a growth chamber at 20°C and 95% RH. Each treatment had 10 replicates. The number of alive, dead or mouldy dead individuals was counted on the 3rd, 5th and 7th day after application. Biotest on plants In a second standardised biotest, seedlings of Ph. vulgaris were singly potted and colonised with T. urticae. Five days after colonisation with spider mites, 3 ml spore suspension per pot were sprayed with a hand sprayer. Two adults of P. persimilis were put on each bean leaf one hour later. 12 pots (replicates) per treatment were put in a plastic cage (40 x 40 x 50 cm). Incubation took place in a growth chamber at 23°C, 60-85% RH and 16 h light. In this experiment, the number of alive, dead, mouldy dead individuals was recorded on the 12th day after application. Spore adhesion on the mite To visualize spore adhesion, fluorescence marked conidia were applied using 1 ml (2x106 sp./ml) spore suspension per leaf. The counting of spores was done at half, one, three and twenty five hours after application.

Treatments are summarized in Table 1.

Table 1. Treatments of L. muscarium on the predatory mite P. persimilis in different conditions and spore densities

Treatment Method Spore density Fluorescence marked conidia of Petri-dish 2x106 sp./ml L. muscarium Spraying suspension Petri-dish 2x106 sp./ml 2x107 sp./ml Potted plant 2x106 sp./ml 2x107 sp./ml Spraying water Petri-dish, Water-treated control Potted plant respectively

111

Results and discussion

Adhesion of spores After spraying of the suspension, individuals of P. persimilis picked up spores from the leaf surface. The number of picked up spores increased up to 3 hours according to the initial spore density. After this time, more spores were lost than new spores adhered to the body. 24 hours after inoculation more than one spore per individual was recorded only in the treatment using 2x107 sp./ml. Numerous individuals did not carry spores at all. Biotest in petri dish Number of dead P. persimilis In all treatments significant more dead individuals of P. persimilis were recorded than in the control. In case of 2x107 sp./ml, death rate of mites was more pronounced than in the treat- ment with 2x106 sp./ml. At the end of the experiment, some predatory mites were also mouldy. Number of living P. persimilis In the course of the experiment, females of P. persimilis laid eggs and the number of offspring increased. In the control, a uniform rising of the population was observed. In the first days after the application of the fungus, more offspring was produced in the treatments than in control, suggesting that the fungus may act as stressor. During the course of the experiment, the effect of fungus was predominant and population density was regressive in case of 2x107 sp./ml. Mortality Mortality increased slowly in consequence of offspring production. The treatment with 2x106 sp./ml did not differ significantly from the control. At higher spore densities, a higher mortality was registered due to lower numbers of offspring. At the end of observation time, mortality of P. persimilis was significantly higher in treatments with 2x107 sp./ml than in the control (Tab. 2). Biotest on plant Number of dead and mouldy P. persimilis In contrast to the treatments in petri dishes, only few individuals of P. persimilis were found dead on the plants. At spore density of 2x106 sp./ml, dead individuals were rare and the difference between control and treatment was not significant. In the treatment with 2x107 sp./ml, somewhat more individuals died than in the treatment with 2x106 sp./ml, but the difference was not significant. In the treatment with 2x107sp./ml up to 75% of the few dead individuals were mouldy, indicating that fungus was the reason of death. Development of population In general, the development of the population on the plants was not so fast and pronounced than in petri dish, probably due to unfavourable humidity conditions for predatory mites on the plants. Nevertheless the population of P. persimilis increased regularly. During the observation time, there was no incidence of a decline in the population due to fungus treatment. Only in the beginning a slight depression in comparison to the control was recognized. Comparative assessment The observed mortality of P. persimilis due to effect of the fungus treatment differed between the testing procedures. On the plant, the mortality was very low and did not increase significantly during the observation period in comparison to the control. In contrast, the difference between observed mortality in petri dish and on plant was significant at the same spore densities (Table 2). 112

Table 2. Corrected mortality of P. persimilis after spraying of spore suspension (2x 106 and 2x107 sp./ml) of L. muscarium in standardised biotest in petri dish and on plant

Spore density Corrected Mortality Biotest method (Sp./ml) % Petri dish 2x106 9,8 2x107 38,1 Water treated control 4,6 Plant 2x106 4,2 2x107 12,7 Water treated control 2,7

Conclusion

Individuals of P. persimilis can pick up spores of L. muscarium from the leaf surface. But due to the high loss of 85 % within 24 hours, only few spores can persist for successful germination. Therefore efficacy depends on the number of spores in suspension and humidity for development of the fungus. Under practical conditions, an indirect contact with spores is more probable for the predatory mite, because their high motility supports spore loss and alternating humidity is unfavourable for the infection process. Nevertheless, the fungus is able to infect and kill the predatory mite. However, results showed that efficacy decreased if the overall conditions approach to the practical situation. Applying practical relevant concentrations of 2x106 and 2x107 sp./ml, seem to create no risk for P. persimilis on the plant. But to confirm this assessment, it is necessary to investigate the long-term effect in further experiments and at greenhouse condition, too.

References

Donka, András 2007: Risikobewertung des Einsatzes von Lecanicillium muscarium (Petch) Zare & Gams (Syn. Verticillium lecanii) – Einflussnahme auf Nichtzielorganismen am Beispiel von Phytoseiulus persimilis Athias-Henriot (Risk assessment of Lecanicillium muscarium (Petch) Zare & Gams (Syn. Verticillium lecanii) – Influence on non-target organisms for instance on Phytoseiulus persimilis Athias-Henriot). Masterarbeit, Humboldt-Universität zu Berlin, 2007. Pesticides and Beneficial Organisms IOBC/wprs Bulletin Vol. 35, 2008 pp. 113-121

Effects of Beauveria bassiana, Heterorhabditis bacteriophora, H. megidis and Steinernema feltiae on the Mediterranean fruit fly Ceratitis capitata and the very sensitive braconid Psyttalia concolor in the lab

Pilar Medina1, Elena Corrales1, Manuel González-Nuñez2, Guy Smagghe3 & Elisa Viñuela1 1 Protección de Cultivos. E.T.S.I.Agrónomos. Universidad Politécnica de Madrid (UPM). 28040-Madrid. Spain. E-mail: [email protected] 2 Dpto. Protección Vegetal, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), Carretera de La Coruña Km 7,5, 28040-Madrid. Spain. 3 Department of Crop Protection, Faculty of Bioscience Engineering, Ghent University, B-9000 Ghent, Belgium.

Abstract: Laboratory experiments were set up to measure the susceptibility of the pest Ceratitis capitata and the braconid Psyttalia concolor (very sensitive to pesticides), to three commercial nematodes available in Spain [maximum field recommended rate (MFRC): 100 infective juveniles (IJ)/cm2]: Steinernema feltiae, Heterorhabditis bacteriophora, H. megidis and the entomopathogenic fungus Beauveria bassiana (MFRC 1000 ml cp/hl; 2.3 x 109 conidia/ml cp). The neurotoxic malathion, used worldwide for the control of C. capitata was used as positive standard (150 ml ai/hl). When nematodes were applied to the pupation medium of C. capitata (vermiculite, 10% humidity), S. feltiae and H. bacteriophora were as effective as malathion, inhibiting practically 100% of adult emergence at a 2-fold MFRC and 75% r.h. Efficacy of nematodes was much higher at 75% r.h. than at 30% r.h. irrespective of the dose used. The three nematodes decreased the progeny size of P. concolor when the parasitoid parasitized C. capitata L3 larvae, treated with the MFRC under 75% r.h., but significant reductions were only scored for S. feltiae. The fungus B. bassiana was tested on adults of the pest and the parasitoid (75% r.h.; 25ºC) by residual contact, topical application and ingestion, as well as by treatment of the oviposition gauze and direct spray on the pest, and vía contaminated hosts on the parasitoid. It did not cause mortality at 3 days or affected reproduction of C. capitata (malathion gave 100% mortality), except when applied at a 10-fold MFRC at the oviposition gauze (significantly decreased fertility by 21.7%). The biopesticide did not cause mortality on P. concolor in contrast with malathion (100%), but it significantly decreased its beneficial capacity by residual contact or vía treated host larvae (lower progeny size) or by ingestion (lower number of attacked hosts).

Key words: Steinernema feltiae, Heterorhabditis bacteriophora, H. megidis, Beauveria bassiana, commercial pesticides, Ceratitis capitata, Psyttalia concolor, exposure methods

Introduction

The Mediterranean fruit fly Ceratitis capitata (Wiedemann) is a cosmopolitan polyphagous pest of more than 250 subtropical and deciduous fruit crops, which threat the economic production of more than 100 of them in several countries, because larvae feed on fruits and reduce yield and quality (Christenson & Foote, 1960). This species is under severe quarantine regulations trying to prevent its establishment in the countries where it is not present (EPPO,

113 114

2007). The control of this pest has traditionally relied worldwide on the use of broad spectrum pesticides, particularly malathion applied as bait spraying (mixture of hydrolysate proteins and insecticide) aiming at controlling adults, because other developmental stages (eggs and larvae) are concealed inside the fruits, and pupation occurs in the first centimetres of the ground. As the organophosphate malathion has not been included in annex I of EU Directive 91/414 (Doce, 2007), there is a high requirement, therefore, for alternative measures of control. Present measures to control C. capitata and other tephritids, include the use of entomo- pathogens as biopesticides, because they are generally considered to provide an environ- mentally benign pest control option. These biopesticides are compatible with other control measures, commercially available in many countries and to date, no significant detrimental effects have been reported (Butt et al., 2001; García Del Pino, 2005). However, microbial control agents could not be entirely safe. So in order to minimise the concerns about their production and application, they are subjected to regulatory approval under the EU Directive 91/414, and an ecological risk assessment procedure is being developed (Hokkanen et al., 2003). The braconid Psyttalia concolor (Szèpligetti) is a parasitoid native to northern Africa and it is commercially available, because in the lab it can be easily mass-reared on the Mediterranean fruit fly C. capitata. Parasitation occurs in the last larval stage, when the larvae jump out of the fruit for pupation in the soil. This parasitoid is supposed to be a good indicator species in the study of pesticides on non-target organisms due to its high sensitiveness to the pesticides compared to other natural enemies (Croft, 1990; Vogt, 2000). It is very common in olive groves all over the Mediterranean region (only in Spain there is more than 2.2 million hectares of olive mainly devoted to the production of high quality oil), because in the field the favourite host of P. concolor is the olive fruit fly Bactrocera oleae (Gmelin). As a consequence, this natural enemy has been repeatedly used in inundative releases aiming at controlling this key pest of olive crops. Entomopathogenic nematodes from the genera Steinernema and Heterorhabditis could attack C. capitata L3 larvae when they emerge from the host and jump out of the fruit for pupating in the ground. Although natural infections of tephritid flies with entomopathogenic fungi seem to be rare, they have greater potential as biocontrol agents for adult fruit flies than bacteria or viruses because they infect their host through the cuticle. Many fungi have been shown to be pathogenic toward adults of fruit flies and they could be also used against mature L3 larvae and puparia in the soil because it provides a favourable environment for their development (Castillo et al., 2000; Quesada-Moraga et al., 2006). Both entomopathogenic nematodes and fungi could affect fruit fly parasitoids when laying eggs on affected L3 mature larvae or when the natural enemies interfere with droplets during the application, ingest contaminated plant fluids or contact residues. In the latest years, a lot of information has been published on effects of entomopatho- genic agents on fruit fly species, included C. capitata, but there is only little information on their effects on the important natural enemies of these flies. Our work aimed at ascertaining the efficacy of the commercial products based on nematodes and fungi registered in Spain in 2006 on the selected pest insect and the natural enemy in comparison to the results using the positive standard malathion. Several exposure methods were studied because the application of entomopathogenic agents will fail if they are not delivered in a manner that enables access to, and infection of the host (Shapiro et al., 2005).

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Materials and methods

Insects Insect rearing and assays, otherwise stated, were carried out in a growth chamber under 25±2ºC of temperature, 75±5% relative humidity and 16:8 (L:D) h photoperiod. The C. capitata and P. concolor specimens used in the tests were obtained from laboratory cultures maintained in Madrid following standard procedures (Jacas & Viñuela, 1994). Biopesticides Effects of three commercial nematodes: Steinernema feltiae Filipjev (Steinernema system®, Biobest), Heterorhabditis bacteriophora Poinar (Larvanem®, Koppert), H. megidis Poinar (Heterorhabditis system®, Biobest) and the entomopathogenic fungus Beauveria bassiana (Bals.) (Naturalis L®, Agrichem) were tested. Biopesticides were applied, otherwise stated, at their respective maximum recommended rate (MFRC): 100 infective juvenile nematodes (IJ)/cm2 and 1000 ml commercial product (cp)/hl for the fungus (2.3 x 109 conidia/ml cp). The positive standard malathion (Malafin® 50, Bayer) was applied at 300 ml cp/hl, corresponding with 150 ml active ingredient (ai)/hl). Application methods Females less than 24-h-old were chosen for the assays because young adults are normally more susceptible to pesticides (Croft, 1990). We only chose one sex for the assays, because regarding the parasitoid the females have a longer life span than the males, their susceptibility towards pesticides is higher and only the mated ones are responsible for the beneficial capacity in the field (Jacas & Viñuela, 1994). Regarding the pest insect, females are responsible for creating the fruit damages. The fungus was tested on the pest and the parasitoid by residual contact (1.8 mg/cm2 residues on glass plates; Potter spraying precision tower; 50 kPa pressure; forced ventilation), topical application (0.5µl droplet/insect delivered with a Burkard microapplicator; CO2 anaesthesia) and ingestion (added to the drinking water), as well as by treatment of the oviposition gauze (immersion in aqueous commercial product concentrations for 30 min) and direct spray on the pest insect under the Potter tower, and via contaminated hosts on the parasitoid [20 mature L3 C. capitata larvae were treated with 2-ml commercial product concentration for 3 min (time enough to contaminate them based on previous tests) and then offered to isolated P. concolor females for parasitization for 2 h during 3 consecutive days]. Nematodes were applied to the pupation medium of C. capitata (vermiculite, 10% humidity) under two environmental relative humidities (30 and 75%) and 2 rates (MFRC and 2-fold MFRC) following Lindregen & Vail (1986). Twenty four hours after substrate treatment, L3 host larvae, ready for pupation, were introduced and left to develop there until adult emergence. To test effects of P. concolor parasitization on contaminated host larvae, we followed a protocol similar to that described for the fungus, and experiments were performed under 75% r.h. and the MFRC. Every experiment consisted of 5 replicates of 15 females. Control units based in water were always used except in the topical treatment where acetone was employed. Statistics Effects of biopesticides were determined by measuring adult mortality at 3 days for the pest and for the parasitoid as well as their reproductive potential [for the pest: fecundity as cumulative number of eggs per female during three days after the onset of oviposition and fertility as percentage of larvae emerged 48h after egg laying; for the parasitoid: percentages of attacked host and progeny size based on Jacas & Viñuela (1994)]. 116

Data expressed in tables as mean±S.E. were analysed by one-way analysis of variance (ANOVA) and means separated by the LSD (P<0.05) test using the Statgraphics program (STSC, 1987). In cases where premises of ANOVA were violated after arsin√x transformation, a non-parametric Kruskall-Wallis test was applied and means were separated using the Box Plot option.

Results and discussion

The nematodes S. feltiae and H. bacteriophora were as effective as malathion, inhibiting nearly 100% of C. capitata adult emergence when the pupation substrate was treated at a 2- fold MFRC at 75% r.h (Figure 1).

% Ceratitis capitata adult emergence at 14 days 100 Maximum rate: 75% R.H; 30% R.H. 90 2-fold Maximum rate, 75% R.H. 80 fg gh gh 70 g gh f 60 e 50 d d 40 c 30 20

10 a a a b a 0 H.Heterorhabditis bacteriophora Heterorhabditis H. megidis Steinernema S. Feltiae Control Agua Malathion M alatión bacteriophora megidis feltiae

Figure 1: Effects of entomopathogenic nematodes on adult emergence in C. capitata when applied to the pupation substrate. Data are mean ± S.E. of 5 replicates. ANOVA and LSD; F= 493.22; df= 4, 10; P<0.0001.

As shown in Figure 1, the relative humidity plays a key factor on the efficacy of commercial nematodes and at the MFRC, the effects against C. capitata were much higher at 75% r.h. than at 30% r.h. irrespective of the concentration used. This was also the case for H. megidis, which is considered to be much more tolerant to desiccation than the others (Liu & Glazer, 2000). At 30% r.h., performance of the three tested species was bad, being S. feltiae the most effective species, which is in agreement with results of Köppler et al. (2005a, b) regarding the host Rhagoletis cerasi L. When the MFRC was doubled and experiments were performed under 75% r.h., the efficacy of H. megidis was not modified, in contrast with results scored in the other two species. Overall from these experiments it was of interest that the efficacy of S. feltiae was significantly similar to that of malathion and adult fly emergence was nearly totally inhibited. 117

The three nematodes decreased the progeny size of P. concolor when females of this natural enemy parasitized on treated C. capitata larvae with the MFRC at 75% r.h., but significant reductions were only scored for S. feltiae (48.4% emergence in controls compared to 9.5% in the nematode treatment) (Figure 2).

100 90 % Psyttalia concolor progeny size 80 75% r.h.; maximum rate= 100 IJ/cm2 70 60 a 50 40 30 ab ab 20 b 10 0 ControlAgua Heterorhabditis H. megidis H.Heterorhabditis bacteriophora Steinernema S. feltiae megidis bacteriophora feltiae

Figure 2: Effects of entomopathogenic nematodes on the progeny size of P. concolor when females parasitized on treated C. capitata L3 host larvae. Data are mean ± S.E. of 5 replicates. Kruskall-Wallis; K= 38.94; P=1.785 E-8. Isolated P. concolor females parasitized 20 host larvae for 2 h during three consecutive days.

The fungus B. bassiana did not cause mortality at 3 days nor affected the reproduction of C. capitata by any of the studied exposure methods (malathion gave 100% mortality), except when applied at a 10-fold maximum dose at the oviposition gauze (significantly decreased fertility by 21.7%) (Table 1). This is in contrast with literature, because several authors have reported effectiveness of this fungus on tephritids. However it should be noted that the efficacy was variable with the LT50 (lethal time) being dependant on the isolate used (Anagnou-Veroniki et al., 2005). Similarly, the biopesticide B. bassiana did not cause mortality in the parasitoid in contrast with malathion (100%), but it significantly decreased its beneficial capacity by residual contact or vía treated host larvae (lower progeny size) or by ingestion (lower number of attacked hosts) (Table 2). This entomopathogenic fungus has been shown to be harmless for many natural enemies in the field even though slight effects were detected on the lab in some cases (Santamaría et al., 1998; Hicks et al., 2001; Vestergaard et al., 2003). As a consequence, we believe that the small negative effect detected here on the parasitic performance of P. concolor, could be attributed to its high susceptibility to pesticides compared to other natural enemies (Croft, 1990; Vogt, 2000), and it will probably disappear under more realistic conditions. In conclusion, based in our lab results, the commercial product of S. feltiae seems to be the most promising entomopathogenic agent for the control of C. capitata among those tested, but future investigations under more realistic conditions should include efficacy evaluations on the pest insect as well as side-effects on natural enemies.

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Table 1: Influence of the exposure method on the susceptibility of Ceratitis capitata adults to Beauveria bassiana

Ceratitis capitata Treatments Concentration* Adult mortality1, Fecundity2** Fertility3*** (ai) 3d (%) (%) Residual contact Control - 3.3 ± 1.3a 48.2 ± 2.7a 60.6 ± 1.7a B. bassiana 1000 ml cp/hl4 4.4 ± 1.1a 53.7 ± 3.3a 60.4 ± 2.4a Malathion 150 ml ai/hl 100 ± 0b - - Topical application (0.5µl droplet/ insect) Control - 2.5 ± 0.8a 65.1 ±3.4a 63.5 ± 2.3a B. bassiana 1000 ml cp/hl4 2.5 ± 0.8a 63.5 ±3.8a 62.2 ± 3.4a Malathion 150 ml ai/hl 100 ± 0b - Ingestion in the drinking water Control - 1.3 ± 0.6a 55.1 ±3.3a 63.6 ± 1.9a B. bassiana 1000 ml cp/hl4 0.3 ± 0.3a 61.3 ±3.5a 63.4 ± 2.1a Malathion 150 ml ai/hl 99 ±0.7b - - Treatment of the oviposition gauze Control - 5.4 ± 2.0a 58.3 ± 2.4a 59.4 ± 3.0a B. bassiana 1000 ml cp/hl4 6.3 ± 1.3a 59.5 ± 3.3a 53.9 ± 2.8ab MFRC 2-fold MFRC 2000 ml cp/hl4 6.1 ± 2.8a 59.5 ± 3.3a 53.9 ± 2.8ab 3-fold MFRC 3000 ml cp/hl4 6.3 ± 3.4a 61.1 ± 2.9a 52.0 ± 3.3ab 10-fold MFRC 10000 ml cp/hl4 5.7 ± 1.9a 58.9 ± 2.8a 46.5 ± 2.9b Malathion 150 ml ai/hl 100±0b - -

Data are mean ± S.E. of 5 replicates. Within columns and treatment, data followed by the same letter are not statistically significant (ANOVA and LSD or Kruskall- Wallis). Residual contact: 1F = 3211.47; df= 2, 12; P< 0.0001; 2F = 411.3, df = 1, 8; P = 0.250; 3F = 0.10; df= l, 8; P = 0.97). Topical application: 1F = 6295.78; df = 2, 12; P < 0.0001; 2F = 0.10; df. = 1, 8; P= 0.7532; 3F = 0.10; df =1,8; P = 0.7492. Ingestion: 1K = 80.28; P = 0.001; 2F = 1.63; df = 1, 8; P = 0.2065; 3F = 0.010; df = 1, 8; P = 0.9346. Treatment of the oviposition gauze: 1F = 1.67, df = 5, 24; P = 0.2014. 2F = 2.46; df = 4, 20; P = 0.0351. 3F = 2.89; df = 4, 20; P = 0.0385. *MFRC as registered in Spain in 2006. **Cumulative number of eggs per female during 3 days after the onset of oviposition. ***Emerged larvae 24-36 h after egg laying. ai= active ingredient. cp= commercial product. 4 2.3 x 109 conidia/ml cp 119

Table 2: Influence of the exposure method on the susceptibility of P. concolor adults to B. bassiana

Psyttalia concolor Treatments Concentration* Adult mortality1, Attacked host2 Progeny size3 (ai) 3d (%) (%) (%) Residual contact Control - 10.4 ± 1.9a 37.2 ± 2.3a 91.2 ± 1.9a B. bassiana 1000 ml cp/hl4 10.8 ± 2.0a 42.8 ± 2.0b 81.2 ± 5.5b Malathion 150 ml ai/hl 100 ± 0b - - Topical application (0.5 µl droplet/ insect) Control - 2.2 ± 0.8a 62.9 ± 2.9a 78.6 ± 2.9a B. bassiana 1000 ml cp/hl4 2.9 ± 0.9a 60.0 ± 2.8a 71.7 ± 2.8a Malathion 150 ml ai/hl 100 ± 0b - - Ingestion in the drinking water Control - 5.0 ± 1.3a 54.2 ± 4.0a 86.6 ± 2.5a B. bassiana 1000 ml cp/hl4 3.6 ± 1.2a 31.9 ± 3.1b 81.7 ± 3.0a Malathion 150 ml ai/hl 100 ± 0b - - Contamination via treated host Control - - 75.4 ± 2.1a 58.6 ± 3.5a B. bassiana 1000 ml cp/hl4 - 69.4 ± 3.5a 48.6 ± 3.4b Malathion 150 ml ai/hl - - -

Data are mean±S.E. of 5 replicates. Within columns and treatment, data followed by the same letter are not statistically significant (ANOVA and LSD or Kruskall- Wallis). Residual contact: 1F = 1062.56; df = 2, 8; P <0.0001. 2K = 5.24457; P = 0.022. 3F = 9.11; df = 1, 8; P = 0.033. Topical application: 1F = 6469.32; df = 2, 12; P < 0.0001. 2F = 0.45; df = 1, 8; P = 0.5036. 3F = 2.76, df = 1, 8; P = 0.0996. Ingestion: 1F = 2797.48; df = 2, 12; P <0.0001. 2F = 19.26; df = 1, 8; P < 0.0001. 3F = 1.53; df = 1, 8; P = 0.2188. Contamination via treated host: 2F = 19.98; df = 1, 8; P = 0.1678. 3F = 4.34; df = 1, 8; P = 0.0455. *MFRC as registered in Spain in 2006. ai= active ingredient. cp= commercial product. 4 2.3 x 109 conidia/ml cp.

Acknowledgements

The authors gratefully acknowledge the research support provided by the Spanish Ministry of Education and Culture to E. Viñuela and M. González (projects AGL2004-07516-C02- 01/AGR and AGL2004-07516-C02-02/AGR) and by the Fund for Scientific Research (FWO- Vlaanderen, Brussels, Belgium) to G. Smagghe. E. Corrales thanks the Crop Protection Unit (ETSI Agrónomos) for assistance in experiments for developing her agricultural engineering final work. Authors also acknowledge the suggestions of Annette Herz (BBA Dossenheim, Germany) concerning the nematode handling.

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Aged-residue method for evaluating toxicity of plant protection products to Stethorus punctillum (Weise) (Coleoptera: Coccinellidae)

Karin M. Nienstedt and Mark Miles Springborn Smithers Laboratories (Europe), Seestrasse 21, 9326 Horn, Switzerland (present address: EFSA, European Food Safety Authority, Largo N. Palli 5a, I-43100 Parma, Italy, [email protected]); Dow AgroSciences, 3 Milton Park, Abingdon OX14 4RN, UK

Abstract: Stethorus punctillum (Weise) (Coleoptera: Coccinellidae) is known as an obligate predator of spider mites. Currently there are no widely recognised laboratory methods for testing the effects of plant protection products (PPP) to this species. Here we present a method for evaluating the toxicity of PPP under extended laboratory conditions or as a persistence (aged residue) study, combining field applications with laboratory bioassays. S. punctillum larvae were exposed to treated apple leaf disks and their development through to pupation and adult emergence monitored. An assessment of reproduction was also performed. Example data corresponding to control, methoxyfenozide and fenoxycarb treatments are presented.

Keywords: Stethorus punctillum, potential effects of plant protection products

Introduction

All known species of the genus Stethorus (Weise) (Coleoptera: Coccinellidae) are predators of tetranychid spider mites (Scriven and Fleschner, 1960, Rott and Ponsonby, 2000). Stethorus punctillum is known as spider mite natural enemy in fruit orchards (Ivancich, 1974) and commercially available as biological control agent. Currently there are no widely recognised laboratory methods for testing the effects of plant protection products (PPP) to this species. Here we present a method to assess potential negative effects of plant protection products to Stethorus sp. under laboratory conditions. The presented method is based on the IOBC-method established for Coccinella septempunctata by Schmuck et al. (2000), which is used for regulatory purposes in the EU (Commission Directive 96/12/EC amending Council Directive 91/414/EEC, Barrett et al. 1994, Candolfi et al. 2001).

Materials and methods

We determined the potential effects of Intrepid® 2F (trademark of Dow AgroSciences LLC, active ingredient methoxyfenozide 23.7 % w/w) and Insegar® DG (trademark of Syngenta, active ingredient fenoxycarb 25 % w/w) applied on a natural substrate (apple trees) on Stethorus punctillum (Weise) (Coleoptera: Coccinellidae). Potted apple trees (Malus domestica Borkh. var. Spartam) were held and treated under field conditions, and thereafter placed under a rain protection in the field. Bioassays exposing Stethours punctillum under laboratory conditions to treated apple tree leaves were started immediately and 7 days after treatment application. Pre-imaginal mortality and the effect on reproduction were used as toxic endpoints.

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Stethorus punctillum culture S. punctillum beetles were obtained from Benfried International b.v., Den Hoorn, The Netherlands, and cultured as described by Walters (1974) providing Tetranychus urticae ad libitum as food. T. urticae was reared in a separate room on bean plants (Phaseolus vulgaris L.) at approximately 25 °C. Fresh bean plants were added to the culture 2 to 3 times per week in order to have a continuous supply of spider mites. Treatment application The present study was designed as an aged residue test. The treatments were applied under field conditions to potted apple trees (Malus domestica Borkh., var Spartan) held under field conditions and watered regularly until test start. No chemical plant protection measures were carried out since start of the growing season, i.e. for 3 months before experimental starting. At treatment application the trees were at BBCH 74. The treatments performed were a control (deionised water), methoxyfenozide at 48 g a.i./ha (10% of the maximum use rate), methoxyfenozide at 480 g a.i./ha (100% of the maximum use rate), methoxyfenozide at 960 g a.i./ha (200% of the maximum use rate), and fenoxycarb at the recommended field rate of 480 g /ha (equivalent to 120 g a.i./ha). Each treatment was applied in deionised water on a tree row of 7 potted trees of approximately 1.65 m height. The trees were located simulating a continuous canopy at 0.6 m distance each from the other (row length of 4.2 m). A 5 m distance between the rows was assumed for calculation of the application rate. The applications were performed with a Birchmeier M125 back-pack- sprayer. The application volume was determined prior to application to be 428.6 L/ha, guaranteeing an optimal wetting of the foliage but avoiding run-off. Each tree row was sprayed twice (once per row side). After treatment application and until the start of the 2nd bioassay, the apple trees were hold under rain cover. During this period, temperature ranged from 16.0 to 32.5 °C and the relative air humidity from 36 to 98 %. Bioassays Bioassays consisted of an exposure and a reproduction phase and were started immediately and 7 days after treatment application. After application of the apple trees, treated leaves were detached immediately after drying of the applied solutions and used as substrate for the exposure of beetle larvae during the 1st bioassay. For the 2nd bioassay, treated leaves were detached 7 days after application and used immediately for the corresponding exposure. Exposure phase test units consisted of a leaf disk (approximate diameter: 30 mm) cut from a treated leaf, resting upside-up on agar (1 % w/v) in a transparent, circular plastic box of Polystyrol (diameter 3.9 cm, height 3.0 cm). Into each test unit, one Stethorus punctillum larva hatched no more than 48 hours ago was transferred together with food (Tetranychus urticae). The test units were then placed top-side down over a mesh fixed on a frame. For each treatment and bioassay, 40 replicates were set up. The status (normal, moribund, missing, pupae, or dead) of the individuals was inspected daily until day 15 of the corresponding bioassay or until hatching of adults. After day 15, the observations were done every 2 to 3 days. The hatched beetles from each treatment were pooled in transparent 1.3 L plastic containers and fed regularly every 2 to 3 days until starting of the reproduction phase. Reproduction phase test units consisted of a circular plastic box of Polystyrol (diameter 3.3 cm, height 1.0 cm). Into each test unit, one Stethorus punctillum adult was transferred together with food (T. urticae). During one week, beetles were transferred every 2 to 3 days to new boxes with fresh food, so that a total of 3 consecutive egg laying samples were obtained by beetle. After removal of the beetles, the eggs in each box were counted. After beginning of larvae hatching, i.e. 2 to 3 days after egg laying, the number of larvae hatched per box was counted during 3 consecutive days removing the hatched larvae on each observation day.

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After the reproduction phase, all adult beetles which did not lay eggs were sexed (beetles which laid eggs were obviously females). During the bioassays, temperatures ranged from 25 ± 2 °C and the relative air humidity from 75 ± 15 %. The light intensity was 1500 to 3000 lux with a photoperiod of 16L: 8D. Cumulative pre-imaginal mortality was determined for the exposure phase. For the reproductive phase, the average number of eggs per female per day and the hatching rate of eggs were calculated. Mortality was analysed by Fisher’s Exact tests. Eggs laid per female and hatching rate were analysed by ANOVA followed by Dunnet t-test.

Results

Control mortality in the present study was 2.5 and 7.5 %. Pre-imaginal mortality in the fenoxycarb treatment was 80 % and statistically significant different from the control (Fisher Exact test: p < 0.001). In this treatment, typical IGR effects were also observed with 20 % of crippled (abnormal) larvae (Table 1). The pre-imaginal mortality in the methoxyfenozide treatments during the 1st bioassay varied between 22.5 and 51.3 % (48 and 960 g a.i./ha methoxyfenozide treatments, respectively) and was statistically significantly different to the control at all the application rates (Fisher Exact Tests: p < 0.05). During the 2nd bioassay, after 7 days of ageing, mortality varied between 4.8 and 45.0 % (48 and 960 g a.i./ha treatments, respectively). A statistically significant difference to the control was observed at the application rate of 960 g a.i. methoxyfenozide/ha (Fisher Exact Test: p < 0.001) but not at 48 and 480 g a.i. methoxy- fenozide/ha (Fisher Exact Tests: p = 0.477 and p = 0.662, respectively) (Table 1).

Table 1. Hatching of adults and pre-imaginal mortality in the 1st and 2nd bioassay exposing Stethorus punctillum to methoxyfenozide and fenoxycarb.

Treatment Bioassay 1 Bioassay 2 (0 days after (7 days after treatment application) treatment application) Hatched Crippled Mortality n Hatched Mortality n adults larvae (%) adults (%) (%) (%) (%) Control 97.5 0.0 2.5 40 92.5 7.5 40 48 g a.i./ha methoxyfenozide 77.5 0.0 22.5 * 40 95.2 4.8 42 480 g a.i./ha methoxyfenozide 82.9 0.0 17.1 * 41 92.5 7.5 40 960 g a.i./ha methoxyfenozide 48.7 0.0 51.3 * 39 55.0 45.0 * 40 120 g a.i./ha fenoxycarb 0 20.0 80.0 * 40 - - - * Statistically significant different from the control (Fisher Exact Test: p < 0.05)

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Table 2. Mean number of eggs laid per female during the reproduction phase of the 1st bioassay exposing Stethorus punctillum to methoxyfenozide (A, B, C refers to egg laying periods of 2 to 3 days respectively).

Eggs per female per Treatment Eggs A Eggs B Eggs C day ns Control Mean 20.4 22.2 31.2 10.5 N 21 21 21 21 Std. Dev. 8.0 6.3 8.1 1.5 48 g a.i./ha Mean 17.6 19.8 26.8 9.2 methoxyfenozide N 14 14 14 14 Std. Dev. 8.6 9.1 11.2 3.2 480 g a.i./ha Mean 17.5 16.9 24.9 8.5 methoxyfenozide N 17 17 17 17 Std. Dev. 10.6 8.3 11.7 3.6 960 g a.i./ha Mean 18.3 18.4 29.4 9.4 methoxyfenozide N 10 10 10 10 Std. Dev. 7.0 12.4 6.8 2.3 ns – no statistically significant differences between the treatments (ANOVA: p = 0.145)

Table 3. Hatching rate of larvae from eggs laid during periods A, B, C, of the reproduction phase of the 1st bioassay exposing Stethorus punctillum to methoxyfenozide.

Hatching Treatment Hatching A Hatching B Hatching C (mean) Control Mean 0.72 0.84 0.84 0.80 N 21 21 21 21 Std. Dev. 0.22 0.20 0.23 0.14 48 g a.i./ha Mean 0.81 0.92 0.79 0.84 methoxyfenozide N 14 14 14 14 Std. Dev. 0.16 0.38 0.27 0.17 480 g a.i./ha Mean 0.73 0.72 0.81 0.76 methoxyfenozide N 15 16 15 14 Std. Dev. 0.22 0.32 0.29 0.21 960 g a.i./ha Mean 0.91 0.93 1.00 0.97 methoxyfenozide N 10 9 10 9 Std. Dev. 0.23 0.17 0.13 0.13 ns – no statistically significant differences between the treatments (ANOVA: p = 0.033 / Dunnett t tests between each test item treatment and control p > 0.05)

The reproduction was assessed for the beetles hatched in the control and methoxyfenozide treatments during the 1st bioassay. A mean of 10.5, 9.2, 8.5, and 9.4 eggs per female per day were observed in the control and the 48, 480 and 960 g a.i./ha methoxyfenozide treatments (Table 2) with no statistically significant differences between these treatments (ANOVA: p = 0.145). The hatching rate was 80, 84, 76, and 97 % in the control and the 48, 480 and 960 g a.i./ha methoxyfenozide treatments (Table 3). No statistically significant differences were observed between these treatments (ANOVA: p = 0.033 / Dunnett t-tests between each methoxyfenozide treatment and control p > 0.05).

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Discussion

Control mortality in the present study was below 10 %. Comparing these mortality values with the trigger value of 30 % control mortality stated in the IOBC guideline for Coccinella septempunctata L. (Coleoptera: Coccinellidae) (Schmuck et al, 2000), the presented method can be considered appropriate. Mortality in the fenoxycarb treatment was above 50 % and statistically significant different to the mortality in the control treatment. Specific IGR effects (crippled / abnormal larvae) were also observed. These effects are typical for the mode of action as non-neurotoxic insect growth regulator with contact and stomach action which inhibits metamorphosis to the adult stage and interferes with moulting of early instar larvae (Tomlin, 1994). As such fenoxycarb demonstrated the sensitivity of the test method and the suitability of the route of exposure, and could be used as positive control (toxic standard) for further tests. Immediately after application of methoxyfenozide, statistically significant effects on preimaginal mortality of Stethorus punctillum exposed under laboratory conditions to the applied apple tree leaves were observed from 48 g a.i./ha onwards. The mortality results obtained with methoxyfenozide showed dose responsiveness. Decay of toxicity with time was observed since after 7 days of residue ageing, statistically significant effects on preimaginal mortality were only observed at 960 g a.i./ha while no effects were observed up to 480 g a.i./ha. Concerning the assessment of reproduction (eggs laid per female per day / hatching rate of larvae) the chosen method also was proven suitable leading to mean values of 9.5 ± 2.8 eggs / female / day and 83 ± 18 % hatching of eggs. No effects were observed on the reproduction of adult beetles exposed as larvae to up to 960 g a.i./ha methoxyfenozide. The method presented in this manuscript gives A) natural mortality values similar to the values stated in guidelines of closely related species (Schmuck et al, 2000), B) reasonable reproduction values, and C) shows sensitivity for different products and application rates. Therefore, we consider it as suitable for detecting side effects of pesticides under laboratory and aged-residue designs.

References

Barrett, K.L., Grandy, N., Harrison, E.G., Hassan, S. & Oomen, P. 1994: Guidance document on regulatory testing procedures for pesticides and non target arthropods. ESCORT Workshop, March 1994. SETAC. 51 pp. Candolfi, M.P., K.L. Barrett, P.J. Campbell, R. Forster, N. Grandy, M-C. Huet, G. Lewis, P.A. Oomen, R. Schmuck & H. Vogt (Eds.) 2001: Guidance document on regulatory testing and risk assessment procedures for plant protection products with non-target arthropods. From the ESCORT 2 Workshop. March 2000. SETAC. Pensacola, USA. 46 pp. European Commission Directive 96/12/EC of 8 March 1996. Official Journal of the European Communities. European Council Directive 91/414/EEC of 15 July 1991. Official Journal of the European Communities. Ivancich G.P. 1974: L'influenza del Typhlodromus italicus Chant (Acarina, Phytoseiidae) e dello Stethorus punctillum Weise (Col. Coccinellidae) sulla dinamica di popolazione degli acari fitofagi del pesco. Boll. Lab. Entomol. Agrar. "Filippo Silvestri" 31: 171-191.

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Rott, A.S. & Ponsonby, D.J. 2000: The effects of temperature, relative humidity and host plant on the behaviour of Stethorus punctillum as a predator of the tow-spotted spider mite, Tetranychus urticae. BioControl 45: 155-164. Scriven, G.T. & Fleschner, C.A. 1960. Insectary Production of Stethorus species. Journal of Economic Entomology 53: 982-985. Schmuck, R., Candolfi, M.P., Kleiner, R., Mead-Briggs, M., Moll, M., Kemmeter, F., Jans, D., Waltersdorfer, A. & Wilhelmy, H. 2000: A laboratory test system for assessing effects of plant protection products on the plant dwelling insect Coccinella septem- punctata L. (Coleoptera: Coccinellidae). In: Guidelines to evaluate side-effects of plant protection products to non-target arthropods. IOBC, BART, and EPPO Joint Initiative. Ed. Candolfi, Blümel, Forster et al.: 45-56. Tomlin, C. (Ed.). 1994: The pesticide manual. 10th Edition. Crop Protection Publications. BCPC and RSC, London: 442-443. Walters, P.J. 1974: A method for culturing Stethorus spp. (Coleoptera: Coccinellidae) on Tetranychus urticae (Koch) (Acarina: Tetranychidae). J. Aust. Ent. Soc. 13: 245-246.

Pesticides and Beneficial Organisms IOBC/wprs Bulletin Vol. 35, 2008 pp. 128-135

Chlorantraniliprole (DPX-E2Y45, DuPont™ Rynaxypyr®, Coragen® and Altacor® insecticide) - a novel anthranilic diamide insecticide - demonstrating low toxicity and low risk for beneficial insects and predatory mites1

Axel Dinter1, Kristin Brugger2, Andrea Bassi3, Niels-Martin Frost4, Michael D. Woodward2 1 DuPont de Nemours Deutschland (GmbH), DuPont Str. 1, D-61352 Bad Homburg v.d.H., Germany (email: [email protected]), 2 DuPont Crop Protection, Stine-Haskell Research Center, 1090 Elkton Road, Newark, DE 19714, USA, 3 DuPont Italia Srl, Via Pietro Gobetti 2/C, 20063 Cernusco sul Naviglio (Mi) Italy, 4DuPont Danmark ApS, Sköjtevej 26, DK-2770 Kastrup, Denmark

Abstract: Chlorantraniliprole (DPX-E2Y45, DuPont™ Rynaxypyr®) is a new anthranilic diamide insecticide with a novel mode of action. Rynaxypyr® activates insect ryanodine receptors causing impaired regulation, paralysis and ultimately death of sensitive species at rates of 10 to 60 g Rynaxypyr®/ha. In worst-case Tier 1 glass plate tests the two indicator species, Aphidius rhopalosiphi and Typhlodromus pyri, were not sensitive to either Coragen® or Altacor® at up to 750 g Rynaxypyr®/ha, the maximum rate tested indicating low risk for non-target arthropods. Low risk for non-target arthropods was confirmed in a wide range of tests with several other species. Overall, DuPont™ Rynaxypyr® and the formulations, Coragen® and Altacor®, were demonstrated to be safe to numerous beneficial non-target arthropod species or to have a rather low and transient impact and therefore will be excellent tools for use in integrated pest management (IPM) programmes.

Key words: Anthranilic diamide insecticide, chlorantraniliprole, DPX-E2Y45, DuPont™ Rynaxypyr®, Coragen®, Altacor®, side-effects, non-target arthropods, Aphidius rhopalosiphi, Typhlodromus pyri, Amblyseius andersoni, Kampimodromus aberrans, Chrysoperla carnea, Coccinella septempunctata, Adalia bipunctata, Episyrphus balteatus, Orius laevigatus, Anthocoris nemoralis, laboratory, extended laboratory, semi-field and field tests

Introduction

Chlorantraniliprole (DPX-E2Y45, DuPont™ Rynaxypyr® insecticide) is a new anthranilic diamide insecticide developed worldwide by E. I. du Pont de Nemours and Company, Inc. with a novel mode of action. Rynaxypyr® activates ryanodine receptors via stimulation of the release of calcium stores from the sarcoplasmic reticulum of muscle cells (i.e. for chewing insect pests) causing impaired regulation, paralysis and ultimately death of sensitive species (Cordova et al. 2006). The differential selectivity Rynaxypyr® has towards insect ryanodine receptors explains the outstanding profile of low mammalian toxicity. Rynaxypyr® is active on chewing pests primarily by ingestion and secondarily by contact and shows good ovi-larvicidal and larvicidal activity. In Europe, Coragen® (200 g Rynaxypyr®/L) and Altacor® (350 g Rynaxypyr®/kg) have been developed for foliar applications in top fruit, vegetable crops, grapes and potatoes at rates of 10 to 60 g Rynaxypyr®/ha, which are highly effective on many

1 DuPont™, Rynaxypyr®, Altacor® and Coragen® are trademarks or registered trademarks of DuPont or its affiliates. Copyright © 2004 E.I. du Pont de Nemours and Company or its affiliates. All rights reserved.

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important pests (Bassi et al. 2007). The purpose of this paper is to summarize the current knowledge on effects of Rynaxypyr® on non-target arthropods.

Material and methods

The studies reported in this paper were designed following the regulatory testing principles for non-target arthropods outlined in the ESCORT II guidance document (Candolfi et al. 2001). Tests were performed according to guidelines to evaluate side-effects of plant protection products to non-target arthropods developed by the IOBC, BART and EPPO Joint Initiative (Candolfi et al. 2000). Effects of two formulations using DuPont™ Rynaxypyr® insecticide, Coragen® (200 g Rynaxypyr®/L; DPX-E2Y45 20SC) and Altacor® (350 g Rynaxypyr®/kg; DPX-E2Y45 35WG), were studied. Aphidius rhopalosiphi: In dose-response laboratory tests effects on mortality and fecundity were assessed following exposure to fresh-dried spray deposits of the formulated products, Coragen® and Altacor® on glass plates (Mead-Briggs et al. 2000). Typhlodromus pyri and other predatory mite species: (1) The sensitivity of T. pyri protonymphs towards Coragen® and Altacor® was studied in dose-response laboratory tests using the open glass plate method (Blümel et al. 2000a). (2) The impact of Rynaxypyr® on natural predatory mite populations was investigated in field trials (3 tests in orchards and one in a vineyard) following guidance by Blümel et al. (2000b). In an apple orchard (Golden Delicious, 5 replicates per treatment) in northern Italy, Coragen® was sprayed with a calibrated knapsack sprayer in mid (BBCH 73) and end (BBCH 75) of June 2004 at 47.5 and 52.5 g Rynaxypyr®/ha at a 13-day spray interval using a spray volume of 1100 and 1200 L/ha, respectively. The initial predatory mite population consisted of T. pyri, Amblyseius andersoni, Kampimodromus aberrans, and Paraseiulus talbii. In 2006 another apple (variety Red Chief) field trial with 4 replicated plots each was conducted with Coragen® in northern Italy. Coragen® was applied in early (BBCH 74) and mid (BBCH 77) July at 60 g Rynaxypyr®/ha each, a 14- day spray interval and with a spray volume of 750 L/ha using a vaporizer. The effects of Coragen® on predatory mites in vineyards were investigated in southern France in 2004 with 2 spray applications at 52.5 g Rynaxypyr®/ha each, a 15-day spray interval and a spray volume of 600 L/ha. The spray applications were performed with a calibrated knapsack sprayer at BBCH 68 and 73 (26 June and 11 July). There were 5 replicates per treatment. In 2006 another replicated field study was conducted in vines in northern Italy. Coragen® was sprayed at 52.5 g Rynaxypyr®/ha twice, a 12-day spray interval and a spray volume of 1500 L/ha (22 June and 4 July, BBCH 73 and 75). Chrysoperla carnea: In a limit test chrysopid larvae were exposed to glass plates treated with tap water or Coragen® at 120 g Rynaxypyr®/ha (Vogt et al. 2000). During the test the larvae were fed ad libidum with aphids (Acyrthosiphon pisum). Orius laevigatus: The effect of Coragen® on the predatory bug, Orius laevigatus was evaluated in an extended laboratory test according to Bakker et al. (2000). The test organisms were exposed to fresh-dried spray deposits of Coragen® on detached dwarf bean leaves (Phaseolus vulgaris) at 0.5, 1.5, 4.4, 13.3, 40 and 120 g Rynaxypyr®/ha. Anthocoris nemoralis: Effects of Coragen® on A. nemoralis were investigated in a pear orchard in northern Italy. A randomised block design with 5 treatments and 4 replicates, each with 6 trees (plot size: 20 m²), was used. Coragen® was compared with an untreated control and 2 reference standards (lambda-cyhalothrin, emamectin-benzoate). Coragen® was applied once or twice at 52.5 g Rynaxypyr®/ha at a 14-day spray interval in June 2006. Applications were performed with a knapsack sprayer at BBCH 74 and 75. To increase the natural A. 130

nemoralis population present in the pear orchard before the applications, 2 releases of laboratory-bred anthocorids were made (13 May and 6 June 2006). Assessments were made by shaking tree shoots and collecting the adults and nymphs in an entomological umbrella (size: 1 m × 1 m, 4 shakings per plot). Coccinella septempuncata and other ladybird beetles: (1) Larvae of C. septempunctata were exposed to fresh-dried spray deposits of Coragen® on detached dwarf bean leaves (Phaseolus vulgaris) at 0.5, 1.5, 4.4, 13.3, 40 and 120 g Rynaxypyr®/ha (Schmuck et al. 2000). To investigate the duration of effects coccinellid larvae were exposed under extended laboratory conditions to detached apple leaves that had been treated and aged under field conditions. For that purpose small potted apple trees were treated twice with Coragen® at 60 g Rynaxypyr®/ha (7-day spray interval) and a spray volume of 1500 L/ha. The treated trees were allowed to age under field conditions for 28 and 78 days before the laboratory bioassays were initiated (Schmuck et al. 2000). (2) The effect of Coragen® on mortality of A. bipunctata L2 larvae was studied in a semi-field trial in an apple orchard in northern Italy. A randomized block design with 5 treatments and 4 replicates, each with 3 treated trees and 7 trees as buffer zone, was used. Coragen® was compared with a control and 2 reference standards. Two spray applications at 14-day interval were made with a knapsack sprayer and a spray volume of 1500 L/ha. Coragen® was also tested with a single application. After the foliage got dry after the 2nd application, 4 cages (40 cm × 15 cm × 15 cm covered with fine net) were assembled around small tree shoots in each plot and 10 larvae of laboratory reared A. bipunctata were introduced. The shoots chosen were naturally infested by aphids and further aphids were added during the exposure period to ensure continuous food supply. Visual mortality assessments were made 1, 3, and 7 days after start of exposure. (3) Effects of Coragen® on a natural coccinellid population was studied in an apple orchard in northern Italy using a randomised block design with 5 treatments and 4 replicates, each with 7 trees (plot size: 50 m²). Coragen® was compared with a control plot and 2 reference standards (lambda- cyhalothrin, azinphos methyl). Coragen® was applied once or twice at 52.5 g Rynaxypyr®/ha, 1500 L spray volume/ha and at a 14-day spray interval during end of June and beginning July 2006. Applications were performed with a knapsack sprayer at BBCH 74 and 75. The coccinellids were assessed by shaking tree shoots and collecting the adults and larvae in an entomological umbrella (size: 1 m × 1 m, 4 shakings per plot). Episyrphus balteatus: (1) The effect of Altacor® and Coragen® on the syrphid, E. balteatus, was evaluated in extended laboratory tests adapted according to Rieckmann (1989). The test organisms were exposed to fresh-dried spray deposits of Altacor® and Coragen® on detached winter rape leaves (Brassica napus) at 0.5, 1.5, 4.4, 13.3 and 40 g Rynaxypyr®/ha. (2) To investigate the duration of effects of both formulations, syrphid larvae were exposed under extended laboratory conditions to detached apple leaves that had been treated and aged under field conditions. For that purpose small potted apple trees were treated twice at 60 g Rynaxypyr®/ha (7-day spray interval) with a spray volume of 1500 L/ha. The treated trees were allowed to age under field conditions for 28 and 42 days before the laboratory bioassays were initiated (Rieckmann 1989).

Results and discussion

Aphidius rhopalosiphi Following exposure to fresh-dried spray deposits on glass plates of Coragen® and Altacor® applied at rates of 1, 50, 100, 200 and 750 g Rynaxypyr®/ha no statistically significant increased mortality or decreased fecundity of the surviving wasps was observed. Therefore the 131

® LR50 and ER50 were estimated to be >750 g Rynaxypyr /ha for both products, indicating low risk for parasitic wasps. Typhlodromus pyri and other predatory mite species ® The LR50 and ER50 on glass plates for T. pyri were determined to be >750 g Rynaxypyr /ha for Coragen® and Altacor® when tested at rates of 1, 50, 100, 200 and 750 g Rynaxypyr®/ha. The effects of Rynaxypyr® on a wider range of predatory mite species were further investigated in field trials conducted in Italy and France. No statistically significant differences in the predatory mite population were observed between the Rynaxypyr® treated plots and the controls during the course of the study in an apple orchard in northern Italy in 2004. The predatory mite population in the toxic reference (deltamethrin) was significantly decreased validating the sensitivity of the test system. In the apple field trial in 2006, the predatory mite population was slightly reduced after the first treatment, but 3 days after the 2nd application a slightly higher population density was found in the Rynaxypyr® treated plots. Therefore it is highly unlikely that the differences after the first treatment were due to Rynaxypyr®. The predatory mite population investigated in the French vineyard in 2004 was a pure T. pyri population and only a slight transient population reduction 2 weeks after the 2nd Rynaxypyr® treatment was found (29.9% lower than the control). However, 55 days after the 2nd treatment, slightly higher predatory mite numbers were found in the treatment group relative to the control. Instead, the toxic reference (methidathion) strongly impacted on the T. pyri population directly after the first application. For all sampling dates (up to 4 weeks after the 2nd application) the predatory mite numbers in the control and Rynaxypyr® treated plots were about the same and not statistically significantly different in the trial conducted in an Italian vineyard in 2006, while the reference treatment with deltamethrin showed a significant effect. Overall, it can be concluded that Rynaxypyr® is harmless to T. pyri under laboratory conditions as well as harmless to numerous other predatory mite species under practical use conditions in orchards or vineyards. Chrysoperla carnea Exposure of C. carnea larvae to 120 g Rynaxypyr®/ha treated glass plates for at least 17 days and fed with aphids for 16 days resulted in a corrected mortality of 24.2 % (control mortality: 7.7 %). No treatment-related effects were observed on fecundity (19 eggs per female per day) and fertility of C. carnea (mean hatching rate of 87.1 %). Therefore the LR50 and ER50 were estimated to be >120 g Rynaxypyr®/ha for Coragen®. Orius laevigatus No adverse effects on mortality or reproduction of O. laevigatus at rates up to 120 g Rynaxypyr®/ha for Coragen® in the extended laboratory test were found. The maximal observed corrected mortality was 8.8 %. The maximal observed reduction in reproduction and in hatching rate were 22.0 % and 26.6 %, respectively. Therefore the LR50 and ER50 was estimated to be >120 g Rynaxypyr®/ha for Coragen®. Anthocoris nemoralis The field population of A. nemoralis nymphs and adults was homogeneous before the first application in all 5 treatments with about 7 to 8 predatory bugs per sample (Table 1). After the 1st and 2nd spray application, slightly higher numbers were found in the control (11 to 12 bugs per sample), while the numbers in the toxic reference, lambda-cyhalothrin, significantly decreased to 1 to 2 bugs per sample, validating the sensitivity of the test system. In contrast, the numbers of predatory bugs collected in both Coragen® treatments were very similar to those in the control plot and not statistically different, demonstrating that Coragen® was safe to A. nemoralis.

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Table 1. Mean numbers of Anthocoris nemoralis (nymphs and adults) collected with an entomological umbrella in a pear orchard in northern Italy in 2006.

Mean number of nymph and adult A. nemoralis Treatment 14 DAA1 0 DBA1 3 DAA2 7 DAA2 14 DAA2 0 DBA2 Control (water) 7.1 a 12.4 a 10.8 a 10.1 a 8.7 a Lambda-cyhalothrin 7.4 a 2.3 b 0.8 b 0.3 b 2.6 b (2 × 22.5 g/ha) Coragen® 7.3 a 10.1 a 12.4 a 8.9 a 6.6 a (1 × 52.5 g Rynaxypyr®/ha) Coragen® 7.9 a 12.1 a 12.5 a 9.4 a 7.9 a (2 × 52.5 g Rynaxypyr®/ha) Emamectin-benzoate + mineral oil 7.8 a 9.9 a 8.4 a 7.6 a 6.4 a (2 × 15 g/ha + 6000 g/ha) Statistics: Tukey’s Test, different letters indicate differences at p < 0.05. DBA = Days before application, DAA = Days after application

Coccinella septempuncata and other coccinellids The maximal corrected mortality observed in the extended laboratory test was 63.3 % at 120 g ® ® Rynaxypyr /ha. The LR50 determined for C. septempunctata exposed to Coragen was 79.5 g ® ® ® Rynaxypyr /ha and the ER50 was 13.3 g Rynaxypyr /ha. Field-aged residues of Coragen applied twice at 60 g Rynaxypyr®/ha in a spray volume of 1500 L/ha and a spray interval of 7 days had no effect on mortality and reproduction of C. septempunctata. Corrected mortalities after larval exposure to 28- and 78-days field-aged residues were 18.6% and -7.8%. The fertility assessments did not indicate any effects due to exposure to Coragen®. When A. bipunctata L2 larvae were exposed to fresh-dried spray deposits resulting from 1 or 2 field applications with Coragen® at 52.5 g Rynaxypyr®/ha in a semi-field test no increased mortality over the 7-day assessment period compared to the control was observed. The toxic references, lambda-cyhalothrin and azinphos methyl, significantly increased mortality (Table 2). The effects of Coragen® on a natural coccinellid population was investigated in an apple orchard in northern Italy consisting of Propylea quatuordecimpunctata (90%), Adalia decempunctata (3%), Adalia bipunctata (7%). In the control a mean of 18 adults and 9 larvae was counted before the first application. The population density decreased during the course of the study (Table 3). Both toxic references, lambda-cyhalothrin and azinphos methyl, showed a statistically significant reduction of the coccinellid population already after the 1st application, while in the plots treated once or twice with Coragen® the coccinellid population followed the same pattern as in the control. Based on the available laboratory, extended laboratory, semi-field and field results it is unlikely that Coragen® will have a negative impact on natural coccinellid populations. Episyrphus balteatus ® Based on the extended laboratory test the LR50 for E. balteatus was 12.6 g Rynaxypyr /ha and ® ® ® the ER50 was 13.3 g Rynaxypyr /ha for Coragen . For Altacor the LR50 for E. balteatus was ® ® 4.6 g Rynaxypyr /ha and the ER50 was estimated to be >4.4 g Rynaxypyr /ha. No negative effects on mortality or reproduction of E. balteatus were observed in the bioassays with 28-day 133

or 42-day field-aged residues on apple leaves of both, Coragen® and Altacor® each applied twice at 60 g Rynaxypyr®/ha in a spray volume of 1500 L/ha and a spray interval of 7 days to potted apple trees. Control mortality of the syrphid larvae in the 1st bioassay was high (64.4%), but in the 2nd bioassay the control mortality was 28%. Corrected mortalities after larval exposure to 28- and 42-days field-aged residues were 43.8% and 33.5% for Coragen® and 25.0% and 3.6% for Altacor®, respectively. Also the reproduction assays did not indicate any effect due to either formulation.

Table 2. Mortality of Adalia bipunctata L2 larvae (%) following exposure to fresh-dried spray deposits in caged tree shoots in an apple orchard in northern Italy in 2006.

Mortality of A. bipunctata larvae (%) Treatment 1 DAA2 3 DAA2 7 DAA2 Control (water) 0.9 b 17.7 c 35.4 c Lambda- cyhalothrin 50.6 a* 100 a* 100 a* (2 × 52.5 g/ha) Coragen® 9.2 b* 24.1 c* 17.7 c* (1 × 52.5 g a.s./ha) Coragen® 17.2 ab* 15.2 c* 6.4 c* (2 × 52.5 g a.s./ha) Azinphos methyl 44.8 a* 82.3 b* 91.9 b* (2 × 780 g/ha) Statistics: Tukey’s Test, different letters indicate differences at p < 0.05. * = mortality (%) corrected for control mortality, DBA = Days before application, DAA = Days after application

Table 3. Mean number of coccinellids (larvae (L) and adults (A)) collected with an entomo- logical umbrella in an apple orchard in northern Italy in 2006.

Mean number of larval and adult coccinellids Treatment L/A 14 DAA1 0 DBA1 1 DAA2 3 DAA2 7 DAA2 14 DAA2 0 DBA2 L 9.0 a 0.8 a 0.8 a 0 a 1.0 a 0 a Control (water) A 18.3 a 10.5 a 8.8 a 8.3 ab 7.5 a 1.5 a Lambda- L 5.0 a 0 a 0 a 0 a 0 a 0 a cyhalothrin (2 × 52.5 g/ha) A 19.8 a 0 b 0.3 b 0 c 0.5 b 0.5 a Coragen® L 6.5 a 1.3 a 1.8 a 0 a 1,0 a 0 a (1 × 52.5 g a.s./ha) A 13.0 a 14.0 a 10.3 a 9.8 ab 9.3 a 3.5 a Coragen® L 4.5 a 1.5 a 1.3 a 1.0 a 1.0 a 0 a (2 × 52.5 g a.s./ha) A 23.3 a 12.5 a 14.5 a 18.8 a 7.3 a 3.0 a Azinphos methyl L 6.0 a 0.3 a 0 a 0 a 1.0 a 0 a (2 × 750 g/ha) A 15.5 a 5.0 ab 4.5 ab 2.8 bc 5.8 a 2.5 a Statistics: Tukey’s Test, different letters indicate differences at p < 0.05. DBA = Days before application, DAA = Days after application 134

Table 4. Overview on the side-effects of Altacor® and Coragen® (active substance: DuPont™ Rynaxypyr® = chlorantraniliprole) on beneficial non-target arthropods.

Test Species Tier Mortality Reproduction substance Aphidius ® 1 LR > 750 g a.s./ha ER > 750 g a.s./ha rhopalosiphi Coragen 50 50 Aphidius ® 1 LR > 750 g a.s./ha ER > 750 g a.s./ha rhopalosiphi Altacor 50 50 Typhlodromus pyri Coragen® 1 LR50 > 750 g a.s./ha ER50 > 750 g a.s./ha Predatory mites: T. pyri, A. andersoni, Coragen® 4 None at 47.5 and 52.5 g a.s./ha at 13-day spray interval K. aberrans, P. talbii Predatory mites: ® 4 None at 2 × 60 g a.s./ha at 14-day spray interval K. aberrans Coragen Predatory mites: A. andersoni, T. pyri, Coragen® 4 None at 2 × 60 g a.s./ha at 14-day spray interval E. finlandicus Typhlodromus pyri Altacor® 1 LR50 > 750 g a.s./ha ER50 > 750 g a.s./ha Predatory mites: ® 4 None at 2 × 52.5 g a.s./ha at 15-day spray interval T. pyri Altacor Chrysoperla carnea Coragen® 1 LR50 > 120 g a.s./ha ER50 > 120 g a.s./ha Orius laevigatus Coragen® 2 LR50 > 120 g a.s./ha ER50 > 120 g a.s./ha Anthocoris nemoralis Coragen® 4 None at 2 × 52.5 g a.s./ha at 14-day spray interval Coccinella ® 2 LR = 79.5 g a.s./ha ER = 13.3 g a.s./ha septempunctata Coragen 50 50 None at 2 × 60 g a.s./ha None at 2 × 60 g a.s./ha Coccinella ® 2+ (7-day spray interval) and (7-day spray interval) and septempunctata Coragen 28-days field-aged residue 28-days field-aged residue None at 2 × 52.5 g a.s./ha Adalia bipunctata ® 3 NA Coragen at 14-day spray interval Coccinellids Coragen® 4 None at 2 × 52.5 g a.s./ha at 14-day spray interval Episyrphus balteatus Coragen® 2 LR50 = 12.6 g a.s./ha ER50 = 8.6 g a.s./ha None at 2 × 60 g a.s./ha None at 2 × 60 g a.s./ha Episyrphus balteatus Coragen® 2+ (7-day spray interval) and (7-day spray interval) and 42-days field-aged residue 42-days field-aged residue Episyrphus balteatus Altacor® 2 LR50 = 4.6 g a.s./ha ER50 > 4.4 g a.s./ha None at 2 × 60 g a.s./ha None at 2 × 60 g a.s./ha Episyrphus balteatus Altacor® 2+ (7-day spray interval) and (7-day spray interval) and 42-days field-aged residue 42-days field-aged residue Tier: 1 = laboratory test with glass plates, Tier 2 = extended laboratory test with natural substrate, Tier 2+ = extended laboratory test with natural substrate plus aging of residues under field conditions, Tier 3 = semi-field test, Tier 4 = field test. NA = Not assessed.

Overall, DuPont™ Rynaxypyr® and its formulated products, Coragen® and Altacor® demonstrated to be safe to numerous beneficial non-target arthropod species or to have a rather low and transient impact (Table 4). As DuPont™ Rynaxypyr® has also proven to be safe to pollinators, i.e. honeybees and bumblebees (unpublished data), Coragen® and Altacor® will be excellent tools in integrated pest management (IPM) programmes. 135

Acknowledgements

Sincere thanks to all co-operators contributing to the development of DuPont™ Rynaxypyr®.

References

Bakker, F.M., Aldershof, S.A., Veire, M.v.d., Candolfi, M.P., Izquierdo, J.I., Kleiner, R., Neumann, Ch., Nienstedt, K.M. & Walker, H. 2000. A laboratory test for evaluating the effects of plant protection products on the predatory bug, Orius laevigatus (Fieber) (Heteroptera: Anthocoridae). In: Candolfi et al. (2000): 57-70. Bassi, A., Alber, R., Wiles, J.A., Rison, J.L., Frost, N.M., Marmor, F.W., Marcon, P.C., 2007. Chlorantraniliprole: a novel anthranilic diamide insecticide. Proceedings of XVI Inter- national Plant Protection Congress 2007, Vol. 1: 52-59. Blümel, S., Bakker, F.M., Baier, B., Brown, K., Candolfi, M.P., Goßmann, A., Grimm, C., Jäckel, B., Nienstedt, K., Schirra, K.J., Ufer, A. & Waltersdorfer, A. 2000a. Laboratory resi- dual contact test with the predatory mite Typhlodromus pyri Scheuten (Acari: Phytoseiidae) for regulatory testing of plant protection products. In: Candolfi et al. (2000):121-143. Blümel, S., Aldershof, S., Bakker, F.M., Baier, B., Boller, E., Brown, K., Bylemans, D., Candolfi, M.P., Huber, B., Linder, C., Louis, F., Müther, J., Nienstedt, K.M., Oberwalder, C., Reber, B., Schirra, K.J., Ufer, A. & Vogt, H. 2000b. Guidance document to detect side effects of plant protection preducts on predatory mite (Acari: Phytoseiidae) under field conditions: vineyards and orchards. In: Candolfi et al. (2000): 145-158. Candolfi, M.P., Blümel, S., Forster, R., Bakker, F.M., Grimm, C., Hassan, S.A., Heimbach, U., Mead-Briggs, M.A., Reber, B., Schmuck, R. & Vogt, H. (eds.) 2000. Guidelines to evaluate side-effects of plant protection products to non-target arthropods. IOBC, BART and EPPO Joint Initiative. IOBC/WPRS, Gent (Belgium). 158 pp. Candolfi, M.P., Barrett, K.L., Campbell, P.J., Forster, R., Grandy, N., Huet, M.-C., Lewis, G., Oomen, P.A., Schmuck, R. & Vogt, H. (eds.) 2001. Guidance document on regulatory testing and risk assessment procedures for plant protection products with non-target arthropods. From the ESCORT 2 workshorp. SETAC. 48 pp. Cordova, D., Benner, E.A., Sacher, M.D., Rauh, J.J., Sopa, J.S., Lahm, G.P., Selby, T.P., Stevenson, T.M., Flexner, L., Gutteridge, S., Rhoades, D.F., Wu, L., Smith, R.M. & Tao, Y. 2006. Anthranilic diamides: A new class of insecticides with a novel mode of action, ryanodine receptor activation. Pestic. Biochem. Phys., 84, 196-214. Mead-Briggs, M.A., Brown, K., Candolfi, M.P., Coulson, M.J.M., Miles, M., Moll, M., Nienstedt, K., Schuld, M., Ufer, A. & McIndoe, E. 2000. A laboratory test for evaluating the effects of plant protection products on the parasitic wasp, Aphidius rhopalosiphi (DeStephani-Perez) (Hymenoptera: Braconidae). In: Candolfi et al. (2000): 3-25. Rieckmann, W. 1989. Auswirkungen von Pflanzenschutzmitteln auf die Schwebfliege Syrphus corollae (FABR.) im Laboratorium. Richtlinie für die Prüfung von Pflanzen- schutzmitteln im Zulassungsverfahren, Teil VI 23-2.1.7.: 13 pp. Schmuck, R., Candolfi, M.P., Kleiner, R., Mead-Briggs, M.A., Moll, M., Kemmeter, F., Jans, D., Waltersdorfer, A. & Wilhelmy, H. 2000. A laboratory test for assessing effects of plant protection products on the plant dwelling insect Coccinella septempuncatat L. (Coleoptera: Coccinellidae). In: Candolfi et al. (2000): 45-56. Vogt, H., Bigler, F., Brown, K., Candolfi, M.P., Kemmeter, F., Kühner, Ch., Moll, M., Travis, A., Ufer, A., Vinuela, E., Waldburger, M. & Waltersdorfer, A. 2000. Laboratory method to test effects of plant protection products on larvae of Chrysoperla carnea (Neuroptera: Chrysopidae). In: Candolfi et al. (2000): 27-44. Pesticides and Beneficial Organisms IOBC/wprs Bulletin Vol. 35, 2008 p. 136

Influence of organic matter on bio-availability of two pesticides and their toxicity to two soil dwelling predators

Louis Hautier1, Nicolas Mabon2, Bruno Schiffers2 & Jean-Pierre Jansen1

1Ecotoxicology Laboratory, Department of Biological control and Plant genetic resources, Walloon Agricultural Research Center, Chemin de Liroux, 2, 5030 Gembloux, Belgium E-mail: [email protected] 2 Phytopharmacy Laboratory, Analytical Chemistry Unit, Faculté Universitaire des Sciences Agronomiques de Gembloux, Passage des déportés, 2, 5030 Gembloux, Belgium

Abstract: In order to determine the influence of soil organic matter content on bioavailability of products applied to the soil and their side-effects on soil dwelling beneficial arthropods, a set of experiments with chlorpropham and carbosulfan as test products, Bembidion lampros and Aleochara bilineata as beneficial insects and pure sand with addition of 3, 6 and 9% compost as substrate was carried out in the laboratory. Additional trials on pure sand were also carried out to complete the data and calculate LR50 on inert surface, in order to compare these values with those obtained on sand + organic content. Both beetle mortality or reduction in onion fly pupae parasitism by rove beetle and pesticide bioavailability were determined and compared. Products were tested at different rates according to standard IOBC methods. Bioavailability of pesticide residues was determined by chemical analysis by HPLC, comparing total extract of the substrate to a CaCl2 aqueous extract that only extract pesticide residues that are not fixed on organic matter complex. Results showed that toxicity was strongly correlated to tested dose and organic content of the substrate. At the maximum recommended field rate, Chlorpropham lead to 96 % mortality of B. lampros and 93 % parasitism reduction by A. bilineata on pure sand. With addition of organic matter, toxicity rapidly decreased and the effects of the herbicide only reached 3% for B. lampros and 0% for A. bilineata with sand + 9% of compost. Similar results were obtained with carbosulfan at 1% of the recommended field rate, with 50% mortality for B. lampros on pure sand and 7% on sand + 9% compost and 100% parasitism reduction for A. bilineata on pure sand and 0% parasitism reduction on sand + 9% compost. Intermediate results were obtained with sand + 3% or 6% compost. Decrease in toxicity appeared to be progressive when organic matter was added to the sand and were indicating a strong relationship between effects, applied doses and organic matter content. Pesticide residue analysis confirm that bioavailable doses were negatively correlated with the addition of organic matter. When expressed in percentage of the dose applied, the bioavailable part was only depending of the organic matter content of the substrate. A comparison of dose-response relationship established on pure sand, when bioavailability was assessed and reached 93.2-98.6% of the dose applied and dose-response relationship established on basis of effects obtained on sand + organic matter and bioavailable doses were indicating that the dose-response were strongly related for the 4 systems (2 products x 2 insects). These results confirm that organic matter is a major component of the soil able to immobilise pesticide residue and reduce their toxicity to beneficial organism. This propriety is discussed in regard of testing scheme for soil beneficial (selection of substrate) and in the global context of pesticide use, as the fixation of pesticide on organic matter has probably also a great impact on efficacy of products and selection of dose to be applied in the field.

136 Pesticides and Beneficial Organisms IOBC/wprs Bulletin Vol. 35, 2008 p. 137

Different methods of application – Different laboratory test strategies

Claudia Norr, Barbara Baier & Detlef Schenke

Julius Kühn Institute, Federal Research Centre for Cultivated Plants, Institute for Ecological Chemistry, Plant Analysis and Stored Product Protection, Königin-Luise-Str. 19, D-14195 Berlin. E-mail: [email protected]

Abstract: Some pesticides can be applied as seed treatment or they can be sprayed. The assessment of effects of seed treatments on beneficial arthropods in the field is usually based on the results of spray applications, if results of seed treatments are not available. However, laboratory tests with the active ingredient imidacloprid using equal imidacloprid rates per ha for seed treatment and spray application showed different effects on the larvae of the carabid beetle Poecilus cupreus. The aim of the investigations was to explain the different effects on the larvae of Poecilus cupreus in extended laboratory tests by residue analyses of the soil. Residues of imidacloprid in soil were obtained using a method developed by Schöning (2001). The effects on larvae of Poecilus cupreus were calculated according to methods of Heise et al. (2004). Residue analyses indicated differences in the distribution of the active ingredient in the soil depending on the application method. Point exposure (coated sugar beet seeds), exposure limited to the seed row (coated winter wheat seeds) and exposure of the whole area (spray application) were detected. Point exposure and exposure limited to the seed row had lower effects on the larvae of Poecilus cupreus than spray application. The results suggest that seed density in the field is one major criterion which has to be considered in tests with plant protection products applied as seed coating in laboratory.

References

Heise, J., Heimbach, U. & Schrader, S. 2004: Influence of insecticide coated seeds on larvae of Poecilus cupreus (L.) (Coleoptera: Carabidae) using different container sizes and quantities of substrate. IOBC/WPRS Bulletin 27 (6): 73-79. Schöning, R. (2001): Pflanzenschutz-Nachrichten Bayer 54 (3): 413-450.

137 Pesticides and Beneficial Organisms IOBC/wprs Bulletin Vol. 35, 2008 pp. 138-142

Assessment of side-effect of water-soluble nitrogen fertilizers applied as foliar spray on the parasitic wasp Aphidius rhopalosiphi (DeStefani- Perez) (Hym.; Aphidiidae)

David Dantinne & Jean-Pierre Jansen Ecotoxicology Laboratory, Department of Biological control and Plant genetic resources, Walloon Centre of Agricultural Research, Chemin de Liroux, 2, 5030 Gembloux, Belgium

Abstract: In several crops, nitrogen fertilizers can be routinely applied as foliar spray at period when beneficial arthropods are active and, thus, exposed to these products in a same way as pesticides. If side-effects of pesticides on beneficial arthropods are well documented, little is know about possible negative impact of nitrogen formulations on beneficial arthropods. In this research, the effects of 3 nitrogen fertilzers applied as foliar spray were tested on the parasitic wasp A. rhopalosiphi in the laboratory on glass plates and on plants. This species was selected because it is very sensitive to pesticides and used as "standard species" for ecotoxicological tests in the context of registration at European level. It is also a key beneficial arthropod for aphid control. The nitrogen formulations tested were a nitrogen solution (nitrate, urea and ammoniac in solution), pearled urea (liquid urea) and Nutriforce®. These fertilizers are widely used in crops such as cereals or potatoes. They were tested at their maximum recommended field rate, corresponding to an application of 15-20 N units/ha according to the product. The nitrogen formulations were first tested on glass plates, according to the IOBC Tier I testing scheme. All formulation exhibit a high toxicity, mainly due to mechanical effects, with re- crystallization of urea and high hygroscopicity of residue. Results clearly showed that Tier I test methodology was not adapted for nitrogen formulation at field rate. Nitrogen formulations were further tested on plants in the laboratory, according to IOBC Tier II testing scheme. Fertilizers were applied on barley seedlings infested with cereal aphids. Both mortality and repellence were followed through a 48h period and aphid mummies were left to developed 10-12 days. They were counted by plants and assessed for parasite emergence. Both lethal (mortality) and sublethal effects (aphid mummies production and emergence) were used to calculate reduction in beneficial capacity, compared to a water-treated control. When they were applied on barley seedlings, the three nitrogen formulation were only slightly toxic for adult wasp, with a minimum of 14% corrected mortality with the nitrogen solution and a maximum of 44% with pearled urea. However, a strong reduction in female capacity was observed with 50.4 aphid mummies/female for control and only 13.4, 9.0 and 17.8 aphid mummies/female with pearled urea, nitrogen solution and Nutriforce®, respectively. Emergence rate of the mummies were comparable to control values. Due to effects on reproduction, the reduction of beneficial capacity were comprised between 69.8% and 85.4%. According to IOBC toxicity classes, Nutriforce® was considered as moderately harmful (class 3) and pearled urea and nitrogen solution as harmful (class 4). Magnitude of the effects was similar than for classical insecticides, indicating that foliar nitrogen application can have a biological signification for beneficial arthropods and probably also on other organisms exposed to foliar spray.

Key words: Aphidius rhopalosiphi, foliar fertilizers, glass plate test, extended lab test

Introduction

Development of IPM programs has promoted the use of pesticides selective for beneficial arthropods, as parasites and predators of agricultural pests (Franz et al., 1980; Hassan et al.,

138 139

1983; 1987; 1988; 1991; 1994; Hautier et al., 2006; Sterk et al., 1999). However, several other products, as nitrogen fertilizers are also routinely applied on crops in a same way as pesticides and the effects of these products on environment, especially beneficial arthropods, are not known. The aim of this study is to assess the possible side-effects of nitrogen fertilizer formulations applied as foliar spray on the parasitic wasp Aphidius rhopalosiphi. This hymenoptera is a key beneficial arthropod for aphid control and a species very sensitive to pesticides, used as "standard species" for ecotoxicological tests in the context of registration at European level.

Material and methods

The nitrogen formulations tested were a nitrogen solution (nitrate, urea and ammoniac in solution), pearled urea (liquid urea) and Nutriforce®. All these commercial formulations are widely used in crops such as cereals or potatoes. They were tested at their recommended field rate, corresponding to 15-20 N units/ha per application, according to the product. Details on the product tested are given in table 1.

Table 1: Details on the nitrogen fertilizers tested (N content, origin, tested rate). Tested rate N units Tested rate (N Composition (kg or l of (N kg/100kg) kg/ha) product/ha) Nitrogen solution 39 50 % urea, 25% 19.5 50 kg/ha in NO3, 25% NH4 400 l of water Pearled urea 46 CON2H4 (urea) 15 32.6 kg/ha in 100% (solid form) 400 l of water Nutriforce® 18 CON2H4 (urea, 19.8 110 l/ha in solution in water) 400 l of water

Nitrogen formulations were tested on barley seedlings in the laboratory with two extended lab-tests: the first (Extended “A” test) was the same as recommended for pesticides (Mead-Briggs et al., 1997) and the second (Extended “B” test) was an upgrade of the first, where aphids used for assessment of sublethal effects are also exposed to tested product (Jansen, 1998). The difference between the two methods were that the “B” method was closer to field conditions but with an higher complexity of several factors and a lower standardisation. A first screening was performed on glass plates according to standardised methods (Mead-Briggs et al., 2001), but results were not presented as several mechanicals problems occurred on the glass plates (crystallisation of urea or nitrogen salts, high hygroscopicity of these compounds and high mortality of the insects due to mechanical problems, trapped or glued on the glass plates). Results showed that glass plates methods were not adapted to test the nitrogen formulations at their maximum recommended field rate. Therefore, only extended laboratory tests on plants were performed. The “extended lab test A” method consists in exposing female parasitic wasps to barley seedlings treated with the test product at the recommended rate. There were 5 replicates of 6 wasps each by object. A sugar solution is applied before product application to simulate aphid honeydew and enhance wasp foraging on the plants. After 48h of exposure, surviving females are collected and confined individually on aphid infested untreated barley seedlings for a 24h- period. Plants and aphids are kept 10-12 days and aphid mummies (parasitised aphids) that 140

developed were counted to assess fertility performance. Aphid mummies are collected and kept 8 more days for the estimation of hatching rate. The “extended lab B” method is similar except that parasitic wasp are directly confined on treated barley seedlings infested with cereal aphids and that the fertility assessment and exposure were performed in the same units. Compare to the “A” method, wasp foraging was only stimulate by aphid honeydew and aphids. In this case, aphid parasite larvae were also exposed to test product. For “A” and “B” test, products were applied in solution in tap water till run-off with the help of a handsprayer, on basis of a 400 l/ha application. Wasps were released in the units 1 to 2 hours after product application, when product residues were dried. Each experiment was performed using five replicates. After approximately 1h and 24h of exposure, each replicate was observed 5 times at 10-15 min interval and wasps were noted as being on the plants (treated) or on the substrate or the unit cells (untreated). There was a total of 50 observations per object. Percentages of wasps foraging on plants were calculated and compare to control to detect possible repellent effects. At the end of the exposure phase, mortality was recorded and corrected according to control values with the Abbott’s formula (Abbott, 1925). Observed mortality and parasitic wasps found on plants were compared to control with the help of a Student t test at p=0.05 level. All experiments were performed in a climatic chamber at 20°C ± 2, 60-90 % RH and with a sodium lamp lighting (7500-15000 lux, L/D 16:8).

Results and discussion

Results of Extended "A" and "B" tests are summarized in table 2 and 3, respectively. With the first test, where a sucrose solution is applied before the fertilizers, high mortality was observed with all formulations, ranging from 53% to 73%. The mortalities were mainly due to mechanical effects, parasitic wasp being glued and trapped when fertilizers crystallized. The Nutriforce ® has also a significant repellent effect, with a 45% reduction of wasp foraging compared to control. An assessment of sublethal effects was planned, but as most of the surviving females were greatly affected by the products, this assessment was cancelled.

Table 2: Effects of nitrogen fertilizers applied as foliar spray on adults of the parasitic wasp A. rhopalosiphi. Results of the Extended lab “A” test on plants using a standard test as for registration of pesticides Proportion of wasps Observed Corrected IOBC on plants mortality mortality Toxicity (%) (%) (%) Class Control 31.1 a 10.0 a - - Nitrogen solution 26.4 a 58.0 b 53.3 3 Pearled urea 25.7 a 76.0 b 73.3 3 Nutriforce ® 17.1 b 61.0 b 56.7 3 Results followed by different letters are statistically different (Student t-test, p=0.05)

With the “B” test, where sugar solution was replaced by aphid and aphid honeydew, lesser mechanical effects could be observed. Repellent effect was increased by 26% with nitrogen solution compared to controls. Maximum mortality was reached with pearled urea (44%). Surviving females were greatly affected and number of parasitised aphids was reduced 141

ranging from 64% to 82% compared to control. However, hatching rate of the aphid mummies were not affected by the products. If results of both mortality and fertility test were combined to estimate reduction in beneficial capacity according to Overmeer-Van Zon formula (Overmeer and Van Zon, 1982), the effects of test products reached at least 70% for Nutriforce®, the less toxic, with a maximum of 87% for Pearled urea, the most toxic one. According to IOBC toxicity classes, these products can be classified as moderately harmful (class 3, Nutriforce®) or harmful (class 4, Pearled urea and Nitrogen solution). Compared to pesticides tested with the similar methods, the toxicity was similar to those of some pyrethroids.

Table 3: Effects of nitrogen fertilizers applied as foliar spray on adults of the parasitic wasp A. rhopalosiphi. Results of the Extended lab “B” test on plants using the standard test for registration of pesticides upgraded for assessment of sublethal effects. Proportion Observed Number of Hatching E IOBC of wasps mortality aphid rate (%) Toxicity on plants (%) mummies/unit (%) Class (%) Control 34.7 a 0.0 a 50.4 a 87.0 a - - Nitrogen solution 43.7 b 14.0 a 9.0 b 86.5 a 84.7 4 Pearled urea 33.4 a 44.0 b 13.8 b 75.0 a 86.8 4 Nutriforce ® 19.4 c 16.0 b 17.8 b 86.9 a 70.4 3 Results followed by different letters are statistically different (Student t-test, p=0.05)

Effects of nitrogen fertilizers on the non-target arthropod A. rhopalosiphi on plants using two different methods were sufficient to reduce the beneficial capacity of the parasitic wasp up to 85.0%, with observation of lethal and sublethal effects. Nitrogen fertilizers can have a direct lethal effect, mainly due to mechanical effects of the products, and a sublethal effect, by reducing the number of aphids parasitised by surviving females. Effects were comparable to those of several insecticides. In our test conditions, no decrease in hatching rate was demonstrated. Repellent effect shown with two fertilizers illustrates a possible loss of aphids control by the parasitic wasps. Further research is needed to determine possible long-term effects of foliar fertilizers in the field on beneficial arthropods.

References

Abbott, S.W. 1925: A method of computing the effectiveness of insecticides. J. Econ. Entomol. 18: 265-267. Franz, J.M., Bogenschütz, H., Hassan, S.A., Huang, P., Ledieu, M.S., Naton, E.; Suter, H. & Viggiani, G. 1980: Results of a joint pesticide test programme by the Working Group "Pesticides and Beneficial Arthropods". Entomophaga 25: 231-236. Hassan, S.A., Albert, F., Bigler F., Blaisinger, P., Bogenschütz, H., Brun, J., Chiverton, P., Edwards, P., Englert, W.D., Huang, P., Inglesfield, C., Naton, E., Oomen, P.A., Overmeer, W.P., Rieckmann, W., Samsøe-petersen, L., Stäubli, A., Tuset, J.J., Vanwetswinkel, G.& Viggiani, G. 1987: Results of the third joint pesticide testing programme by the IOBC/WPRS-Working Group "Pesticides and Beneficial Arthropods". J. Appl. Entomol. 103: 92-107. 142

Hassan, S.A., Bigler, F., Bogenschütz, H., Boller, E., Brun, J., Chiverton, P., Edwards, P., Mansour, F., Naton, E., Oomen, P.A., Overmeer, W.P., Polgar, L., Rieckmann, W., Samsøe-petersen, L., Stäubli, A., Sterk, G, Tavares, K., Tuset, J.J., Viggiani, G. & Vivas A.G. 1988: Results of the fourth joint pesticide testing programme by the IOBC/WPRS- Working Group "Pesticides and Beneficial Arthropods". J. Appl. Entomol. 105: 321-329. Hassan, S.A., Bigler, F., Bogenschütz, H, Boller, E., Brun, J., Calis, J.N., Chiverton, P., Coremans-Pelseneer, J., Duso, C., Lewis, G.B., Mansour, F., Moreth, L., Oomen, P.A., Overmeer, W.P., Polgar, L., Rieckmann, W., Samsøe-petersen, L., Stäubli, A., Sterk, G., Tavares, K., Tuset, J.J. & Viggiani, G. 1991: Results of the fifth joint pesticide testing programme carried out by the IOBC/WPRS-Working Group "Pesticides and Beneficial Arthropods". Entomophaga 36: 55-67 Hassan, S.A., Bigler, F., Bogenschütz, H., Boller, E., Brun, J., Calis, J.N., Coremans- Pelseneer, J., Duso, C., Grove, A., Heimbach, U., Helyer, N., Hokkanen, H., Lewis, G.B., Mansour, F., Moreth, L., Polgar, L., Samsøe-petersen, L., Sauphanor, B., Stäubli, A., Sterk, G., Vainio, A., Van de Veire, M., Viggiani, G. & Vogt, H. 1994: Results of the sixth joint pesticide testing programme of the IOBC/WPRS-Working Group "Pesticides and Beneficial Arthropods". Entomophaga 39: 107-119 Hassan, S.A., Bigler, F., Bogenschütz, H., Brown, J.U., Firth, S.I., Huang, P., Ledieu, M.S., Naton, E., Oomen, P.A., Overmeer, W.P., Rieckmann, W., Samsøe-petersen, L., Viggiani, G. & Van Zon, A.Q. 1983: Results of the second joint pesticide testing programme by the IOBC/WPRS-Working Group "Pesticides and Beneficial Arthropods". J. Appl. Entomol. 95: 151-158. Hautier, L., Jansen, J-P., Mabon, N., Schiffers, B., Deleu, R. & Moreira, C. 2006: Building selectivity list of plant protection products on beneficial arthropods in open field: a concrete example with potato crop. IOBC wprs Bull. 29(10): 21-32. Jansen, J-P. 1998: Effects of wheat foliar fungicides on the aphid endoparasitoid Aphidius rhopalosiphi DeStefani-Perez (Hym., Aphidiidae) on glass plates and on plants. J. Appl. Entomol. 123: 217-223. Mead-Briggs, M. & Longley, M. 1997: A standard extended laboratory test to evaluate the effects of plant protection products on adults of the parasitoid Aphidius rhopalosiphi (Hymenoptera, Braconidae). Internal method, unpublished Mead-Briggs, M.A., Brown, K., Candolfi, M.P., Coulson, M.J., Miles, M., Moll, M., Nienstedt, K., Schuld, M., Ufer, A. & McIndoe, E. 2001. A laboratory test for evaluating the effects of plant protection products on the parasitic wasp, Aphidius rhopalosiphi (DeStefani-Perez) (Hymenoptera: Braconidae). In: Guidelines to evaluate side-effects of plant protection products to non-target arthropods, an IOBC, BART and EPPO Joint Initiative”, eds. Candolfi, M.P., Blümel, S., Forster, R., Bakker, F.M., Grimm, C., Hassan, S.A., Heimbach, U., Mead-Briggs, M.A., Reber, B., Schmuck, R., and Vogt, H.: 13-26. Overmeer, W.P. & Van Zon, A.Q. 1982: A standardized method for testing the side effects of pesticides on the predacious mite Amblyselus potentillae (Acarina; Phytoseiidae). J. Econ. Entomol. 93: 1-11. Sterk, G., Hassan, S.A., Baillod, F., Bakker, F., Bigler, F., Blumel, S., Bogenschütz, H., Boller, E., Bromand, B., Brun, J., Calis, J.N., Coremans-Pelseneer, J., Duso, C., Garido, A., Grove, A., Heimbach, U., Hokkanen, H., Jacas, J., Lewis, G. B., Moreth, L., Polgar, L., Rovesti, L., Samsøe-petersen, L., Sauphanor, B., Schaub, L., Stäubli, A., Tuset, J.J., Vainio, A., Van de Veire, M., Viggiani, G., Vinuela, E. & Vogt, H. 1999: Results of the seventh joint pesticide testing programme of the IOBC/WPRS-Working Group "Pesticides and Beneficial Organisms". BioControl 44: 99-117. Pesticides and Beneficial Organisms IOBC/wprs Bulletin Vol. 35, 2008 p. 143

Field toxicity of four acaricides on the predatory mites Amblyseius andersoni (Chant) and Euseius stipulatus (Athias-Henriot) (Acari: Phytoseiidae) in apple orchard at Northwest of Portugal

J. Raul Rodrigues & Laura M. Torres

* Escola Superior Agrária de Ponte de Lima, Refóios do Lima, 4990-706 Ponte de Lima, Portugal; **Universidade de Trás-os-Montes e Alto Douro - Quinta de Prados, 5000-911 Vila Real, Portugal. E-mail: [email protected]

Abstract: The complex of species of predatory mites dominated by Amblyseius andersoni (Chant) and Euseius stipulatus (Athias-Henriot) (Acari: Phtyoseiidae) has been recognized as highly important in regulating phytophagous mites in apple orchards in northwest region of Portugal. In order to utilize these species in integrated pest management programs in apple orchards, it is essential to obtain information about the field toxicity of commonly used pesticides on these predators. During the period of 2002-2004, three bioassay experiments were undertaken to assess the field toxicity of four acaricides Vertimec® (abamectin), Dinamite® (fenpyroximate), Magister® (fenazaquin) and Envidor® (spirodiclofen), on phytoseiids A. andersoni and E stipulatus, in apple orchard in the region of Braga, Portugal, according to the standard guidelines of IOBC/wrps Working Group “Pesticides and Beneficial Organisms”. Vertimec® was found to be harmless to slightly harmful, Dinamite® and the novel acaricide Envidor® were assessed as slightly to moderately harmful to the phytoseiid mites, while Magister® revealed to have a poor selectivity to these predators, that was moderately harmful to harmful.

Key-words: apple tree; integrated pest management; predatory mites; acaricides, side-effects

143 Pesticides and Beneficial Organisms IOBC/wprs Bulletin Vol. 35, 2008 pp. 144-145

Influence of teflubenzuron residues on the predation of thrips by Iphiseius degenerans and Orius laevigatus 1

Alison Scott Brown, Monique Simmonds and Wally Blaney Royal Botanic Gardens, Kew, Surrey, TW9 3AB, UK.

Abstract1: Nomolt® is commonly prescribed as a control for western flower thrips; Frankliniella occidentalis (Pergande) (Anonomyus 1998), on ornamentals and was recommended for use in the glasshouses at The Royal Botanic Gardens, Kew, to combat infestations of Heliothrips haemorrhoidalis (Bouché). The active ingredient teflubenzuron (a benzoylphenyl urea compound) disrupts the formation of chitin, preventing larval stages developing into adult thrips. Control is achieved through a slow decline of the pest population which is ensured by additional applications of the compound over a specified period of time. The biological control agents used against thrips in the glasshouses at Kew include a phytoseiid mite, Iphiseius degenerans (Berlese), and an anthocorid bug, Orius laevigatus (Fieber). Teflubenzuron was initially reported to be harmless to beneficials (Agrow 1990) , however, more recent studies have demonstrated that benzoylureas affect a range of predators including anthocorids (Angeli et al. 2000, Sterk et al. 1999). A short duration (24-h) leaf-disc bioassay was used to determine the effects of teflubenzuron residues on the predation levels of I. degenerans and O. laevigatus, foraging on immature life stages of F. occidentalis and H. haemorrhoidalis on a range of different species of plants. The rationale behind the experiments was to investigate why immediately after spraying infested plants with the recommended concentration of teflubenzuron, thrips populations declined rapidly on some species of plants but not on others. It was unclear as to whether the differences observed after spraying could be because the insecticide was affecting the thrips or weakening the predators and to what extent the plant host effected these interactions. In summary, contact with teflubenzuron residues caused an increase in thrips mortality during the 24-h bioassay; it was more active against H. haemorrhoidalis than F. occidentalis. Teflubenzuron did not cause significant mortality within 24 hours to either species of predator, although the foraging effectiveness of both predators was reduced in the presence of teflubenzuron. The teflubenzuron- predator interaction was also influenced by the species of plant. Thus the observations in the glasshouse at RBG Kew that populations of thrips on some species of plants decreased soon after spraying with teflubenzuron could be associated with these interactions or to the fact that Nemolt® formulation was itself able to reduce thrips numbers even after the short exposure time. Further research into the effect that teflubenzuron has on the foraging behaviour of predators and the behaviour of thrips and over a longer exposure time would assist to evaluate its suitability for use as part of an IPM system for ornamentals.

Key words: Iphiseius degenerans; Orius laevigatus; Frankliniella occidentalis; Heliothrips haemorrhoidalis; host plants; teflubenzuron

1 Full publication: Scott Brown, A.S., Simmonds, M.S.J. and Blaney, W.M. 2003: Influence of a short exposure to teflubenzuron residues on the predation of thrips by Iphiseius degenerans (Acari: Phytoseiidae) and Orius laevigatus (Hemiptera: Anthocoridae). Pest Management Science 59: 1255-1259.

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References

Agrow 1990: Insect growth Regulators, Agrow Report (February), Agrow, Surrey, UK. Angeli, G., Forti, D. and Maines, R. 2000: Side-effects of eleven insect growth regulators on the predatory bug Orius laevigatus Fieber (Heteroptera: Anthocoridae). IOBC/WPRS Bull. 23 (9): 85-92. Anonomyous 1998: Information Sheet, Applied Horticulture, Fargro Ltd, UK. Sterk, G., Hassan, S.A., Baillod, M., Bakker, F., Bigler, F., Blümel, S., Bogenschütz, H., Boller, E., Bromand, B., Brun, J., Calis, J.N.M., Coremans-Pelseneer, J., Duso, C., Garrido, A., Grove, A., Heimbach, U,, Hokkanen, H., Jacas, J., Lewi,s G., Moreth, L., Polgar, L., Rovesti, L,, Samsøe-Peterson, L., Sauphanor, B., Schaub, L., Stäubli, A., Tuset, J.J., Vainio, A., Van de Veire, M., Viggiani, G., Viñuela, E. and Vogt, H. 1999: Results of the seventh joint testing programme carried out by the IOBC/WPRS-Working group ‘Pesticides and Beneficial Organisms’. Biocontrol 44: 99-117.

Pesticides and Beneficial Organisms IOBC/wprs Bulletin Vol. 35, 2008 pp. 146-151

Study on the side-effects of three pesticides on the predatory mite, Phytoseius plumifer (Canestrini & Fanzago) (Acari: Phytoseiidae) under laboratory conditions

S. Noii, K. Talebi, A. Saboori, H. Allahyari, Q. Sabahi and A. Ashouri Department of Plant Protection, College of Agriculture, University of Tehran, Karaj 31584, Iran.

Abstract: The predatory mite, Phytoseius plumifer is one of the most abundant natural enemies of phytophagous pests and mites especially in the north of Iran. Experiments were carried out to assess the compatibility of commonly used pesticides against phytophagous pests in order to determine pesticides which have the least side-effects on the predator and are more suitable for using in integrated control programs. In this study the side-effects of three pesticides (abamectin, malathion and phosalone) were evaluated in laboratory. The laboratory tests were done using the ‘detached leaf’ method. Percentages of predator mortality and oviposition rate were assessed. The effect of the pesticides at the maximum field rates on P. plumifer adults was above the upper tolerance threshold. All three tested pesticides caused 100% mortality within 24 hours after treatment and were classified as harmful for the predator. Therefore they are not compatible with the predator within an integrated control program. Effects of abamectin, malathion and phosalone even at 0.1 recommended field rates were above the upper tolerance threshold. The residue test revealed that these pesticides caused 100% mortality within 3, 10 and 15 days after treatment. According to a dose-response test, the LR50 of phosalone amounts to 1.48 µg a.i./cm2 for the adult predator. The rate of fecundity decreased as the rate phosalone increased. These results suggested that P. plumifer can be used as a biological indicator in the safe shelters.

Key words: side-effects, Phytoseius plumifer, abamectin, malathion, phosalone

Introduction

The predatory mite, Phytoseius plumifer (Canestrini & Fanzago) (Acari: Phytoseidae) is one of the most abundant natural enemies of phytophagous pests in natural habitat, especially in the north of Iran (Hajizadeh et al, 2002). This mite that mostly occurs in fig trees is a generalist predator and feeds on all stages (except resting stages) of fig spider mite, Eotetra- nychus hirsti Pritchard & Baker and other genera of tetranychid mites. This predator can also feed on different species of thrips and other minute sucking pests. Studying the side-effects of pesticides on natural enemies, including predaceous mites is an important task in pest management program, however, the use of pesticides remains necessary due to inadequate control achieved by natural enemies. The combination of biological and chemical control as an IPM program is only possible when the side-effects of pesticides on natural agents are well known. In this study the side-effects of commonly used pesticides against P. plumifer were evaluated under laboratory conditions.

146 147

Materials and methods

This study was carried out to assess the compatibility of the predator with three pesticides, including abamectin, malathion and phosalone in order to determine pesticides with the least side-effects on P. plumifer and as suitable compound in Integrated Pest Management Programs. We used "detached leaf" method based on sequential decision making scheme presented by Oomen (1988). Test organisms and rearing methods The predatory mite P. plumifer was collected from fig trees in the Botanic Garden of the Agriculture Campus at University of Tehran. The predator had not been exposed to pesticides previously. The collected individuals were placed in the rearing units which contained a detached fig leaf. The leaf placed under side up on a water-soaked cotton pad in a cut-out Petri dish (8 cm diameter of cut-out circle) and the dish set into another bigger one (10 cm diameter) as a water supply for the first one. A roll of water-soaked cotton was placed around the leaf to prevent escaping of mites from the leaf. Rearing units were kept under laboratory conditions at 27±2°C, 50±5% RH and photoperiod of 16L:8D. Juvenile stages (mostly larval stage) of the two-spotted spider mite, maize pollen and diluted solution of sugar were added to rearing units as daily food. Pesticides and application The three tested pesticides were used at recommended field rates: Abamectin (Vertimec®) 1.8 g/ha of 1.8 EC; Malathion (Malathion®) 570.0 g/ha of 57 EC; Phosalone (Zolone®) 262.5 g/ha of 35 EC. Test units were treated with pesticides solution, using a potter tower (Burckhard Manufacturing) to get 2 mg wet deposit per square centimeter and left to dry. Tap water was used as control. Residual initial toxicity Rearing units were used as test units and treated with the pesticides in four replicates. Since preliminary tests revealed that mortality of the tiny and sensitive stages (larva and protonymphs) in the control treatments was 100%, the experiments were carried out with adults of P. plumifer. Some hundred larvae of spider mites and fifteen 3- days-old adults of P. plumifer were placed in each test unit and mortality of the mites was assessed. Optional route test This trial is the same as the residual initial toxicity test, but was carried out with 0.1 recommended field rates whenever total effect of the recommended rate was above 99%. Residual persistence test According to the procedure when the results of sequential decision-making scheme of the IOBC are not decisive (E>50%), the residual persistence test should be accomplished. Mortality of predatory mite on the first and third day after treatment, also the number of escaped predators and eggs per unit were counted and the food was added as needed. Statistics and calculation of the total effects Total effects of the pesticides were calculated by the following formula:

E = 100 %-( 100%-Ma) × Er in which E is the total effect, Ma is corrected mortality calculated using Abbott formula (Abbott, 1925) and Er is the effect of pesticides on reproduction. Based on total effects, hazard classification of pesticides was evaluated according to the IOBC evaluation categories (Hassan, 1991).

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LR50 of phosalone In order to determine the LR50 (Lethal Rate) of phosalone on P. plumifer, different rates of the insecticide were used plus water as control. Five rates were selected based on bracketing test results plus control unit in four replications. The rates were: 0.70, 0.98, 1.40, 1.98 and 2.89 µg a.i./cm2. For achieving these rates on the leaf, it was needed to use solutions of the formulated product (Zolone® 35 EC) at concentrations of 0.35, 0.49, 0.70, 0.99 and 1.44 mg/ml respectively, and to apply 2 mg/cm2 of each solution. Each test unit was sprayed and then 10 adults of P. plumifer as well as sixty larvae of the spider mite placed on each dried unit. Percentages of mortality and numbers of eggs on each unit were measured within five days after treatment. An estimate of the LR50 and the regression equation for the rate-mortality line was obtained using software (LeOra Software, 1987).

Results and discussion

Effects on mortality Residual initial effect of the pesticides on mortality of P. plumifer is presented in table 1. All three compounds caused 100% mortality at recommended rates and thus were classified as harmful insecticides. Using insecticides at 0.1 the recommended field rate, lead to 100% mortality of P. plumifer. These results are presented in Table 2. Exposing of the predator to 3- day old residues of insecticides caused 100% mortality. Table 3 showed these results. Results of the probit analysis for the effect of phosalone on P. plumifer are shown in Table 4. Effects on reproduction Effect of Phosalone at different rates on the oviposition of P. plumifer is presented in Table 5. Mean oviposition increased with decrease in rate of Phosalone, so that at 2.89 µg/cm2 the mean was 1.7, whereas it was 24,7 at 0,7 µg/cm2. There were significant differences among treatments at p<0.05. Mortality is the clearest effect of a pesticide. It is easily measurable and has been applied in many studies. On the other hand pesticides have sublethal effects and these effects can alter fecundity, development period, longevity and behavior repellent effects (Croft, 1990, Stark et al., 1998). Total effect (E) of the three pesticides, malathion, phosalone, and abamectin at the recommended rates in the residual initial toxicity test on P. plumifer was above 99%. Therefore these pesticides were classified as harmful, based on the categories suggested by IOBC (Table 1). Oomen et al. (1991) reported the side effects of 100 pesticides on P. persimilis, and concluded that in 83% of experiments pesticides could be categorized based on the first toxicity test. In spite of this, one optional test with 0.1 the recommended field rate showed total effect of this rate was above 50% for all three pesticides (Table 2). Therefore further tests were unnecessary. Results of 3-day old residues showed a total effect above 30% (Table 3), so it was shown that even 3-day old residues were too dangerous for P. plumifer. Even the effects of 10 and 15-day old residues test were the same as 3-day old residues, and 100% mortality of P. plumifer were observed in all treatments. Nadimi (2006) reported that abamectin and fenpyroximate (at recommended field rate) caused 100% mortality of the predatory mites, Phytoseiulus persimilis and P. plumifer. Pesticides can have different effects on the oviposition rate of predatory mites. Kavousi (2003) showed that Heptenophos increased the fecundity rate of the predatory mite Phytoseiulus persimilis at recommended field rates. Results of Nadimi (2006) showed, that hexythiazox had significant effect on increasing fecundity rate of P. persimilis at the residual initial toxicity rate of 0.5 and 0.25 of the recommended field rate.

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Table 1. Residual initial effect of the pesticides at recommended rate on survival of P. plumifer (adult stage).

Treatment Rate1 Mortality Classification (µg/cm2) (%) (IOBC) Control - 15 - Abamectin 0.018 100 4* Malathion 5.70 100 4 Phosalone 2.63 100 4 *: harmful

Table 2. Mortality of P. plumifer at 0.1 rate of recommended field rate.

Rate1 Mortality Classification Treatment (µg/cm2) (%) (IOBC) Control - 5 Abamectin 0.0018 100 4* Malathion 0.570 100 4 Phosalone 0.263 100 4 *: harmful

Table 3. Mortality of P. plumifer (adults) after exposure to 3-day old residues of insecticides.

Treatment Rate Mortality Classification (µg/cm2) (%) (IOBC) control - 5 - Abamectin 0.018 100 4* Malathion 5.70 100 4 Phosalone 2.63 100 4 *: harmful

Table 4. Probit analysis for the effects of phosalone on P. plumifer (adults) and LR50 (Lethal rate) determination.

2 Numbers Numbers slope LR50 χ df 95%FL SE of the of the rates (µg/cm2) predators 240 6 3.05 1.48 12.40 18 1.22-1.78 0.54

1 For achieving these rates on the leaf, it is needed to use solutions of the formulated products at concentrations of 0.0036 g/l for abamectin, 1.14 g/l for malathion and 0.525 g/l for phosalone and apply 2 mg/cm2 of each solution.

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Table 5. Average oviposition for P. plumifer in sublethal concentrations of phosalone.

Rate Mean±SE (µg/cm2) Control 61.7 ± 5.1 a 0.70 24.7 ± 2.8 b 0.98 29.5 ± 4.3 b 1.40 8.7 ± 1.8 c 1.98 5.0 ± 1.1 c 2.89 1.7 ± 0.7 d Mean±SE followed by the same letter were not significantly different based on Duncan´s multiple range test (p<0.05).

In our study it was not possible to assess the effects of the pesticides on fecundity in residual initial toxicity and residual persistence test due to 100% mortality in the first 24 hours of the test, but effects of sub lethal rates of phosalone showed a significant decrease in average oviposition of P. plumifer. It is suggested to use sensitive organisms to assess the safeness of pesticides (Samsøe-Petersen, 1990). Results of the toxicity tests suggest that P. plumifer can be used as a biological indicator for the safe shelters. In order to recognize the efficiency of this predatory mite in IPM programs, further research on biology, feeding and predatory behavior (as insufficient information exists in these fields) and side-effects of other pesticides are recommended

Acknowledgement

This research project was founded by the Centre of Excellence in Plant Protection, Ministry of Science and Technology, Iran.

References

Abbott, W. 1925: A method of computing the effectiveness of an insecticide. J. Econ. Entomol. 18: 265-267. Croft, B.A. 1990: Arthropod biological control agents and pesticides. John Wiley & Sons, New York, USA: 110-125. Hajizadeh, J., Hosseini, R., & McMurtry, J.A. 2002: Phytoseiid mites (Acari: Phytoseiidae) associated with eriophyid mites (Acari: Eriophyidae) In: Guilan province of Iran. Internat. J. Acarol. 28(4): 373-377. Hassan, S.A., Bigler, F., Bogenschütz, H., Boller, E., Brun, J., Calis, J.N.M., Chiverton, P., Coremans-Pelseneer, J., Duso, C., Lewis, G.B., Mansour, F., Moreth, L., Oomen, P.A., Overmeer, W.P.J., Polgar, L., Rieckmann, W., Samsøe-Petersen, L., Stäubli, A., Sterk, G., Tavares, K., Tuset, J.J. & Viggiani, G. 1991: Results of the fifth joint pesticide testing programme carried out by IOBC/WPRS-working group Pesticides and beneficial organisms . Entomophaga 36(1): 55-67. Kavousi, A. & Talebi, K. 2003: Side-effects of three pesticides on the predatory mite, Phytoseiulus persimilis (Acari: Phytoseiidae). Exp. Appl. Acarol. 31: 51-58. LeOra Software 1987: POLO-PC: A user's guide to Probit or Logit analysis. LeOra Softwarte, Berkeley, California.

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Nadimi, P.A. 2006: Study on side-effect of three acaricides on two predatory mites, Phytoseiulus persimilis A.-H. and Phytoseius plumifer (Canestrini & Fanzago) (Acari: Phytoseiidae) under laboratory conditions. MSc. thesis. Tarbiat Modares University., Tehran.: 76 pp. Oomen, P.A. 1988: Guideline for the evaluation of side-effects of pesticides on Phytoseiulus persimilis A.-H. Sequential scheme: Laboratory tests: 1-residual contact test, 2 – persistence test; field test. IOBC/WPRS Bulletin 11 (4): 51-63. Oomen, P.A., Romeijn, G. & Wiegers, G.L. 1991: Side-effects of 100 pesticides on the predatory mite Phytoseiulus persimilis, collected and evaluated according to the EPPO Guideline. OEPP/EPPO Bull. 21 (4): 701-712. Samsøe-Petersen, L., Bigler, F., Bogenschütz, H., Brun, J., Hassan, S.A., Helyer, N.L., Kühner, C., Mansour, F., Naton, E., Oomen, P.A., Overmeer, W.P.J., Polgar, L., Rieckmann, W. & Stäubli, A. 1990: Laboratory rearing techniques for 16 beneficial arthropod species and their prey/hosts. Z. Pflanzenkr. Pflanzenschutz 96 (3): 289-316. Stark, J.D., Banken, J.A.O. & Walthal, W.K. 1998: The importance of the population perspective for the evaluation of side-effects of pesticides on beneficial species. In: Haskell, P.T. and McEwen, P. (eds.). Ecotoxicology: Pesticides and beneficial organisms. Kluwer Academic Publishers, Dordrecht, The Netherlands: 348-359.

Pesticides and Beneficial Organisms IOBC/wprs Bulletin Vol. 35, 2008 pp. 152-156

The need for taxonomists of pest and beneficial organisms - results of an inquiry at the meeting of the IOBC working group “Pesticides and Beneficial Organisms” in Berlin in October 2007

Günther Schmitt Schmitt Faunistic Studies, Friedensstr. 23, D-18190 Sanitz (Germany). E-mail: [email protected]

Abstract: An inquiry has been carried out on the meeting of the IOBC working group “Pesticide and Beneficial Organisms” in October 2007 to record the international need for taxonomists of pest and beneficial organisms by specialists on phytopathology and biocontrol. The questionnaire consisted of 5 questions on 19 proposed taxonomic groups at different taxonomic level. The outcome of the inquiry includes a list of the taxonomic groups ranked by the numbers of asked persons working with and showing on which groups taxonomic support is needed. Most of the respondents require the support of professional taxonomists from external institutions. According to the statements, the presence of taxonomists has been summarized for each of the countries the respondents came from. Overall, taxonomists were frequently missed or not known, and the majority of the asked persons agreed with the claim for more permanent positions for taxonomist.

Key words: taxonomist, taxonomic support, determination, verification, pests, beneficials

Introduction

It is not new to biological science that specialists on a wide variety of taxonomic groups are becoming rare. Especially there is a lack of experienced taxonomists holding a permanent position in museums or other related institutions and who can assume long-termed responsi- bility for a specific taxonomic group. At present, a few campaigns are under way (e.g. the “Global Taxonomy Initiative” as a programm of the “Convention of the Biological Diversity”, the “Buffon Declaration” promoting natural history institutions, and a petition towards the establishing of endowed professorships in the field of botany, zoology and mycorrhiza in Germany) to counteract the loss of taxonomists. None of the campaigns give emphasize on specialists in the field of pest organisms or taxa groups important to biological control so far. The intention of the presented survey was to state for which taxonomic group of pest and beneficial organisms (additional) taxonomists are recently most urgent needed. The outcomes of the inquiry may contribute to finding demand-driven arguments for decision-makers towards the establishment of positions for taxonomists.

Material and methods

Questionnaire The participants of the meeting of the IOBC WG “Pesticides and Beneficial Organisms” have been asked to answer five questions. The questionnaire included the notation of some person’s data including the home country of the contributors. An anonymous answering was possible. Five questions were to be answered:

152 153

1) Are you working with this taxonomic group? Answering options: yes – no 2) Do you need an expert of this taxonomic group for determination or verification? Answering options: never – sometimes – often 3) Are there experts of this group present in your country (internals or from outside of your institution)? Answering options: yes – no – Don’t know 4) If you contact/need a specialist in your country: Is it a professional or a private person (hobby)? Answering options: professional in your institution – professional in other institution – private (hobby) 5) Do you think there is a demand to establish (more) permanent public position(s) for specialists of this group (e.g. in museums) Answering options: yes – no The questions have been applied to 19 proposed taxa at different taxonomic levels (see Figure 1; Table 1). The participants were also welcome to add taxa important to them.

Participants on the inquiry In total, 22 persons out of 11 countries representing a cross-section of the attended nationalities responded on the call for information. The highest number of replies came from German participants (7) followed by participants from Belgium and Greece (3 respectively) and Spain (2). Also, one participant each from Croatia, England, France, Netherlands, Portugal, Switzerland, and Turkey gave information on the situation in their countries. 4 contributors answered anonymously.

Results and discussion

The outcomes of the inquiry reflect the experiences of the participants and were evolved from their field of duties that may include laboratory, seminatural or field studies or the exclusive work with specific taxa. Thus, the following conclusion should be treated as a rough overview based on a restricted database and reflecting the judgement of the respondents.

The importance of the proposed taxonomic groups to the participants of the inquiry The results refer to the first and second questions. Comments on several taxonomic groups have been possible. Figure 1 shows a ranked listing of the proposed taxonomic group in accordance to their importance to the asked persons. Among the most prominent taxonomic groups were Acari, Aphidina, Heteroptera, Braconidae, Thysanoptera, and Araneae. More than a half of the respondents are working on these taxa and require taxonomic support. The ranking list ends up with the soil dwelling Collembola and Nematoda. The great majority of the respondents need sometimes or often the assistance of taxonomists. In the case of 11 out of the 19 supposed taxonomic groups none of the persons are able to determine the taxa without any help. Conversely, the Carabidae were stated as a taxon with a low need of external taxonomic support. Some of the respondents added taxonomic groups of pests and beneficials important to themselves. The Curculionoidea (Curculion resp.), Chalcidoidea, Chrysopidae, Gastropoda, Forficulidae were noted once each. 2 persons went into detail and cited the Phytoseiidae and Tetranychidae among the proposed comprehensive taxonomic group of Acari. Taxonomic support was stated to be essential for all of the added groups. 154

Acari Aphidina Heteroptera Braconidae Thysanoptera Araneae Ichneumonidae Coccina Cecidomyiidae “Microlepidoptera” Carabidae Auchenorrhyncha Aleyrodina Staphylinidae Chloropidae Agromyzidae often Mymaridae sometimes Collembola never Nematoda

0 5 10 15 20

Figure 1. The ranking list of the proposed taxonomic groups the asked persons working with and the manner (often, sometimes, never) taxonomic support is needed

The presence of taxonomists in the home countries of the participants The outcomes of question 3 are summarized in Table 1. The knowledge on the existence of taxonomists of the proposed taxa tended to increase with the number of contributing persons coming from the same country. Even the replies of single national representatives have been useful to complement the presented overview. Dissimilar answers for a single country were weighted and condensed to a single outcome. The inferior answers were additionally noted. Table 1 illustrates the dilemma of the present situation on taxonomic support. Firstly, dissimilar answers and the high number of the asked persons who stated that they do not know a specialist of the taxonomic group they are working with reflects the need for a closer cooperation between taxonomists and scientists in the field of biological control. The second conclusion refers to the overall lack of specialists. None of the countries the replies came from hold experts for each of the proposed taxa. Merely a few countries such as France, Germany, and Spain have experts for the majority of the proposed taxa, though several asked persons noted that the expertise of the specialists they know is restricted to specific taxa within the proposed and often species rich groups. Concerning single taxa, the Acari and Thysanoptera have been the only groups, for which the respondents stated that taxonomists are relatively widespread. However, the 155

Coccina, the Cecidomyiidae, and also parasitoid wasps such as the Braconidae, Ichneumonidae or Mymaridae were taxonomic groups, where none or merely some single taxonomists exist at all.

Table 1. The presence of taxonomists in the home countries of the asked persons according to their experiences due to the groups they are working with. - Dissimilar answers were weighted and condensed to one outcome. Divergent answers were added as superior characters. Yes: +, no: -, Don’t know: ?, in brackets the number of replies out of the country, the arrangement of the taxa refers to the ranking list (see Figure 1)

Belgium (3) Croatia (1) England (1) France (1) (7) Germany Greece (3) Netherlands (1) Portugal (1) Spain (2) Switzerland (1) Turkey (1)

Acari ? - + +? + + + ? + Aphidina -? + + +? -? - + ? Heteroptera ? + ? +? -? + + ? - Braconidae -? - ? -+ -? ? + ? - Thysanoptera ? + + + + +?,- + + Araneae - - ? + - -? ? + Ichneumonidae - - -+ ? ? + - Coccina ? - + -? -? +? - Cecidomyiidae ? - + -+ -? ? -? - “Microlepidoptera” - +- +- ? + - Carabidae + + ? + -+ + ? - Auchenorrhyncha - + +? -? ? ? Aleyrodina ? + ? - -? + Staphylinidae - ? ? + - - Chloropidae - + +? - - - Agromyzidae + +? ? -? - Mymaridae - -+ -? -? - Collembola - + - -? ? Nematoda + - + - +? ? -

The status of the contacted specialist in the home countries of the participants: professional or private (hobby) In total, 166 statements have been made by the respondents on question 4 on the 19 proposed taxonomic groups they are working with. The outcomes are summarized without noting any details on each single taxon. 156

The majority of the asked persons needed the assistance of professional taxonomists outside of their institutions (about 80%). Professional external support was always necessary for the Cecidomyiidae, Mymaridae, Chloropidae, Staphylinidae, and Nematoda. Approximately 20% of statements referred to contacts to private persons carrying out the determination and verification of specimen of taxa they are specialized in as a hobby. The most contacts to private persons within a country (about a third of all of those statements) were done in Germany. Taxonomic assistance within the same institutions the respondents were working encompassed less than 3% of the statements and merely refer to the Acari, Aphidina, and “Microlepidoptera”.

The demand to establish (more) permanent public position(s) for taxonomists 122 statements on question 5 were done to those taxonomic group the persons working with. Approximately 90% of the statements agreed with the claim for establishing (more) permanent public positions for taxonomists. None of the respondents rejected the establishing of positions for the following taxonomic groups: “Microlepidoptera”, Chloropidae, Auchenor- rhyncha, Staphylinidae, Agromyzidae, Nematoda, and Collembola. Statements refusing the demand have been made by persons coming from Spain, Belgium, and in the case of 2 statements from Germany.

Acknowledgements

The author thanks the asked participants of the meeting of the IOBC working group “Pesticides and Beneficial Organisms” for their contribution to the inquiry.

Cited and useful links

Buffon Declaration (promoting of natural history institutions): www.diversitas-international.org/ uploads/File/BuffonDeclarationFinal.pdf European Distributed Institute of Taxonomy (EDIT): www.e-taxonomy.eu Global Biodiversity Information Facility (information about natural history collections, library materials, databases): www.gbif.org Global Taxonomy Initiative (GTI): www.cbd.int/programmes/cross-cutting/taxonomy/ German National GTI Focal Point: www.gti-kontaktstelle.de German campaign towards endowed professorships on taxonomy: www.taxonomie-initiative.de Pest Directory of ISPI (International Society for Pest Information): www.pestinfo.org/pestdir.htm World taxonomist database: www.eti.uva.nl/tools/wtd.php