Working Group „Pesticides and Beneficial Organisms“ · GARCIA GARCIA, Maria del Mar,...
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IOBC / WPRS Working Group „Pesticides and Beneficial Organisms“ OILB / SROP Groupe de Travail „Pesticides et Organismes Utiles“ Proceedings of the meeting at Castelló de la Plana, Spain 18 - 20 October, 2000 editors: Heidrun Vogt, Elisa Viñuela & Josep Jacas IOBC wprs Bulletin Bulletin OILB srop Vol. 24 (4) 2001
Transcript of Working Group „Pesticides and Beneficial Organisms“ · GARCIA GARCIA, Maria del Mar,...
IOBC / WPRS
Working Group „Pesticides and Beneficial Organisms“
OILB / SROP
Groupe de Travail „Pesticides et Organismes Utiles“
Proceedings of the meeting
at
Castelló de la Plana, Spain
18 - 20 October, 2000
editors:
Heidrun Vogt, Elisa Viñuela & Josep Jacas
IOBC wprs Bulletin Bulletin OILB srop Vol. 24 (4) 2001
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 2001
The Publication Commission of the IOBC/WPRS: Horst Bathon Federal Biological Research Center for Agriculture and Forestry (BBA) Institute for Biological Control Heinrichstr. 243 D-64287 Darmstadt (Germany) Tel +49 6151 407-225, Fax +49 6151 407-290 e-mail: [email protected]
Luc Tirry University of Gent Laboratory of Agrozoology Department of Crop Protection Coupure Links 653 B-9000 Gent (Belgium) Tel +32-9-2646152, Fax +32-9-2646239 e-mail: luc.tirry@ rug.ac.be
Address General Secretariat: INRA – Centre de Recherches de Dijon Laboratoire de recherches sur la Flore Pathogène dans le Sol 17, Rue Sully, BV 1540 F-21034 DIJON CEDEX France ISBN 92-9067-133-5
SPONSORS
The meeting and the printing of this Bulletin
were supported by:
Ministerio de Ciencia y Tecnología
(Acción Especial AGL2000-1967-E)
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Preface The IOBC/WPRS Working Group „Pesticides and Beneficial Organisms“ held its annual meeting at the Universitat Jaume I, Castelló de la Plana, Spain, from 18th to 20th October 2000. With almost 100 experts from 12 countries the number of participants was again very high, underlining the importance and actuality of the topic. A lot of colleagues from Spain took the opportunity to meet the WG the first time and to present their research activities. In total 28 contributions were given, and 17 of these are published in the present volume.
Several expert groups from the Joint Initiative of IOBC, EPPO (European and Mediterranean Plant Protection Organisation in collaboration with the Council of Europe) and BART (Beneficial Arthropod Regulatory Testing Group) to develop and validate test methods to assess side-effects of pesticides on non-target arthropods for registration purposes met during the meeting. Meanwhile, eleven test methods have been finalised and published as an IOBC booklet: Guidelines to evaluate side-effects of plant protection products to non-target arthropods. IOBC/WPRS, Gent. 2000.
The meeting was again an excellent forum for presenting and discussing actual topics of research about side-effects of pesticides, test method development, interpretation of results and their implementation into Integrated Pest Management, as well as focusing on future objectives. The excellent facilities of the University contributed to a stimulating atmosphere and the meeting was characterized by fruitful discussions and intensive exchange of experience and ideas.
The WG expresses many thanks to the local organizer, Dr. Josep Jacas, and to his colleagues, who supported him, especially Dr. Aurelio Gómez, for the excellent local arrangements, including a half-day excursion to a citrus packing house, to citrus and olive orchards as well as to typical villages of the region. Heidrun Vogt (Convenor) Dossenheim, .June 2001
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List of participants 1. ABDELGADER, Hayder, Dr., Agricultural Research Corporation, Crop Protection Research
Center, Entomology Section, P.O. Box 126, Wad Medani, Sudan, e-mail: [email protected]
2. ALDERSHOF, Saskia, Universiteit van Amsterdam, Dept. of Pure & Applied Ecology, Sect. Population Biology / MITOX, Kruislaan 320, NL-01098 SM Amsterdam, e-mail: [email protected]
3. ANGELI, Gino, Dr., Istituto Agrario S. Michele, Adige, Via E. Mach n° 1, I-38010 S. Michele a/A Trento, e-mail: [email protected]
4. ARDIZZONI, Marco, Dr., Institute of Entomology "G. Grandi", University of Bologna, Via F. Re 6, I-40123 Bologna, e-mail: [email protected]
5. AZNAR, Vicente, Dr., Trialcamp, C/Pedro de Luna 9 1e-Izq., E-46017 Valencia, e-mail: [email protected]
6. BAIER, Barbara, Dr., Biologische Bundesanstalt für Land- und Forstwirtschaft (BBA), Institut für Ökotoxikologie im Pflanzenschutz, Stahnsdorfer Damm 81, D-14532 Kleinmachnow, e-mail: [email protected]
7. BAKKER, F.M., Dr., Universiteit van Amsterdam, Dept. of Pure & Applied Ecology, Sect. Population Biology/MITOX, Kruislaan 320, NL-1098 SM Amsterdam, e-mail: [email protected]
8. BARTH, Markus, BioChem agrar GmbH, Am Wieseneck 7, D-04451 Cunnersdorf, e-mail: [email protected]
9. BERNARDO, Umberto, Dr., Centro Studi CNR, Technice di Lotta Biologica, Via Universita 133, I-80055 Portici (NA), e-mail: [email protected]
10. BIELZA, Pablo, Dr., Universidad Politecnica de Cartagena, (E.T.S.I.), Departamento de Produccion Agraria, Paseo Alfonso XIII 52, E-30203 Cartagena, e-mail: [email protected]
11. BIGLER, Franz, Dr., Swiss Federal Research Station for Agroecology & Agriculture, Reckenholzstr. 191, CH-8046 Zürich, e-mail: [email protected]
12. BLÜMEL, Sylvia, Dr., Bundesamt und Forschungszentrum für Landwirtschaft, Institut für Phytomedizin, Spargelfeldstr. 191, A-1226 Wien, e-mail: [email protected]
13. BOAVIDA, Conceiçao, Dr. Ing., Impactest, Av. Almirante Reis, 204-7° Dto., 1000-056, Lisbon, Portugal, e-mail: [email protected]
14. BONAFOS, Romain, ENSA-M /INRA, UFR d'Ecologie animale et Zoologie agricola, 2, Place Pierre Viala, F-34060 Montpellier Cedex 1, e-mail: [email protected]
15. BOYERO GALLARDO, Juan Ramón, Centro de Investigación y Formación Agraria de Málaga (CIFA), Cortijo de la Cruz s/n, E-29140 Churriana
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16. BROWN, Kevin, Ecotox Ltd., Tavistock, P.O.Box 1, Devon PL19 0YU, U.K., e-mail: [email protected]
17. BUTTLE, Victoria, Ecotox Ltd., Tavistock, P.O.Box 1, Devon PL19 0YU, U.K., e-mail: [email protected]
18. BYLEMANS, Dany, Dr., Royal Research Station of Gorsem, Brede Akker 13, B-3800 Sint-Truiden, e-mail: [email protected]
19. CANDOLFI, Marco, Dr., Syngenta AG, WRO-1058.3.64, CH-4002 Basel, e-mail: [email protected]
20. CARLI, Guido, Dr, Centro Ricerche Produzioni Vegetali (CRPV), Via Vicinale Monticino, 1969, I-47020 Diegaro di Cesena (FC), Italy, e-mail: [email protected]
21. CAROLI, Luigi, Dr., Centro Ricerche Produzioni Vegetali (CRPV), Via Vicinale Monticino, 1969, I-47020 Diegaro di Cesena (FC), Italy, e-mail: [email protected]
22. CAVAZZA, Christian, Dr., Institute of Entomology "G. Grandi", University of Bologna, Via F. Re 6, I-40123 Bologna, e-mail: [email protected]
23. COLE, John F.H., Zeneca Agrochemicals, Ecological Risk Assessment, Jealott's Hill Research Station, Bracknell, Berkshire RG42 6ET, U.K., e-mail: [email protected]
24. CONTRERAS GALLEGO, Josefina, Departamento de Producción Agraria, Universidad Politécnica de Cartagena, Paseo Alfonso XIII, 52, E-30203 Cartagena
25. COULOMB, Philippe, VITI - SARI, au capital de 50 250 F, RCS Montpellier B 349 697 144, 101, Impasse des Capitelles- Chemin des Combes Noires, F-34400 Villetelle, e-mail: [email protected]
26. COULSON, Mike, Dr., Zeneca Agrochemicals, Environmental Sciences Dept., Jealott's Hill Research Station, Bracknell, Berkshire RG42 6ET, U.K., e-mail: [email protected]
27. DINTER, Axel, Dr., Du Pont Crop Protection, Du Pont de Nemours GmbH, DuPont Str. 1, D-61352 Bad Homburg, e-mail: [email protected]
28. DREXLER, Andrea, IBACON, Institut für Biologische Analytik und Consulting GmbH, Arheilger Weg 17, D-64380 Roßdorf, e-mail: [email protected]
29. DRIJVER, Cora, Plant Protection Service, Postbus 9102, NL-6700 Wageningen, e-mail: [email protected]
30. ENGELHARDT, Margrit, Chemin Notre - Dame des Anges, F-26170 Mollans, e-mail: [email protected]
31. FAYEL, Olivier, Serres, F-26570 Montbrun, e-mail: [email protected]
32. FEUERRIEGEL Furtes, Fernando, GAB, Biotechnologie GmbH, Eutinger Str. 24, D-75223 Niefern-Öschelbronn, e-mail: [email protected]
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33. FORSTER, Rolf, Dr., Biologische Bundesanstalt für Land- und Forstwirtschaft (BBA), Abt. für Pflanzenschutzmittel, Fachgrupe Biologische Mittelüprüfung, Messeweg 11/12, D-38104 Braunschweig, e-mail: [email protected]
34. GARCIA GARCIA, Maria del Mar, Departamento de Sanidad Vegetal, Delegacion De Agricultura y Pesca de Almeria, C/Hermanos Machado N° 4 3 Planta, E-04071 Almeria, e-mail: [email protected]
35. GATHMANN, Achim, Dr., Institute of Plant Diseases and Plant Protection, University of Hannover, Herrenhäuser Str. 2, D-30419 Hannover, e-mail: [email protected]
36. GEUIJEN, Ine, NOTOX B.V., P.O. Box 3476, NL-5231 DD 's-Hertogenbosch, e-mail: [email protected]
37. GRAY, Adrian, Dr., Syngenta AG, Environmental Saftey Assessments and Contracting, R-1058.8.42, CH-4002 Basel, e-mail: [email protected]
38. HALSALL Nigel, Dr., Mambo-Tox Ltd., Biomedical Sciences Building, Bassett Crescent East, Southampton SO16 7PX, U.K., e-mail: [email protected]
39. HARWOOD, Robert, Dr., Inveresk Research, Tranent EH33 2NE, U.K., e-mail: [email protected]
40. HEIMBACH, Udo, Dr., Biologische Bundesanstalt für Land- und Forstwirtschaft (BBA), Institut für Pflanzenschutz in Ackerbau und Grünland, Messeweg 11/12, D-38104 Braunschweig, e-mail: [email protected]
41. HELLAL, Hamadi, Dr., Société ERRAFRAF -Recherches Agronomiques, 7, Rue du Caire, Raf-Raf-Plage 7045, Aïn-Chiquoua, Tunesia, Fax: 002162441558
42. HUTCHINGS, Matt, Astrazeneca, Brixham Environmental Laboratory, Freshwater Quarry, Brixham, Devon, U.K., e-mail: [email protected]
43. IZQUIERDO, Josep, Dr., Bayer Hispania SA., Division Agro, Pau Claris 196, E-08037 Barcelona, e-mail: [email protected]
44. JACAS Miret, Josep Anton, Dr., Departament de Ciències Experimentals, Campus de Riu Sec, E-12071 Castelló de la Plana, e-mail: [email protected]
45. JANSEN, Jean-Pierre, Dr., Dept. Biological Control, Agricultural Research Centre, Chemin de Liroux 2, B-5030 Gembloux, e-mail: [email protected]
46. KNAEBE, Silvio, GAB, Biotechnologie GmbH, Eutinger Str. 24, D-75223 Niefern-Öschelbronn, e-mail: [email protected]
47. KOCH, Heribert, Dr., Landesanstalt für Pflanzenschutz und Pflanzenbau, Essenheimer Str. 144, D-55128 Mainz, e-mail: [email protected]
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48. KOLLMANN, Stefanie, Dipl.-Ing. agr., Springborn Laboratories (Europe) AG, Seestr. 21, CH-9826 Horn, e-mail: [email protected]
49. LAFUENTE Fernandes, Maria, GAB, Biotechnologie GmbH, Eutinger Str. 24, D-75223 Niefern-Öschelbronn, e-mail: [email protected]
50. LEWIS, Gavin, Dr., JSC International Ltd., Osborn House 20, Victoria Avenue, Harrogate North Yorkshire HG1 5QY, U.K., e-mail: [email protected]
51. LUNA, Francisco, Dr. Insetec, C/Partida del Olivar s/n, E-46165 Burgarr-Valencia, Spain, e.mail: [email protected]
52. MARTIN, Sabine, Dr., Umweltbundesamt, Postfach 33 00 22, D-14191 Berlin, e-mail: [email protected]
53. MAUS, Christian, BAYER AG, Landwirtschaftszentrum Monheim, Gebäude 6220, D-40789 Monheim, e-mail: [email protected]
54. MEAD, Ian, Huntingdon Life Sciences (HLS), U.K. 55. MEAD-BRIGGS, Mike, Dr., Mambo-Tox Ltd., Biomedical Sciences Building, Bassett
Crescent East, Southampton SO16 7PX, U.K., e-mail: [email protected]
56. MENDEL, Renate, Dr., Feinchemie Schweda GmbH, Eupener Str. 150, D-50933 Köln e-mail: [email protected]
57. MOLL, Monika, Dr., IBACON, Institut für Biologische Analytik und Consulting GmbH, Arheilger Weg 17, D-64380 Roßdorf, e-mail: [email protected]
58. MOLLA Inglada, Jose Pablo, C/Canoa n° 1, E-46009 Valencia 59. MÜTHER, Jutta, Dr., GAB, Biotechnologie GmbH, Eutinger Str. 24, D-75223 Niefern-
Öschelbronn, e-mail: [email protected]
60. NIENSTEDT, Karin, Dr., Springborn Laboratories (Europe) AG, Seestr. 21, CH-9326 Horn, e-mail: [email protected]
61. NOACK, Udo, Dr., Dr. U. Noack-Laboratorium, Käthe-Paulus-Str. 1, D-31157, Sarstedt, e-mail: [email protected]
62. PASQUALINI, Edison, Dr., Universita degli Studi Bologna, Istituto di Entomologia "G. Grandi", Via Filippo Re, 6, I-40123, Bologna, e-mail: [email protected]
63. POULLOT, Delphine, 23 rue Mme de Sevigne, F-78960, Voisins-le-Bretonneux, e-mail: [email protected]
64. REBER, Beat, Syngenta AG, Postfach, CH-4002 Basel, e-mail: [email protected]
65. RIEDL, Helmut, Dr., Mid-Columbia Agricultural Research & Extension Center, Oregon State University, 3005 Exp. Station Drive - Hood River, Oregon 97031-9512, USA, e-mail: [email protected]
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66. RODRIGUEZ Rodriquez, Maria Dolores, CIFA, E-04071 Almeria, e-mail: [email protected]
67. RODRIGUEZ Rodriquez, Maria Paz, Departamento de Sanidad Vegetal, Delegacion De Agricultura y Pesca de Almeria, C/Hermanos Machado N° 4 3 Planta, E-04071 Almeria e-mail: [email protected]
68. RÖHLIG, Uta, BioChem agrar GmbH, Am Wiseneck 7, D-04451 Cunnersdorf, e-mail: [email protected]
69. RUANO, Francisca, Dr., Estacion Experimental del Zaidin, Departamento de Agroecologia y Proteccion Vegetal, Consejo Superior de Investigaciones Cientificas, C/Profesor Albareda n° 1, E-18008 Granada, e-mail: [email protected]
70. SCHIRRA, Karl-Josef, Dr., SLFA, Neustadt an der Weinstraße, Breitenweg 71, D-67435 Neustadt, e-mail: [email protected]
71. SCHMIDT, Hans-Werner, Dr., Bayer AG, Pflanzenschutz/Entwicklung, Agricultural Research Centre Monheim, D-51368 Leverkusen, e-mail: [email protected]
72. SCHMITZER, Stephan, IBACON, Institut für Biologische Analytik und Consulting GmbH, Arheilger Weg 17, D-64380 Roßdorf, e-mail: [email protected]
73. SCHULD, Michael, GAB, Biotechnologie GmbH, Eutinger Str. 24, D-75223 Niefern-Öschelbronn, e-mail: [email protected]
74. SCHULTE, Christoph, Dr., Umweltbundesamt, Postfach 33 00 22, D-14191 Berlin, e-mail: [email protected]
75. SECHSER, Burkhard, Dr., Bodenacker 73, CH-3065 Bolligen, e-mail: [email protected]
76. SERRANO, Carlos, Dr., Trialcamp, C/Pedro de Luna 9 1e-Izq., E-46017 Valencia (Spain), e-mail: [email protected]
77. SERVAJEAN, Elisabeth, Dr., Envirotests, 14 rue Paul Bert, F-64000 Pau, e-mail: [email protected]
78. SHARPLES, Amanda, Huntingdon Life Sciences (HLS), PO Box 2, Huntingdon, Cambridge PE18 6ES, U.K.
79. SOLER FELIU, José Mª, Aventis CropScience España, S.A., Políg. Industrial El Plá, Parc. 30, E-46290 Alcácer (Valencia, e-mail: [email protected]
80. STAAIJ, M.van der, Research Station for Floriculture and Glasshouse Vegetables, Kruisbroekweg 5, Postbus 8, NL-2670 AA Naaldwijk, Niederlande, e-mail: [email protected]
81. URBANEJA García, Alberto, Departamento de Investigación y Desarrollo, Koppert Biological Systems, Finca Labradorcico del Medio s/n, Apartado de correos 286, E-30880 Aguilas (Murcia), e-mail: [email protected]
82. TAKEUCHI, Hiroaki, Crop Entomology Lab, Dept. of Plant Protection, National Agriculture Research Center, 3-1-1, Kannondai, Tsukuba, Ibaraki 305, Japan, e-mail: [email protected]
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83. TELLEZ Navarro, Maria del Mar, CIFA, E-04071 Almeria, e-mail: [email protected]
84. TESSIER, Céline, Promovert, ZI du Haut Ossau, rue d'Aste Béon, BP 27, F-64121 Serres Castet, e-mail: [email protected]
85. TORNIER, Ingo, Dr., GAB, Biotechnologie GmbH, Eutinger Str. 24, D-75223 Niefern-Öschelbronn, e-mail: [email protected]
86. TORRES Macias, Maria del Mar, Departamento de Sanidad Vegetal, Delegacion De Agricultura y Pesca de Almeria, C/Hermanos Machado N° 4 3 Planta, E-04071 Almeria e-mail: [email protected]
87. TRAVIS, Andrea, Zeneca Agrochemicals, Ecological Risk Assessment, Jealott’s Hill Research Station, Bracknell, Berkshire RG42 6ET, United Kingdom e-mail: [email protected]
88. UFER, Andreas, Dr., BASF AG, Agrarzentrum Limburgerhof, Postfach 120, D-67114 Limburgerhof, e-mail: [email protected]
89. VAN DER BLOM, Jan, Dr., Departamento de Investigación y Desarrollo, Koppert Biological Systems, C/ Vicente Aleixandre, 15, Las Cabañuelas. Apartado de Correos, 38, E-04738 Vicar (Almería), e-mail: [email protected]
90. VAN DE VEIRE, Marc, Dr., University of Gent, Laboratory of Agrozoology, Coupure Links 653, B-9000 Ghent, e-mail: [email protected]
91. VERGNET, Christine, Dr., Structure Scientifique Mixte -INRA/DGAL, Route de Saint-Cyr, F-78026 Versailles Cedex, e-mail: [email protected]
92. VIGGIANI, G., Prof. Dr., Universita' di Napoli "Federico II", Dipartimento di Entomologia e Zoologia Agraria, Via Universita 100, I-80055 Portici (NA), e-mail: [email protected]
93. VIÑUELA, Elisa, Prof. Dr., Unidad de Proteccion de Cultivos, E.T.S.I. Agrónomos, Universidad Politécnica de Madrid, E-28040 Madrid, e-mail: [email protected]
94. VOGT, Heidrun, Dr., Biologische Bundesanstalt für Land- und Forstwirtschaft (BBA), Institut für Pflanzenschutz im Obstbau, Schwabenheimerstr. 101, D-69221 Dossenheim, e-mail: [email protected]
95. WALKER, Hazel, Ecotox Ltd., Tavistock, P.O.Box 1, Devon PL19 0YU, U.K., e-mail: [email protected]
96. WALTERSDORFER, Anna, Dr., Aventis CropScience GmbH, Industriepark Höchst, H872, D-65926 Frankfurt, e-mail: [email protected]
97. WILHELMY, Hermann, Neudorff GmbH KG, An der Mühle 3, D-31860 Emmerthal, e-mail: [email protected]
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Table of Contents Preface........................................................................................................................................ i List of participants..................................................................................................................... iii Non-Target Arthropod Testing – The current German interim procedure based on proposals made at the ESCORT 2 workshop. .............................. ............................................. 1 Forster, R. & Martin, S. Influence of leaf substrates on the toxicity of selected plant protection products to Typhlodromus pyri Scheuten (Acari: Phytoseiidae) and Aphidius rhopalosiphi DeStefani Perez (Hymenoptera: Aphidiidae)............................................................................ 7 Ternes, P., Candolfi, M.P., Ufer, A. & Vogt, H. Toxicity of insecticides used in wheat to adults of Aphidius rhopalosiphi DeStefani Perez (Hymenoptera: Aphidiidae) with field treated plants.................................................... 17 Jansen, J.P. Comparison of side-effects of spinosad, tebufenozide and azadirachtin on the predators Chrysoperla carnea and Podisus maculiventris and the parasitoids Opius concolor and Hyposoter didymator under laboratory conditions............................................ 25 Viñuela, E., Medina, Mª.P., Schneider, M., González, M., Budia, F., Adán, A. & Del Estal, P. Influence of the host density on the reproduction of Aleochara bilineata Gyll. (Coleoptera: Staphylinidae)..................................................................................................... 35 Nienstedt, K.M. & Galicia, H.F. Effects of Quassia products on predatory mite species............................................................ 39 Baier, B. Effects of Quassia products on Chrysoperla carnea (Stephens) (Neuroptera, Chrysopidae) ........................................................................................................................... 47 Vogt, H. Extended laboratory methods to determine effects of plant protection products on two strains of Amblyseius andersoni Chant and their resistance level.................................... 53 Angeli, G., Forti, D. & Finato, S. Development of a laboratory test method to determine the duration of pesticide-effects on predatory mites ....................................................................................................... 61 Van de Veire, M., Cornelis, W. & Tirry, L.
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A semi-field test for evaluating the side-effects of plant protection products on the aphid parasitoid Aphidius rhopalosiphi (DeStefani-Perez) (Hymenoptera, Braconidae) – First results....................................................................................................... 67 Moll, M. & Schuld, M. A sequential testing program to assess the side effects of pesticides on Tricho-gramma cacoeciae Marchal (Hym., Trichogrammatidae) ...................................................... 71 Hassan, S. & Abdelgader, H. Effects of Confidor 20 LS and Nemacur CS on bumblebees pollinating greenhouse tomatoes .................................................................................................................................. 83 Bielza, P., Contreras, J., Guerrero, M.M., Izquierdo, J., Lacasa, A. & Mansanet, V. Pre-sampling for Large Field Trials – a valuable instrument to chose the ‘perfect’ site?.......................................................................................................................................... 89 Knäbe, S., Cole, J.F.H. & Waltersdorfer, A. Side effects of some pesticides on predatory mites (Phytoseiidae) in citrus orchards............ 97 Viggiani, G. & Bernardo, U. Side-effects of pesticides on selected natural enemies occurring in citrus in Spain ............. 103 Jacas Miret, J.J. & Garcia-Marí, F. Impact of pesticides on beneficial arthropod fauna of olive groves ..................................... 113 Ruano, F., Lozano, C., Tinaut, A., Peña, A., Pascual, F., García, P. & Campos, M. Selectivity of Lufenuron (Match ®), Profenofos and mixtures of both versus cotton predators ................................................................................................................................ 121 Sechser, B., Ayoub, S. & Monuir, N.
Pesticides and Beneficial Organisms IOBC/wprs Bulletin Vol. 24 (4) 2001
pp. 1 - 6
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Non-Target Arthropod Testing - The current German interim procedure based on proposals made at the ESCORT 2 workshop a) Rolf Forster1) & Sabine Martin2) 1) Federal Biological Research Centre for Agriculture and Forestry (BBA), Braunschweig,
Germany; 2) Federal Environmental Agency (UBA), Berlin, Germany Abstract: Six years of experiences with the testing procedure for pesticides according to ESCORT 1b) indicated the need to update the decision-making scheme for regulatory purposes in the light of the current scientific knowledge. Consequently an ESCORT 2 workshop was held to discuss a new guidance for non-target arthropod (NTA) testing, in Wageningen in March 2000. The new scheme was elaborated by the Federal Biological Research Centre for Agriculture and Forestry (BBA) together with the Federal Environmental Agency (UBA) covering ESCORT 2 proposals as far as justified, in the light of the current scientific knowledge. Major concerns focus on the proposed Hazard Quotient (HQ)-values: 1) very high effects on both species are reported at their proposed HQ-values (Aphidius spp. 98 % at HQ 8.3; T. pyri 95 % at HQ 12.1), 2) there is uncertainty due to test methods, because different HQs can be found due to different methods used for T. pyri for the same product, 3) the data base is very limited, only about half a dozen products were analyzed in both of the validations, 4) recovery of univoltine species is not sufficiently addressed in the validation, 5) there is a lack of transparency in the data set, as not all names of the products used or their active substances are known to national authorities. Key words: non-target arthropod testing, plant protection products, regulatory testing, ESCORT 2 Introduction The testing procedure according to ESCORT 1 was no longer considered up-to-date, in the light of the current scientific knowledge. Consequently an update was organized, and an ESCORT 2 workshop was held to agree on a new guidance for NTA testing, in Wageningen in March 2000. However, in January 2000 National policy makers in Germany induced the BBA together with the UBA to revise the testing procedure until May 2000. This new scheme covers ESCORT 2 proposals as far as justified, in the light of the current scientific knowledge. The new scheme ESCORT 2 At ESCORT 2 needs for a change of ESCORT 1 principles were identified: - The objectives of the ESCORT 1 testing scheme were not clear e.g. it does not precisely
discriminate between non-target arthropods in an ecological context and beneficial arthropods in an agricultural or IPM context.
a) Workshop on European Standard Characteristics of Non-Target Arthropod Regulatory Testing, Wageningen
2000 b) Workshop Wageningen 1994
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- The trigger value for 1st tier data (30 %) produces too many false positives (i.e. wrongly classified as harmful) and consequently requires excessive higher tier testing.
- The single-dose laboratory data generated do not allow a satisfactory risk assessment e.g. for in-crop and off-crop habitats.
- Uncertainty about data requirements, testing methodology and evaluation especially for a) multiple application products, where currently life span, spraying interval and fate are ignored and b) for off-crop habitats, where exposure scenarios and risk assessment procedures were not defined.
- There were conflicting objectives with respect to the denial of authorizations as laid down in Directive 91/414/EEC compared to the SETAC/ESCORT Guidance Document (Barrett et al., 1994).
The new scheme Data requirements for active substances cover 2 sensitive species at tier 1 (Figure 1). Two sensitive indicator species, the cereal aphid parasitoid Aphidius rhopalosiphi (Hymenoptera: Braconidae) and the predatory mite Typhlodromus pyri (Acari: Phytoseiidae) are tested at the tier 1 level. Selection of these two organisms was based on analyses of species/test system sensitivity performed by BART (Candolfi et al., 1999) and IOBC (Vogt, 2000).
Dose-response tests are required at tier 1 to allow for a more detailed risk assessment. At tier 1 laboratory toxicity studies with two indicator species are performed. LR50 (Lethal Rate 50, the application rate causing 50 % mortality of the test organisms) data are to be generated for two sensitive species which subsequently will be used to calculate TERs (Toxicity-Exposure-Ratios) for in-field and off-field exposure scenarios. The reason for generating lethal effects at this stage is because fecundity assessments for non-target arthropods have been associated with significant technical difficulties such as an extreme variability (Schmuck et al., 1996). For the time being we consider a conservative HQ or TER in-field of 1 as an appropriate value to trigger further testing. Since the species sensitivity analyses were mainly based on a comparison of in-field species which represent a lower species diversity than expected within off-field habitats, an uncertainty (safety) factor of 10 was built into the off-field TER-calculation to account for uncertainty with the applicability of T. pyri and A. rhopalosiphi as indicator species for off-field non-target arthropods.
The value triggering higher tier testing was set to 50 % (i.e. LR50), because for many test methods a lack of power was found to reliably detect differences of less than 50 % (30 %) and too many false positives were produced inducing unnecessary higher tier testing.
Spray-drift-values are corrected for 3-D by a factor of 2 for 2-D-tests (an additional Leaf area index (LAI) factor might be introduced as appropriate). Spray drift is the most relevant exposure route for NTA in off-field areas. Usually, the overall 90th percentile drift data published by BBA (2000) are used to estimate off-field drift deposition. However, while drift was measured on 2-D collectors, the off-field area is typically vegetated and will cause a spatial distribution of the pesticide drift. Therefore, the drift values should be corrected appropriately for off-field situations, including the leaf area if supported by appropriate data.
Drift reducing technique and a no spray zone may be required, if unacceptable effects (e.g. ecological significant impacts) to non-target arthropod populations inhabiting off-field areas are likely to occur for certain pesticides, depending on the local agricultural and environmental situation.
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exposure of
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assess lethal effects (LR50) in lab tests on glass plates using Aphidius rhopalosiphi and Typhlodromus pyri
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plus 1species if TERin-field is < 1 or plus 2 species if TERoff-field is < 10
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no authorization granted authorization possible
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Figure 1: Decision-making scheme for non-target arthropods
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Differences of the scheme compared to ESCORT 2 proposals TER vs HQ-approach Currently the TER concept rather than the HQ concept is implemented, in order to emphasize that the HQ-values of 12 and 8 as proposed by Campbell et al. (2000) are currently not accepted for a number of reasons:
At ESCORT 2 a threshold value of 40 % effects in the field was agreed for validation. The most important fact is that very high effects on both species were reported at their proposed HQ-values (Aphidius spp. 98 % at HQ 8.3; T. pyri 95 % at HQ 12.1) which indicates a considerable data gap with respect to the recommendations made at ESCORT 2.
According to the data provided, different HQs can be found due to different test methods used for T. pyri for the same product (e.g. 5.5 and 34.3) inducing a considerable uncertainty into 1st tier risk assessment. The particular product is known to have little to moderate effects on mites in the field, and therefore might indicate a borderline case as recomended (i.e. effects < 40 %).
In addition, for multiple applications products, field trials with populations of phytoseiid mites strongly indicate to considerably reduce the HQ value.
Field tests of the 4th IOBC ring test for phytoseiid mites (i.e. Amblyseius finlandicus) indicate that at an HQ of 2.4, unacceptable effects still cannot be ruled out as required according to Annex VI of directive 91/414/EEC.
For Aphidius spp. a detailed scrutiny of the pesticides analyzed was not possible because products or active substances were not known to the national authorities, indicating an unacceptable lack of transparency in this data set.
In addition, according to the data made available, it is obvious from field data that there is not much margin of safety within the Aphidius data set.
Additional information provided indicates that only about half a dozen products have been used in both of the validations. Obviously the data base is very limited.
Furthermore recovery of univoltine species is not sufficiently addressed in the validation.
Additional data used for regulatory purposes by BBA and UBA Another critical issue is the question whether individual sensitivity based on mortality alone is a good measure for population sensitivity or population vulnerability, especially with respect to multiple applications.
When data used for evaluation of effects on phytoseiid mites by national authorities in Germany were checked, a number of pesticides were found with HQs less than 12 for T. pyri (lowest HQ 1.8) especially with multiple application products (fungicides) with moderate effects in the field on predatory mites. These data again indicate that the consequences of multiple applications may be different from the consequences of a single treatment at a rate covering the max. number and max. field rate which might be attributable aspects of population ecology.
Exposure assessment In the new interim scheme spray drift values are usually corrected (i.e. reduced) by a factor of 2 for spatial distribution (i.e. upper and lower leaf surfaces) of pesticides within the off-field vegetation. A default value of 10 as proposed at ESCORT 2 based on a proposal made by ECPA (Gonzalez-Valero et al. 2001) is currently not accepted for the following reasons:
- Proposed LAI-values were measured to the time of the maximum leaf area development for a limited number of crops (i.e. 8).
- Seasonal growth is not addressed.
5
- Data for natural vegetation are not available.
According to the data presented max. LAIs differ between different crops and peak LAIs are found at different times during the season, which is not very surprising, earliest in April and latest in July. Overall average of max. LAIs is 5.7 with a range of 2 to 12. A default of 10 as proposed at ESCORT 2 does however not take into account seasonal development and heterogeneity of the off-field vegetation. A conservative estimate based on these data might be advisable, e.g. a default of 2 with the option to increase this value for late applications. Discussion In general the basic philosophy of ESCORT 2 was adopted for the German interim procedure whereas issues which are under scientific debate are not yet implemented and therefore the scheme is deemed to produce less false positives compared to the former procedure according to ESCORT 1 while in the same time reducing the risk to produce false negatives compared to ESCORT 2. This is in compliance with the General Principles of Annex VI of Directive 91/414/EEC:
Annex VI, B. Evaluation, 1. General Principles, Point 4: ”In interpreting the results of evaluations, experts of the national authorities and the companies, shall take into consideration possible elements of uncertainty in the information obtained during the evaluation, in order to ensure that the chances of failing to detect adverse effects or of under-estimating their importance are reduced to a minimum.”
The best Member States can possibly do is to support a testing procedure that reduces possible elements of uncertainty, in order to ensure that the chances of failing to detect adverse effects or of under-estimating their importance are reduced to a minimum.
We believe that the limitations outlined must be taken into account before a final decision on the HQ values can be taken. The German authorities suggest to adopt HQ-values lower than those currently discussed by Campbell et al. (2000). If sufficiently supported by data the HQ-values might be adjusted appropriately. Acknowledgements The authors like to thank all colleagues who joint the discussions and contributed to the finalisation of the scheme. References Barrett, K.L., Grandy, N., Harrison, E.G., Hassan, S. & Oomen, P. (eds.) 1994: Guidance
document on regulatory testing procedures for pesticides with non-target arthropods. SETAC Europe, Brussels. ISBN 0 9522535 2 6.
BBA (Federal Biological Research Centre for Agriculture and Forestry, Germany) 2000: Bekanntmachung des Verzeichnisses risikomindernder Anwendungsbedingungen für Nichtzielorganismen. Bundesanzeiger 100: 9878-9880.
Campbell, P.J., Brown, K.C., Harrison, E.G., Bakker, F., Barrett, K.L., Candolfi, M.P., Cañez, V., Dinter, A.; Lewis, G., Mead-Briggs, M., Miles, M., Neumann, P., Romijn, K.,
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Schmuck, R., Shires, S., Ufer, A. & Waltersdorfer, A. 2000: A Hazard Quotient approach for assessing the risk to non-target arthropods from Plant Protection Products under 91/414/EEC: Hazard Quotient trigger value proposal and validation. J. Pest Science (in press).
Candolfi, M., Bakker, F., Cañez, V., Miles, M., Neumann, C., Pilling, E., Priminani, M., Romijn, K., Schmuck, R., Storck-Weyhermüller, S., Ufer, A. & 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.
Council of the European Union, 1991: Council Directive 15 July 1991 concerning the placing of plant protection products on the market (91/414/EEC). Official Journal of the European Communities L230: 1-32.
Council of the European Union, 1996: Commission Directive 96/12/EC of 8 March 1996 amending Council Directive 91/414/EEC concerning the placing of plant protection products on the market. Official Journal of the European Communities L65: 20-37.
Gonzalez-Valero, J.F., Campbell, P.J., Fritsch, H.J., Grau, R. & Romijn, P. 2001: Exposure assessment for terrestrial non-target arthropods. J. Pest Science (in press).
Schmuck, R., Mager, H., Künast, Ch., Bock, K.D. & Storck-Weyhermüller, S. 1996: Variability in the reproductive performance of beneficial insects in standard laboratory toxicity assays – Implications for hazard classification of pesticides. Ann. Appl. Biol. 128: 437-451.
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 Bulletin 23 (9): 3-15.
Pesticides and Beneficial Organisms IOBC/wprs Bulletin Vol. 24 (4) 2001
pp. 7 - 15
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Influence of leaf substrates on the toxicity of selected plant protection products to Typhlodromus pyri Scheuten (Acari: Phytoseiidae) and Aphidius rhopalosiphi DeStefani Perez (Hymenoptera: Aphidiidae) Pia Ternes1, Marco P. Candolfi2, Andreas Ufer3 & Heidrun Vogt1
1 BBA Dossenheim, Germany; 2 Syngenta Crop Protection AG, Basel, Switzerland; 3 BASF AG, Limburgerhof, Germany Abstract: The toxicity of selected plant protection products to T. pyri and A. rhopalosiphi under extended laboratory conditions (exposure of the arthropods on different natural substrates, namely treated leaves) was compared with worst case Tier 1 laboratory results (exposure of the arthropods on glass plates). Six different leaf substrates were selected and tested for their suitability for use in extended laboratory tests. Used plant substrates were apple (Malus domestica; Rosaceae), grapevine (Vitis vinifera; Vitaceae), bush bean (Phaseolus vulgaris; Fabaceae), bamboo (Sasa palmata; Poaceae), cockspur grass (Echinochloa grus galli; Poaceae) and cast iron plant (Aspidistra elatior; Liliaceae). Protonymphs of T. pyri and adult wasps of A. rhopalosiphi were exposed to fresh dried residues of plant protection products. For each test carried out, LR50-values of the applied plant protection products were determined. In general all substrates used in this project proved to be suitable as natural substrate from a technical point of view. Phaseolus vulgaris has certain advantages for general use. Bamboo was preferred, when testing "difficult" compounds like herbicides. Taking the variability of non target arthropod tests systems into consideration, no relevant differences in the rate-response curves of T. pyri or A. rhopalosiphi could be found, when different leaf substrates were used. Currently used plants like bush bean, apple or vine are suitable test substrates for registration tests with plant protection products. Keywords: side-effects, Typhlodromus pyri, Aphidius rhopalosiphi, laboratory test, extended laboratory test, plant protection products Introduction The predatory mite Typhlodromus pyri Scheuten (Acari: Phytoseiidae) and the aphid parasitoid Aphidius rhopalosiphi DeStefani Perez (Hymenoptera: Aphidiidae) were selected for this study since they are recommended standard test organisms for assessing side-effects of plant protection products for registration in the European Union (Barrett et al., 1994; Candolfi et al., 2001).
Main aim of the study was to investigate the influence of different plant substrates on the variability of results and the level of the toxic response. Rate response data on inert substrate (glass) were used as reference. The project should give an indication if the effects and their respective variability found using the most commonly used plant species in extended laboratory tests supports the current sequential testing scheme going from Tier 1 studies on inert substrate to higher tiers on natural substrate until reaching field trials with increasing level of "practical relevance".
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Materials and methods Test organisms – Typhlodromus pyri (Scheuten) (Acari: Phytoseiidae) Mites in the protonymph stage (4 days old) were used in the test and obtained from synchronised in-house cultures. – Aphidius rhopalosiphi (DeStefani-Perez) (Hymenoptera: Aphidiidae) The parasitoids were obtained from a commercial supplier (Katz Biotech Services, Welzheim, Germany). The test insects were 36 to 44 h old at test initiation. Test plants – Bush-bean Phaseolus vulgaris (Fabaceae) ‘Caruso’ – Apple Malus domestica (Rosaceae) ‘Priam’ and ‘Querina’ – Grapevine Vitis vinifera (Vitaceae) ‘Regent’ and ‘Blauer Portugieser’ – Cockspur grass Echinochloa grus galli (Poaceae) – Bamboo Sasa palmata ‘Nebulosa’(Poaceae) – Cast iron plant Aspidistra elatior (Liliaceae) Bean, apple and grapevine were selected because of their widespread use in extended laboratory tests for registration purpose. Monocotyledon plants (cockspur grass, bamboo and cast iron) were selected as potential test plants for herbicides affecting dicotyledons. All plants except the field cultured grapevine ‘Blauer Portugieser’ were cultured and kept in the greenhouse. Test substances and treatment application One insecticide, two fungicides and one herbicide were selected as representative of the three major groups of plant protection products: – Insecticide: Dimethoate 400 g/L EC; Perfekthion® – Fungicides: Mancozeb 750 g/kg WG; Dithane Ultra® Chinomethionate 260 g/kg WP; Morestan® – Herbicide: Bentazone 600 g/kg SG, Basagran Super® All studies included a deionized water control and five rates of each test substance. The test substances rate selection was based on preliminary range-finder tests. The test items were diluted in deionised water for an application at a volume rate of 200 l/ha. Treatments were applied using a laboratory sprayer (Potter Tower, Burkard, U.K) calibrated to deliver a deposit of 2 ± 0.2 mg/cm2 onto the glass surface or the upper side of detached leaves or leaf discs. The Tables 1 and 2 indicate for which combinations of test organism, test substrate and test substance LR50 values were determined. In the case of A. rhopalosiphi the toxicity of Dithane Ultra® was too low to assess a reliable LR50-value and therefore this fungicide was replaced by Morestan®. Experimental procedures T. pyri Tier I laboratory studies were conducted according to Blümel et al. (2000 a) and Louis & Ufer (1995). Extended laboratory studies were conducted according to Grimm et al. (2001). Protonymphs of T. pyri were exposed to fresh dried residues of plant protection products for a
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period of seven days. The test units consisted of a perforated Petri dish, a punched leaf disc (diameter 3,5 cm) placed on a wet cotton wool pad or a glue-bordered glass surface of the same size. Each study included six treatment groups: a control and five test substance rates. Six replicates, containing ten protonymphs each, were established for each treatment group. The total number of T. pyri per test was 360. The mites were fed with pollen of Pinus sp. and the experiments were carried out at 25 ± 2°C, 75 ± 15 % relative humidity and under long-day condition (16 h light, 8 h dark, 5000-6000 lux). Mortality (including escaped mites) recorded 7 days after test initiation was used as toxic endpoint. Table 1. Test substances and test substrates to assess LR50-values for T. pyri
Substrate Perfekthion Dithane Ultra Basagran Super Glass X X X Apple ‘Priam’ X – – Apple ‘Querina’ – X – Grapevine ‘Regent’ X X – Bean ‘Caruso’ X X – Echinochloa X – X Sasa – – X Aspidistra – – X
Table 2. Test substances and test substrates to assess LR50-values for A. rhopalosiphi
Substrate Perfekthion Morestan Glass X X Apple ‘Priam’ X – Apple ‘Querina’ X X Grapevine ‘Regent’ X – Grapevine ‘Blauer Portugieser’ – X Bean ‘Caruso’ X X
A. rhopalosiphi Tier I laboratory studies were conducted according to Mead-Briggs (1992) and Mead-Briggs et al. (2000) however, using smaller test units. The exposure cages consisted of two treated square glass plates and an untreated acrylic glass ring (diameter 5 cm, height 1,5 cm) in between, held together with two rubber bands. In the case of exposure on plant surface (extended laboratory studies), two treated leaves, the upper part treated, were placed between the glass plates and the ring. Adult wasps of A. rhopalosiphi were exposed to the fresh dried residues of plant protection products for a period of 48 hours. During this time the wasps were fed with 25% fructose solution and the test units were connected to a ventilation system and set up in a controlled environment room at 20 ± 2 °C, 75 ± 15 % RH and a 16 h photoperiod (5000-6000 lux). Each study included six treatment groups: a control and five test substance rates. Six replicates, containing five adult wasps of mixed sex each, were established for each treatment group. The total number of wasps per experiment was 180. Mortality recorded 48 h after test initiation was used as toxic endpoint.
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Data analysis For each test carried out, LR50-values (application rate, which is estimated to result in 50 % mortality of the exposed test organisms) and 95% confidence limits were determined by Standard Probit analysis. Studies were only considered valid when the ratio between the upper confidence limit and the lower confidence limit was smaller or equal to five (Grimm et al., 2001). The computer programme used to perform all statistical analysis was SAS v. 6.12.
LR50-values were considered to be significantly different when the 95 % confidence intervals did not overlap.
In order to compare the LR50-values, each LR50-value obtained on plant surfaces was divided by the LR50-value determined on glass for the respective product. The result of this calculation is described as ratio plant : glass. In the same way, the ratios between different plant substrates (plant : plant) were calculated for each product. Results
The generation of LR50-data using leaves proved to be feasible and the data quality, considering the confidence limits, was high. The LR50 values determined on the various plant substrates mostly do not differ significantly (Fig. 1-5). Significant differences are found only in some cases: a) T. pyri, Perfekthion, apple leaf vs. the other plant substrates (Fig. 1), indicating a higher mite mortality on the apple leaves than on the other plant leaves; b). T pyri, Dithane Ultra, bean leaf vs. the other plant substrates (Fig. 2) and c) A. rhopalosiphi, Perfekthion, apple ‘priam’ vs. apple ‘querina‘, vine ’regent‘ and one study with bean ‘caruso‘ (Fig. 4). However, in case c), the LR50-value obtained on apple ‘priam’ does not differ significantly from the second value on bean ‘caruso’. This is an indication for the inherent variability of these sensitive biological test systems. Taking the small differences between all the LR50 values obtained for A. rhopalosiphi with Perfekthion on leaf substrates into consideration as well as the very narrow confidence limits for the result with apple ‘priam’, this difference should not be overestimated.
Comparing the maximum ratios plant : plant, it is obvious that the differences from plant to plant substrate are rather small. For A. rhopalosiphi the maximum ratios range from 1.1 to 1.7, for T. pyri from 1.4 and 2.2 (Fig. 6).
The LR50-values determined on leaves were always higher than on glass and with only one exception (T. pyri, Dithane Ultra, bean leaf ) the differences between glass and leaf substrates are significant (Fig. 1-5). In the case of T. pyri, Perfekthion, the LR50-values obtained on glass plates in two experiments also differed significantly. The maximum ratios plant : glass for all tests were mostly between 2 and 3, and reached 6 for Perfekthion (Fig. 6).
Discussion
In the majority of the cases significant differences of the LR50-values between the plant species or varieties had not been found. But there was a trend, that Perfekthion resulted in higher mortalities on apple leaves than on other leaf substrates (Fig. 1 and 4). This was observed for both, T. pyri and A. rhopalosiphi. However, these differences do not seem to indicate a continuous phenomenon, because the study performed with T. pyri and Dithane Ultra on bean leaf resulted in a significant lower LR50-value compared to those obtained with the other plant substrates (Fig. 2); the LR50-value on beans was similar to the LR50-value on glass (Fig. 2). On the other hand, there was also a significant difference between the results of two experiments on glass performed with T. pyri and Perfekthion (Fig. 1).
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Figure 1: Determined LR50-values for Typhlodromus pyri and Perfekthion and confidence limits on glass and leaves from different plants. Figure 2: Determined LR50-values for Typhlodromus pyri and Dithane Ultra and confidence limits on glass and leaves from different plants.
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Figure 3: Determined LR50-values for Typhlodromus pyri and Basagran Super and confidence limits on glass and leaves from different plants. Figure 4: Determined LR50-values for Aphidius rhopalosiphi and Perfekthion and confidence limits on glass and leaves from different plants.
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Figure 5: Determined LR50-values for Aphidius rhopalosiphi and Morestan and confidence limits on glass and leaves from different plants. Figure 6: Comparison of the maximum ratios of the LR50-values calculated for both species, all tested plant protection products and plant surfaces.
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With the exception of bean leaf, T. pyri and Dithane Ultra (Fig. 2) all leaf substrates caused a significant decrease of toxicity when compared with the respective LR50-values obtained from exposure on the inert substrate glass. The maximum ratios plant : plant lay between 1.1 and 2.2 and they were for each plant protection product smaller than the ratios plant : glass (Fig. 6). This indicates that the step from glass to leaf is more relevant than from one leaf substrate to another one. The results reveal, that going from Tier I studies on inert substrate to a higher tier on natural substrate results in decreasing effects, in our study in most cases by a factor between 2 and 3 and only for Perfekthion by a factor of 6.
While all leaf substrates used in the experiments proved to be suited for the extended laboratory tests, the bean leaves had some advantages from the technical point of view. Phaseolus vulgaris has a short growth period. Its primary or young leaves are of a sufficient size and rather homogenous. The bean plants do not need any pesticide treatment prior to the use of the leaves to control pest or diseases. However, this may be necessary under certain circumstances when apple or vine leaves are used. Furthermore, as the bean leaves are softer and have thinner veins than the apple and vine leaves, their leaf discs stay better in horizontal position during the treatment with the potter tower. This is essential to obtain the correct and homogenous target residue. Because the used bean variety has burr-like hairs on the under-surface, it sticks well on the cotton wool, what prevents the mites’ escape from the leaf discs. Leaves of apples have more protruding ribs on the under-surface than primary leaves of beans. When the border of the apple leaves are bended upwards, these ribs provide hiding places for the mites and prevent the exposure to the plant protection product.
For the herbicide experiments bamboo was suitable because it is insensitive to the used herbicide and an easy-care plant with broad leaf blades. The cockspur grass was also suitable, but the handling was more difficult, because two leaf blades had to be stuck together, in order to reach the recommended size of the leaf discs. Conclusions • The project showed that currently used dicotyledon plants like bush bean, apple or vine as
well as the tested monocotelydons are suitable test substrates for extended laboratory studies. From the technical point of view Phaseolus vulgaris has certain advantages for general use. Bamboo was preferred, when testing "difficult" compounds like herbicides with activity against dicotyledons.
• Taking the variability of non-target arthropod tests systems into consideration, no relevant
differences in the toxic response of T. pyri or A. rhopalosiphi could be found, when different plant leaves were used as test substrate. However, it cannot be excluded that studies with further plant protection products may result in other ratios between various plant substrates.
• Despite all variability produced by the biological test systems themselves and by different
plant substrates, all results measured with natural substrates were found between the expected toxicity on glass and the range of effects known from field experiments (Blümel et al. 2000 b).
Acknowledgements This study was supported by the Industrieverband Agrar, Frankfurt; Referat Technik und Umwelt.
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References Barrett K.L., Grandy N., Harrison E.G., Hassan S. & Oomen P. (eds.), 1994: Guidance
Document on Regulatory Testing Procedures for Pesticides with Non-Target Arthropods. SETAC Europe, Brussels.
Blümel S., Bakker F., Baier B., Brown K., Candolfi M.P., Goßmann A., Grimm C., Jäckel B., Nienstedt K., Schirra K.J., Ufer A. & Waltersdorfer A., 2000 a: Laboratory residual contact test with the predatory mite Typhlodromus pyri Scheuten (Acari: Phytoseiidae) for regulatory testing of plant protection products. In: 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): Guidelines to evaluate side-effects of plant protection products to non-target arthropods. IOBC/WPRS, Gent, 121-143.
Blümel, S., Aldershof, S., Bakker, F., Baier, B., Boller, E., Brown, K., Bylemans, D., Candolfi, M., Louis, F., Linder, C., Müther, J., Nienstedt, K., Oberwalder, C., Reber, B., Schirra, K.J., Ufer, A. & Vogt, H. 2000 b: Guidance document to detect side effects of plant protection products on predatory mites (Acari: Phytoseiidae) under field conditions: vineyards and orchards. In: 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): Guidelines to evaluate side-effects of plant protection products to non-target arthropods. IOBC/WPRS, Gent: 145-158.
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. SETAC Europe, Brussels (in press).
Grimm, C., Schmidli, H., Bakker, F., Brown, K., Campbell, P., Candolfi, M.P., Chapman, P., Harrison, E.G., Mead-Briggs, M., Schmuck, R. & Ufer A., 2001: Use of standard toxicity tests with Typhlodromus pyri and Aphidius rhopalosiphi to establish a dose-response relationship. J. Pest Science, in press.
Louis, F. & Ufer, A., 1995: Methodical improvements of standard laboratory tests for determinating the side- effects of agrochemicals on predatory mites (Acari: Phytoseiidae). Anz. Schädlingskde, Pflanzenschutz, Umweltschutz 68: 153-154.
Mead-Briggs, M. 1992: A laboratory method for evaluating the side-effects of pesticides on the cereal aphid parasitoid Aphidius rhopalosiphi (DeStefani-Perez). Aspects of Applied Biology 31: 179-189.
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 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): Guidelines to evaluate side-effects of plant protection products to non-target arthropods. IOBC/WPRS, Gent: 13-25.
SAS Institute Inc. 1990: SAS Procedures Guide, Version 6, Third Edition. SAS Institute Inc. 1989: SAS/STAT User’s Guide, Version 6, Fourth Edition, Volume I and
II.
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Pesticides and Beneficial Organisms IOBC/wprs Bulletin Vol. 24 (4) 2001
pp. 17 - 24
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Toxicity of insecticides used in wheat to adults of Aphidius rhopalosiphi DeStefani-Perez (Hymenoptera: Aphidiidae) with field treated plants
J.P. Jansen Laboratoire d’Ecotoxicologie, Ministry of Agriculture, Agricultural Research Centre, Gembloux, Department of Biological control and Plant genetic resources, Gembloux, Belgium Abstract: Effects of 8 insecticides used in wheat to control cereal aphids were assessed on adults of the parasitic wasp A. rhopalosiphi using a semi-field test design. Products were applied at their maximum field rate recommended in Belgium on small plots of wheat. Directly after treatment, plants were sampled randomly and brought back to the laboratory to form the exposure units where adult wasps were released. Units were placed outdoor and surviving females were collected 24 h later and assessed for fecundity in the laboratory. Mortality and fecundity were used to estimate the reduction of beneficial capacity, compared to control. Products were rated according IOBC standards. In order to assess duration of harmful effects, experiments with products that were initially toxic were repeated in the same way sampling plants either 1 or 3 days after pesticide application.
Exposure of wasps to plants treated with cyfluthrin, cypermethrin, fluvalinate, lambdacyhalothrin and pirimicarb lead to less than 25% control corrected mortalities. Mortalities higher than 25% were obtained for bifenthrin, deltamethrin and esfenvalerate. Mortalities were reduced to less than 25% with deltamethrin and esfenvalerate one day after treatment, but were still higher than 25% for bifenthrin. However, no effects were detected with this compound 3 days after treatment.
From these results, it can be concluded that exposure of wasps to field treated ear and last leaves of wheat was either harmless or short persistent. Harmful effects observed at day 0 did not last for more than 1 or 3 days. Keywords: Aphidius rhopalosiphi, bifenthrin, cyfluthrin, cypermethrin, deltamethrin, esfenvalerate, fluvalinate, lambdacyhalothrin, pirimicarb, semi-field test, wheat, persistence. Introduction Aphidiid wasps are one of the major aphid natural enemies in different crops. Their action greatly contributes to the natural control of aphid populations (Stary, 1988). In wheat, several authors have reported their beneficial activity (Jones, 1972, Powell, 1983, Latteur & Moens, 1990, Oakley et al., 1996). A. rhopalosiphi is the commonest species found in wheat in Belgium (Latteur, 1973, Latteur & Destain, 1980) and one of the most important ones in Western Europe (Stary, 1970, Dedryver et al., 1985).
Several laboratory studies showed that adults of A. rhopalosiphi were highly sensitive to pesticides and especially to insecticides (Kühner et al., 1985, Krespi et al., 1991, Borgemeister et al., 1993). The effects of products used in Belgium to control cereal aphids were previously assessed in the laboratory on both adult wasps and aphidiid nymphs protected inside mummified aphids (Jansen, 1996). Insecticides were found to be toxic to adults by contact on both glass plates and maize leaves previously treated, but their effects were limited when products were applied on aphid mummies. Thus, although field application of these insecticides could affect adult parasitoid populations, adults emerged from treated mummies could restore aphidiid populations within a few days provided that pesticide residues rapidly
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loose their toxicity. In this context, determination of harmful activity of insecticides is of particular importance.
The objective of this study was to test the toxicity and duration of harmful activity of insecticides used in wheat to control cereal aphids when adult wasps were exposed to field treated plants under conditions close to farmer practises. Methods used for this study are quite similar to those previously used to test wheat fungicides against the same species (Jansen, 2000). Material and methods Chemicals All insecticides tested in this study are used in wheat to control aphid populations at the end of spring, beginning of summer. They were tested at the maximum field rate recommended in Belgium. Details of trade name, formulation, active ingredients and tested rate are given in table 1. These insecticides were previously tested in the laboratory and were selected for this study because they were toxic for adults of A. rhopalosiphi on glass plates test and detached maize test (Jansen, 1996).
Table 1: Insecticides applied in the field and tested on A. rhopalosiphi
trade name formulation active ingredient tested dose (g a.i./ha)
Baythroid EC050 EC cyfluthrin 10.0 Cymbush DG DG cypermethrin 15.0 Decis EC 2,5 EC deltamethrin 5.0 Karate EC lambdacyhalothrin 5.0 Mavrik 2 F SC fluvalinate 36.0 Pirimor G WG pirimicarb 125.0 Sumi Alpha EC esfenvalerate 5.0 Sumi Alpha EC esfenvalerate 5.0 Talstar Flo SC bifenthrin 7.5
Application of chemicals Insecticides were applied in the field on rectangular wheat plots (3 m x 10 m) at the end of flowering (GS 69, Zadoks et al., 1974). Plots were delimited in a wheat field (cv “Pajero”) conducted according to normal agricultural practices, except that they were not treated with fungicides. Test products were applied with a 3 m ramp bearing 6 Azo 110° nozzles at 50 cm spacing, connected to a knapsack sprayer. Work pressure was 2.5 b and products were diluted and applied in a volume equivalent to 300 l of water/ha. Products were applied in 2 different sets of 4 products each. Each treatment included one plot per product and for the control (untreated).
Exposure units 30 minutes to 1 h after the treatment, when residues were dry, 30 to 40 stems of wheat with ear, first and second leaves were randomly selected from the central part of the plot (2 m x
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8 m). Plants were put in plastic trays and immediately brought back in the laboratory located nearby the field.
In the laboratory, wheat plants were carefully inspected and aphids, aphidiid mummies and aphid predators (syrphid eggs or larvae, ladybirds) were removed. Terminal sections of wheat stem, with last leaf and ear were immediately used to form exposure units. Exposure units were made of 4 stems of about 30cm long planted in pots (∅: 12cm) containing moistened vermiculite (Sibli, grade 5). 5 units were assembled for each product and 5 for control. Vermiculite was directly covered with sand (1cm height) to form a uniform untreated surface and a cylindrical Perspex cage (∅: 12cm, h:25 cm) was added on each unit. Top of cage and two rectangular cutaways were covered with nylon netting for ventilation. Ear and last leaves of wheat were selected to represent a worst-case field exposure in the laboratory. According to Cilgi & Jepson (1992) and Longley & Jepson, (1997b), these leaves received a greater amount of products that the rest of the plants. Conduct of trials Immediately after unit assembling, six 2-48h old adults of A. rhopalosiphi (3 males, 3 females) were introduced and left undisturbed for a 24h period. Wasps were obtained from a laboratory colony established in 1993. Details of the rearing are given in Jansen (1996 and 1998). Plants used for the test were covered with natural honeydew produced by aphids in the field. At the moment of treatment, aphid population reached a mean of 3.5-4.0 aphids/tiller. No artificial manipulations, such as sucrose solution application to stimulate wasp foraging, were applied.
During exposure, wasps were observed 10 times in each unit (50 observations per product and control) and the number of wasps found on plants or on sand and perspex cage was noted. Mean percentages of wasps found on plants were calculated and compared to control to detect possible repellent effects of products. The percentage of wasps found on plants were compared to control and a Student t-test was applied(Dagnelie, 1974).
24 h after wasp release, units were disassembled and surviving wasps counted. Missing wasps were considered as dead. Mortalities were calculated in each object and corrected with the mean mortality observed in the control units (Abbott, 1925). Exposure of wasps to treated plants was made outdoor under a plastic shelter for rain protection.
Surviving wasps were collected, sexed and females were used to assess effects on fertility. Ten surviving females per product and control were individually confined in fertility assessment units, similar to those described in previous publications (Jansen, 1998). Females were removed from the units 24h later and aphid mummies were left to develop and counted 10-12 days later. Mean number of mummies produced in each unit were compared to control and a Student t-test (Dagnelie, 1974) applied. Fertility assessments were made in the laboratory in climatic chamber at 20°± 2°C and 50-90% RH.
Reduction in beneficial capacity (E) was calculated according to Overmeer-Van Zon (1982) whose formula combines mean mortality and reproductive performance results. According to the E value, products were classified in one of the four IOBC categories for extended-lab test and semi-field tests:
1 – “harmless”, E < 25% 2 – “slightly harmful”, 25% < E < 50% 3 – “moderately harmful”, 50 % < E < 75 % 4 – “harmful”, E > 75 %
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To determine duration of harmful activity of insecticides, products resulting in more than 25% corrected mortality, experiments were repeated as described but plants were taken from the same plots 1 or 3 days after treatment, until mortality decreased below 25%. Climatic conditions Temperature and rainfall measured during experiments are summarised in table 2. Results were provided by the automatic meteorological station of Gembloux-Ernage, which is located approximately 3 km away from the experimental site. Table 2: Minimum, maximum and mean (minimum+maximum/2) temperatures, and rainfall recorded during the assay.
Temperature (°C) Rainfall minimum maximum mean (mm)
Set 1 exposure day 0 12.3 20.0 16.2 (protected) day 1 10.0 20.6 15.3 (protected) day 3 11.0 16.1 13.6 (protected) field day 0-1 10.0 20.6 15.3 0.0 mm day 1-3 6.3 20.6 13.5 6.4 mm
Set 2 exposure day 0 11.8 20.9 16.4 (protected) day 1 12.3 20.0 16.2 (protected) field day 0-1 12.3 20.0 2.4mm
Results Results of the 2 sets of experiments are listed in table 3. Control mortalities ranged from 6.7% to 10.0%. In set 1, corrected mortalities with cyfluthrin, cypermethrin and lambdacyhalothrin were lower than 25% and these products did not affect fertility capacity of surviving females. According to reduction of beneficial capacity and IOBC rating, these products were considered as harmless. Bifenthrin was more toxic, with mortalities reaching 40%. Repetition of the experiments one day after treatment did not reduce observed mortalities but no harmful effects were detected 3 days after treatment. In the second set of experiments, fluvalinate and pirimicarb were harmless while deltamethrin and esfenvalerate were toxic, with corrected mortalities of 46.4% for both products. No harmful effects were detected with deltamethrin and esfenvalerate one day after treatment.
Observations of wasps during exposure showed that some of the insecticides had repellence effects on adult wasps. Adult wasps were observed less frequently on plants treated with cyfluthrin, cypermethrin (set 1), deltamethrin and esfenvalerate (set 2) in a statistically significant way compared to control. However, no relation between repellence and mortality can be established. Discussion Results obtained in these experiments show that when adults of A. rhopalosiphi are exposed to insecticide field treated plants, effects observed directly after treatment are generally low.
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Furthermore, for products that were initially toxic, duration of harmful activity is limited in time, with no detectable effects 3 days after treatment. Because methods used in this study greatly differ from previously used ones, comparison of results is difficult. However, effects observed during this study are surprisingly low. These results could be partly explained as follows. Table 3: Initial toxicity (day 0) and determination of duration of harmful activity (day 1-3) of selected insecticides to adults of A. rhopalosiphi. Observed and corrected mortalities, wasp activity during exposure, mummy production, and IOBC rating for semi-field tests.
trade observed corrected % wasp # mummies/ E IOBC name mortality
(%) mortality
(%) on plants unit (%) class
set 1 – day 0 control 10.0 - 24.8a 19.8a - - bifenthrin 46.7 40.8 27.2a 21.5a 35.7 2 cyfluthrin 13.3 3.7 15.3b 17.7a 13.9 1 cypermethrin 13.3 3.7 16.1b 21.2a -3.1 1 lambdacyhalothrin 20.0 11.1 23.5a 22.9a -2.8 1
set 1 – day 1 control 10.0 - 29.9a 22.3a - - bifenthrin 40.0 33.3 28.0a 19.7a 41.1 2
set 1 – day 3 control 6.7 - 31.0a 23.7a - bifenthrin 10.0 3.6 28.4a 26.8a -9.0 1
set 2 – day 0 control 13.3 - 36.2ab 42.6a - - deltamethrin 50.0 46.4 17.3d 43.9a 44.8 2 esfenvalerate 50.0 46.4 26.9c 23.8a 69.8 3 fluvalinate 16.7 10.7 37.3a 45.4a 4.8 1 pirimicarb 23.3 17.9 29.8bc 50.8a 2.1 1
set 2 – day 1 control 10.0 - 32.7a 33.1a - - deltamethrin 26.7 18.5 28.0a 31.2a 23.2 1 esfenvalerate 23.3 14.8 29.0a 30.5a 21.5 1
Within a column, figures followed by the same letter are not significantly different (Student t-test at p=0.05 level).
Contact toxicity on plant is generally lower than on glass plates. Pesticides are less available to insects on leaves than on inert, nonporous surfaces (Longley & Jepson, 1997a). Furthermore, when products are applied in the field, repartition of pesticide residues is more heterogeneous than in the laboratory and generally lower pesticide concentrations can be found on treated substrates. Chemical analysis of residues of field applied pesticides have
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shown that ear and last leaf of wheat receive more or less 20 to 25% of the field rate, the rest of the dose being distributed on stem, other part of plants and soil (Cilgi & Jepson, 1992, Longley & Jepson, 1997b). Upperside of leaves generally receive a greater amount of pesticide and very low levels of pesticide residues are found on the underside of leaves (Longley & Jepson, 1997b). Thus, application of pesticides in the field with spraying equipment similar to that used by farmers leads to a more heterogeneous distribution of residues and a reduction of doses as compared to results obtained when laboratory spraying equipment is used.
Low toxicity values observed in this study can also be explained by wasp behaviour during exposure. With either glass plate or detached leave tests, adult wasps are released for a certain period of time in an artificial environment and contact between treated surfaces and insects is forced and non-natural. On plants, females wasps exhibit a typical pattern sequence in search for aphids to parasitize (Ayal, 1987, Cloutier & Bauduin, 1990). This pattern is generally repeated several times and separated by flight activity. Thus, contact between parasitoid and plant in natural environment is not continuous but disrupted, as contact with pesticide residues when plants are treated. Furthermore, detection of pesticide residues by adult wasps can modify the behaviour of this insect by repellency effects (Kühner et al., 1985), adult wasps spending less time on treated plants than on untreated ones (Jansen, 1999, Jansen, 2000). Adult wasps are also attracted by aphid honeydew cover which is linked to the presence and abundance of aphids. Longley and Jepson (1997b) have shown that cereal aphids, such as Sitobion avenae and Metapolophium dirhodum preferred underside of leaves as feeding site. As pesticide residues are mostly distributed on the upperside of leaves, adult wasps spend most of the time in contact with limited concentration of pesticides. Thus, exposure of adult wasps to insecticides applied on plants cannot be considered continuous as in laboratory tests but discontinuous. Furthermore, wasp behaviour tends to diminish contact with parts of plants receiving highest amounts of pesticides.
Climatic conditions observed during experiments can also partly explain the results obtained. Some rainfall occurred after insecticide applications and plant sampling 1 and 3 day after treatment. These rains probably reduced pesticide concentration on plants and subsequently observed toxicity. These climatic conditions were not considered as unusual for the season, but repetition of experiments in different climatic conditions could be of high interest to determine influence of the rain on duration of harmful activity of tested insecticides. Conclusions According to the results obtained in this study, it can be concluded that exposure of adult wasps to insecticide field treated plants at their maximum recommended field rate did not lead to marked toxic effects. When significant toxicity was observed with freshly applied insecticide residues, effects were short-lived and no harmful effects could be detected 3 days after treatments. Because the absence of effects of these products on aphidiid nymphs was already known, these results can lead to the general conclusion that the insecticides tested could have a very limited effect on aphidiid field populations. The low toxicity of these products to A. rhopalosiphi could be explained by parameters such as heterogeneity of pesticide deposits on plants, real amount of pesticide residues in contact with adult wasps when products are field applied, wasp behaviour and climatic conditions. Repetition of these experiments to encounter other climatic conditions could be helpful to confirm these results.
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References Abbott, S.W. 1925: A method of computing the effectiveness of insecticides. Journal of
Economic Entomology 18: 265-267. Ayal, Y. 1987: The foraging strategy of Diaeretiella rapae. I. The concept of the elementary
unit of foraging. Journal of Animal Ecology 56: 1057-1068. Borgemeister, C., Poehling, H.-M., Dinter, A. & Höller, C. 1993: Effects of insecticides on
life history parameters of the aphid parasitoid Aphidius rhopalosiphi (Hym.: Aphidiidae). Entomophaga 38: 245-255
Cilgi, T. & Jepson, P. 1992: The use of tracers to estimate the exposure of beneficial insects to direct pesticide spraying in cereals. Annals of Applied Biology 121: 239-247.
Cloutier, C. & Bauduin, F. 1990: Searching behaviour of the aphid parasitoid Aphidius nigripes (Hymenoptera: Aphidiidae) foraging on potato plants. Environmental Entomology 19: 222-228.
Dagnelie, P. 1974: Théorie et méthodes statistiques, Vol. 2, ed. by Duculot SA, J., Gembloux, Belgium.
Dedryver, C.A., Ankersmit, G.W., Basedow, T., Bode, E., Carter, N., Castanera, P., Chambers, R., Dewar, A., Keller, S., Latteur, G., Papierok, B., Powell, W., Rabbinge, R., Sotherton, N.W., Sunderland, K., Wilding, N. & Wratten, S.D. 1985: Rapport de synthèse sur les activités du sous-groupe «Ecologie des pucerons des céréales». IOBC/WPRS Bulletin 8(3): 57-104.
Jansen, J.-P. 1996: Side effects of insecticides on Aphidius rhopalosiphi (Hym., Aphidiidae) in laboratory. Entomophaga 41:37-43.
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. Journal of Applied Entomology 123: 217-223.
Jansen, J.-P. 2000: Toxicity of fungicides to adults of Aphidius rhopalosiphi DeStefani-Perez (Hymenoptera: Aphidiidae) in laboratory tests with field treated wheat. IOBC/WPRS Bulletin 23(9): 73-80.
Jones, M. 1972: Cereal aphids, their parasites and predators caught in cages over oats and winter wheat crops. Annals of Applied Biology 72: 13-25.
Krespi, L., Rabasse, J.M., Dedryver, C.A. & Nenon J.P. 1991: Effect of three insecticides on the life cycle of Aphidius uzbekistanicus Luz. (Hym.: Aphidiidae). Journal of Applied Entomology 111: 113-119.
Kühner, C., Klingauf, F. & Hassan, S.A. 1985: Development of laboratory and semi-field methods to test the side-effect of pesticides on Diaeretiella rapae (Hym: Aphidiidae) Med. Fac. Landbouww. Rijksuniv. Gent 50/2b: 531-538.
Latteur, G. & Destain, J. 1980: Etude de l’action des champignons et des hyménoptères parasites inféodés aux populations de Sitobion avenae (F.) et de Metopolophium dirhodum (Walk.) dans le champ expérimental de Milmort en 1978 et 1979. IOBC/WPRS Bulletin 1980/III/4: 11-17.
Latteur, G. & Moens, R. 1990: Rôle clef de la lutte biologique naturelle dans la lutte intégrée contre les pucerons des céréales en été. Fumure et protection phytosanitaire des céréales, 1990. Ed. Faculté Sciences Agronomiques de Gembloux et Centre de Recherches Agronomiques de l’état, Gembloux, Belgique.
Latteur, G. 1973: Etude de la dynamique des populations des pucerons des céréales - Premières données relatives aux organismes aphidiphages en trois localités différentes. Parasitica 29: 134-151.
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Longley, M. & Jepson, P. 1997a: Effects of life stage, substrate, and crop position on the exposure and susceptibility of Aphidius rhopalosiphi De Stefani-Perez (Hymenoptera: Braconidae) to deltamethrin. Environmental Toxicology and Chemistry 16(5): 1034-1041.
Longley, M. & Jepson, P. 1997b: Cereal aphid parasitoid survival in a logarithmically diluted deltamethrin spray transect in winter wheat: field-based risk assessment. Environmental Toxicology and Chemistry 16(8): 1761-1767.
Oakley, J.N., Walters, K.F., Ellis, S.A., Green, D.B., Watling, M. & Young, J.E. 1996: Development of selective aphicide treatments for integrated control of summer aphids in winter wheat. Annals of Applied Biology 128: 423-436.
Overmeer, W.P.J. & van Zon, A.Q. 1982: A standardised method for testing the side-effects of pesticides on the predacious mite Amblyseius potentillae Garman (Acarina: Phytoseiidae). Entomophaga 27: 357-364.
Powell, N. 1983: The role of parasitoids in limiting cereal aphid populations. In: Aphid antagonists, proceedings of a meeting of the EC experts group, Portici, Italy, 1983. R. Cavalloro (ed.), Balkema, Rotterdam.
Stary, P. 1970: Biology of Aphid parasitoids (Hymenoptera: Aphidiidae) with respect to integrated control. Series Entomologica, Dr. W. Junk Publ., Den Haag.
Stary, P. 1988: Natural enemies: Parasites. In: World Crop Pests, Aphids, their biology, natural enemies and control. A.K. Minks and P. Harrewijn (eds.), Vol. 2B: 171-188.
Zadoks, J.C., Chang, T.T. & Konzak, C.F. 1974: A decimal code for the growth stages of cereals. Weed Research 14: 415-421.
Pesticides and Beneficial Organisms IOBC/wprs Bulletin Vol. 24 (4) 2001
pp. 25 - 34
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Comparison of side-effects of spinosad, tebufenozide and azadirachtin on the predators Chrysoperla carnea and Podisus maculiventris and the parasitoids Opius concolor and Hyposoter didymator under laboratory conditions Viñuela, E., Medina, Mª.P., Schneider, M., González, M., Budia, F., Adán, A. & Del Estal, P. Protección de Cultivos, E.T.S.I. Agrónomos, E-28040-Madrid, Spain Abstract: Side-effects of spinosad, tebufenozide and azadirachtin have been evaluated against pupae of the predator Chrysoperla carnea and the parasitoid Hyposoter didymator, and adults of the predators C. carnea and Podisus maculiventris, and the parasitoid Opius concolor. Insecticides were applied with different techniques: topically to pupae, and via residual contact, topical application and ingestion, to adults. Azadirachtin was harmless for the enemies except for the braconid O. concolor. Reductions in progeny size when adults were exposed to fresh residues, and both in mortality and reproductive parameters when applied via ingestion, occurred. Tebufenozide was harmless for the enemies irrespective of the mode of application or the developmental stage studied. Spinosad was the most harmful insecticide but it was safe for pupae and adults of the predator C. carnea at the maximum field rate. Key words: Chrysoperla carnea, Podisus maculiventris, Opius concolor, Hyposoter didymator, spinosad, tebufenozide, azadirachtin, pupae, adults, side-effects, laboratory Introduction Amid the interesting insecticides for Integrated Pest Management (IPM) and Integrated Production (IP) programmes, based on their selectivity against different enemies or certain developmental stages of them, are spinosad, tebufenozide and azadirachtin. Spinosad is a macrocyclic lactone obtained from a soil actinomycete, which depolarizes insect neurons by activating the postsynaptic nicotinic acetylcholine receptors, leading to tremors of most muscles in the body (Salgado, 1997). Tebufenozide is a moulting accelerating compound, which stimulates directly the ecdysteroid receptors inducing a premature and lethal larval moult in insects (Smagghe et al., 1996, 1997). Azadirachtin is a triterpenoid pesticide obtained from Azadirachta indica A. Juss, which acts by inhibiting the releasing of prothoracicotropic hormones and allatotropins (Banken & Stark, 1997).
In this work and with the goal of increasing our knowledge on side-effects of these pesticides, we have evaluated their effects applied with different techniques (residual contact, ingestion and topical application), on certain developmental stages of some relevant natural enemies: Hyposoter didymator (Thunberg) pupae (Hymenoptera, Ichneumonidae), Opius concolor (Szèpligeti) adults (Hymenoptera, Braconidae), Chrysoperla carnea (Stephens) pupae and adults (Neuroptera, Chrysopidae), and Podisus maculiventris (Say) adults (Heteroptera, Pentatomidae).
H. didymator is a solitary and non-paralysing ichneumonid endoparasitoid of several species of noctuid larvae of economic importance belonging to genuses Helicoverpa, Spodoptera, Autographa, Chrysodeixis, etc (Schneider & Viñuela, 1999). This enemy is
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currently found in many Spanish regions on a great variety of crops: alfalfa, cotton, melon, sunflowers, tomato (Bahena et al., 1999). O. concolor is a braconid endoparasitoid of the olive fruit fly Bactrocera oleae (Gmelin). The fly seriously threatens olive production in the Mediterranean region, where losses to its action may account for up to 60% of the total insect damage to olive groves, with more than 2.2 million cultivated hectares in Spain (González & Viñuela, 1997). C. carnea is a cosmopolitan polyphagous predator, widely used in biocontrol of aphids in greenhouses, and very common in a wide range of natural, agricultural and forestry habitats (Greeve, 1984; Thompson, 1992). P. maculiventris is also a generalist predator, but this pentatomid mainly feeds on larvae of lepidopterans and it is also used in greenhouses, but for the control of noctuid pests (Viñuela et al., 2000). Material and methods Rearings Both enemies and hosts, were maintained in controlled environment cabinets, at 25ºC, 75% relative humidity and a 16L:8D photoperiod, and reared following standard procedures. O. concolor was reared in the substitution host, the Mediterranean fruit fly Ceratitis capitata (Wiedemann) (Albajes & Santiago-Álvarez, 1980; Jacas & Viñuela, 1994). C. carnea larvae were fed Sitotroga cerealella (Oliver) eggs and adults Hassan’s diet (Vogt et al., 2000). H. didymator was reared on Spodoptera littoralis (Boisduval) larvae following a procedure adapted from Harrington et al. (1993) (Schneider et al., 2000). P. maculiventris was also reared on S. littoralis (De Clerq et al., 1988; Viñuela et al., 1998).
Insecticides The commercial products Mimic®(24% tebufenozide), Tracer® (48% spinosad) and Align® (aqueous formulation with 3.2% of azadirachtin) were used in the assays. Depending on the application technique, aqueous or acetone fresh solutions were prepared prior the experiments. In some assays insecticides were applied in a wide range of concentrations: 0.1 to 1000 mg/l a.i. In others, we only studied the maximum field rates of tebufenozide (180 mg/l a.i.) and azadirachtin (47.5 mg/l a.i.) and several rates of spinosad: 96 g/ha a.i., present maximum recommended rate in orchards; 120 g/ha a.i., former maximum recommended rate in 1999; a very high rate of 400 g/ha a.i and a fifth of it, 80 g/ha a.i. In every case at least three replicates were performed.
Topical application assays Young pupae and adults of C. carnea, young pupae of H. didymator and young adults of O. concolor were topically dosed with 0.5µl droplets of acetone solutions of the three insecticides using a Burkard manual microapplicator. In pupal treatments, six to seven different dose levels per insecticide, ranging from 0.1 to 1000 mg/l a.i. (0.005 to 50 µ/g for C. carnea pupae and 0.003 to 25.5 µ/g for H. didymator pupae), were used. The weight of C. carnea and H. didymator pupae averaged 10.02 ± 0.002 and 19.6 ± 0.003 mg, respectively. In adult treatments, only maximum field rates were used. The weight of C. carnea and O. concolor adults averaged 7.47 ± 0.92 and 2.18 ± 0.38 mg, respectively.
Residual contact assays Young adults of C. carnea, P. maculiventris and O. concolor were exposed to fresh residues of the three insecticides in glass surfaces, applied at the maximum field rates. Dismountable glass cages designed for O. concolor tests and forced ventilation were used (Jacas & Viñuela, 1994).
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Ingestion assays Recently emerged adults of C. carnea were offered continuosly during the preoviposition period (4 days) aqueous solutions of the three insecticides. Less than 24-h-old adults of O. concolor were supplied the insecticides ad libitum via the drinking water during the life span. Maximum field rates were studied
Assessments Evaluation of results depended on the kind of experiment. Pupal and adult mortality, average adult longevity (number of dead insects was scored at regular intervals, which were variable with the pesticides, until the last insect died), percentages of adult emergence and beneficial capacity of adult females were scored.
For parasitoids, beneficial capacity was studied by determining number of hosts attacked and progeny size using ventilated plastic round cages (9 cm in diameter) whose floor had a round hole (3 cm in diameter) covered by a gauze. In the case of O. concolor, females were isolated and during the following 3 days, 20 fully-grown C. capitata larvae were offered for parasization during 2 hours and number of emerging O. concolor and C.capitata were recorded (Viñuela et al., 2000). In the case of H. didymator, five 3-d-old, presumably mated females, were isolated and during the following 5 days, ten L3 S. littoralis larvae were offered for parasitization during 1 hour (Schneider et al., 2000). For predators, fecundity and fertility were evaluated (see De Clerq et al., 1995 and Viñuela et al., 2000).
Data were analysed by one-way analysis of variance using Statgraphics (STSC, 1987). Means were separated by the LSD multiple range test (P<0.05) or Bonferroni test when the F value from ANOVA was not significant. When the premises of ANOVA were violated, the Kruskal Wallis test was applied. Results Topical treatment of pupae The three pesticides were harmless for pupae of C. carnea. Both adult emergence, fecundity (scored as mean number of eggs per female in 7 days) and fertility (% egg hatch) were similar in control and treated units (Table 1).
For H. didymator, adult emergence was not significantly different from that of the controls. However, the three insecticides affected adult longevity at certain doses (Table 2). Azadirachtin and tebufenozide, did not significantly decrease longevity from the dose of 0.026 µ a.i./g pupae onwards. However, azadirachtin exhibited significant effectsat the dose of 12.8 µ a.i./g pupae. One factor that might have accounted, is that at this dose 65% of the emerged adults were males, because in this ichneumonid their longevity is much more variable than in females (from 5 to 25 days). For tebufenozide, significant reductions between 40.4% and 52% occurred from 5.2 µ a.i./g pupae onwards. The most drastic effect was observed with spinosad. At concentrations as low as 10 mg/l a.i., longevity was approximately 32% lower than that of the controls, and from 100 mg/l a.i. onwards, insects only survived for 5 days. However, beneficial capacity of H. didymator survivors did not significantly differ from that of the controls (Table 2).
28
Table 1. Toxicity of spinosad, tebufenozide and azadirachtin to the predator Chrysoperla carnea when topically applied to young pupae
Chrysoperla carnea Topical treatment of pupae
Insecticides Doses µ a.i./g pupae (Concentrations mg a.i./l)
% Adult emergence a
Eggs/female,7 days b
% Eclosionc IOBCclass
Control 0 199.5 ± 54.9a 85.0 ± 1.5a –
Spinosad 0.005 (0.1) 85 ± 5.0a 217.1 ± 29.9a 95.0 ± 3.2a 1
0.05 (1) 100 ± 0.0a – – 1
0.5 (10) 95 ± 5.0a 202.5 ± 38.6a 90.3 ± 1.3a 1
5.0 (100) 100 ± 0.0a 241.5 ± 19.7a 97.6 ± 2.3a 1
25.0 (500) 100 ± 0.0a 202.5 ± 7.9a 92.0 ± 4.1a 1
50.0 (1000) 90 ± 5.7a – – –
Tebufenozide 0.005 (0.1) 90 ± 5.7a 208.7 ± 22.6a 86.3 ± 2.3a 1
0.05 (1) 100 ± 0.0a – – –
0.5 (10) 100 ± 0.0a 240.6 ± 10.2a 86.3 ± 4.0a 1
5.0 (100) 100 ± 0.0a 241.2 ± 16.9a 89.6 ± 0.3a 1
25.0 (500) 95 ± 5.0a 248.7 ± 7.5a 92.0 ± 3.5a 1
50.0 (1000) 95 ± 5.0a – – –
Azadirachtin 0.005 (0.1) 95 ± 5.0a 234.0 ± 8.3a 95.0 ± 2.4a 1
0.05 (1) 90 ± 5.7a – – –
0.5 (10) 95 ± 5.0a 201.1 ± 22.5a 85.6 ± 7.3a 1
5.0 (100) 90 ± 5.7a 188.6 ± 14.2a 91.3 ± 2.0a 1
25.0 (500) 100 ± 0.0a 208.1 ± 12.9a 86.3 ± 3.1a 1
50.0 (1000) 95 ± 5.0a – – –
Within the same column, data followed by the same letter are not significantly different (P= 0.05; Bonferroni mean separation). a F= 1.06; df= 24,77; P=0.4104. b F= 0.69; df= 12,26; P= 0.7438. c F= 1.55; df= 12,26; P= 0.1685.
29
Table 2. Toxicity of spinosad, tebufenozide and azadirachtin to the parasitoid Hyposoter didymator when topically applied to young pupae
Hyposoter didymator Topical treatment of pupae
Insecticides Doses µ a.i./g pupae
(Concentrations mg a.i./l)
% Adult emergence a
Longevity, days b
% Attacked hosts c
% Progeny size d
IOBCclass
Control 0 95.0 ± 5.0a 21.7 ± 2.9ab 86.0 ± 4.0a 98.0 ± 1.2a –
Spinosad 0.0026 (0.1) 80.0 ± 8.2a 22.1 ± 2.7a 86.0 ± 1.0a 97.0 ± 2.0a 1
0.026 (1) 80.0 ± 8.2a 18.0 ± 1.9abc 83.4 ± 1.9a 97.0 ± 1.2a 1
0.26 (10) 80.0 ± 8.2a 14.8 ± 2.0abcde 83.0 ± 2.5a 97.0 ± 2.0a 2
2.6 (100) 90.0 ± 5.8a 3.3 ± 0.4f 83.3 ± 4.4a* 91.7 ± 8.3a* 3
5.2 (200) 75.0 ± 5.0a 1.8 ± 0.2g 85.0 ± 2.9a* 87.5 ± 2.5a* 3
12.8 (500) 80.0 ± 8.2a 1.6 ± 0.2g 80.0 ± 0.0a* 87.5 ± 2.5a* 3
25.5 (1000) 90.0 ± 5.8a 1.4 ± 0.1g – – 4
Tebufenozide 0.0026 (0.1) 85.0 ± 5.0a 21.5 ± 1.8a 82.0 ± 2.5a 97.0 ± 2.0a 1
0.026 (1) 80.0 ± 8.2a 19.1 ± 2.1ab 83.0 ± 2.0a 97.0 ± 1.2a 1
0.26 (10) 80.0 ± 8.2a 21.8 ± 2.4ab 85.4 ± 2.1a 97.0 ± 2.0a 1
2.6 (100) 75.0 ± 9.6a 19.4 ± 2.4ab 81.0 ± 3.3a 87.0 ± 5.4a 1
5.2 (200) 80.0 ± 8.2a 10.4 ± 1.2e 86.8 ± 2.4a 93.0 ± 2.5a 2
12.8 (500) 75.0 ± 5.0a 11.2 ± 1.2cde 83.0 ± 2.0a 96.0 ± 1.9a 2
25.5 (1000) 75.0 ± 9.6a 12.9 ± 1.5bcde 86.6 ± 3.4a 95.0 ± 1.6a 2
Azadirachtin 0.0026 (0.1) 80.0 ± 8.2a 21.1 ± 2.4ab 83.0 ± 2.0a 97.0 ± 2.0a 1
0.026 (1) 85.0 ± 5.0a 16.9 ± 2.0abcd 84.0 ± 2.9a 97.0 ± 1.2a 1
0.26 (10) 85.0 ± 5.0a 16.3 ± 2.1abcde 84.8 ± 2.0a 97.0 ± 2.0a 1
2.6 (100) 80.0 ± 8.2a 17.6 ± 1.7abc 85.0 ± 2.2a 87.0 ± 5.4a 1
5.2 (200) 90.0 ± 5.8a 15.7 ± 1.7abcde 85.02± 2.8a 94.0 ± 1.9a 1
12.8 (500) 85.0 ± 5. a 11.7 ± 1.6de 83.4 ± 1.9a 96.0 ± 1.9a 2
25.5 (1000) 80.0 ± 8.2a 15.3 ± 1.5abcde 87.6 ± 2.7a 94.0 ± 1.9a 1
Within the same column, data followed by the same letter are not significantly different (P= 0.05; a,c,d Bonferroni mean separation. b Kruskal-Wallis). a F= 0.55; df= 21,66; P=0.93. b K=184.02; P=0.00. c F=0.55; df=20,72; P=0.93. d F=0.84; df=20,72; P=0.65. * Measured for less than 5 days. Residual contact assays with Podisus adults The results of our study indicated that azadirachtin and tebufenozide, applied at the maximum field rates, were harmless for this pentatomid, and no mortality was observed after exposure of adults to fresh insecticide residues. Nevertheless, Spinosad was very deleterous for the predator and resulted in a direct mortality in adults, which increased along time: 20% at 24h, 84% at 48h and 100% at 72h (Figure 1).
30
Figure 1. Adult mortality in Podisus maculiventris (%) when young adults were exposed to fresh residues of spinosad, tebufenozide and azadirachtin in glass surfaces for 24-, 48- and 72-h.
Reproductive studies showed that treated insects with both insecticides, had fertility and fecundity values slightly lower than those of the controls. Nevertheless, only a statistically significant reduction of 40.8% in the average number of eggs per female, was recorded for tebufenozide (Table 3). Table 3. Effects of spinosad, tebufenozide and azadirachtin on the reproduction of young adults of Podisus maculiventris, in residual contact treatments.
Podisus maculiventris Adults exposed via residual contact
Insecticides Concentrations mg a.i./l
% Adult mortality, 72 h
Eggs/female 1 month a**
% Eclosion b IOBC class
Control 0 0 359.6± 31.1a 70.3± 6.5a – Spinosad 120 100 - - 4 Tebufenozide 180 0 213.0± 39.9b 68.8± 5.5a 2 Azadirachtin 48 0 283.9± 28.8ab 53.4± 5.7a 1
Within the same column, data followed by the same letter do not differ significantly. (P=0.05; a LSD mean separation; b Bonferroni mean separation). a F=4.09; df=2,18; P=0.03. b F=2.14; df=2,14; P=0.16. ** Data represent the mean of 8 replicates of pairs kept separately. Residual, ingestion and topical application assays with Opius adults The results obtained with adults of the braconid O. concolor when insecticides were applied at the maximum field recommended rates with different techniques (Table 4), showed that spinosad was very harmful to the enemy whereas tebufenozide totally harmless irrespective of the mode of application. However, azadirachtin, when ingested, gave a large reduction
Control Spinosad Azadirachtin Tebufenozide0
20
40
60
80
100
%
24h48h72h
b
b
ADULT MORTALITY
180 mg/l48 mg/l
max field recommended rates
Podisus maculiventris
31
(84.3%) in the longevity of treated wasps, that did not apparently show any change on behaviour. However, this drastic effect might be related to the reported antifeeding effect of this product, because liquids intake is a limiting factor for the survival of this braconid (González & Viñuela, 1997). Table 4. Influence of the mode of application on the susceptibility of Opius concolor young adults to spinosad, tebufenozide and azadirachtin
Opius concolor Adult treatments
RESIDUAL Insecticides Concentrations
mg a.i./ l Longevity,
days a % Attacked
hosts b % Progeny size c IOBC
class Control 0 21.6±0.5a 88.8±6.3a 75.1±5.8a – Spinosad 120 0.2±0.4b – – 4 Tebufenozide 180 22.2±1.1a 88.8±2.0a 71.3±2.9a 1 Azadirachtin 48 21.2±1.8a 86.4±2.8a 43.7±5.0b 2
INGESTION* Insecticides Concentrations
mg a.i./ l Longevity,
days d % Attacked
hosts e % Progeny size f IOBC
class Control 0 31.4±0.7a 87.5±4.8a 65.0±7.9a – Spinosad 120 0.5±0.1b – – 4 Tebufenozide 180 26.3±0.4a 91.3±3.1a 70.0±5.4a 1 Azadirachtin 48 4.9±0.1b 64.1±8.2b 43.9±4.8b 3
TOPICAL APPLICATION Insecticides Doses µg a.i./ g adult
(Concentrations mg a.i./ l)
Longevity, days g
% Attacked hosts h
% Progeny size i IOBC class
Control 0 28.1±0.5a 92.5±2.8a 70.0±6.3a – Spinosad 27.39 (120) 0b – – 4 Tebufenozide 41.10 (180) 27.8±1.0a 88.3±2.2a 73.9±3.4a 1 Azadirachtin 10.96 (48) 28.8±0.4a 96.5±1.7a 71.5±4.7a 1
Within the same column and treatment, data followed by the same letter are not significantly different (P=0.05; a,c,d,e,f LSD mean separation; b,h,i Bonferroni mean separation). a F=90.20; df=3,12; P=0.00. b Data transformed to arcsin√x/100. F= 0.55; df= 2,25; P=0.584. c F= 12.35; df= 2,25; P=0.002. d F=1758.8; df=3,12; P=0.00. e F=6.53; df=9,2; P=0.018. f F=5.03; df=9,2; P=0.034. g F=434.66; df=7,24; P=0.00. h F=0.70; df=2,22; P=0.51. i F=0.16; df=2,22; P=0.85. *Insecticides were supplied in water to adults during the life span.
The beneficial capacity of O.concolor females was only impaired by azadirachtin applied via residual contact or ingestion. Via residual contact a 40.5% reduction in the progeny size was detected, whereas when ingested, the number of attacked hosts and progeny size decreased by 26.7% and 32.5% respectively.
Residual, ingestion and topical application assays with Chrysoperla adults
32
Tebufenozide and azadirachtin applied at the maximum field recommended rates were totally harmless to C. carnea adults, irrespective of the mode of application: residual contact, ingestion via drinking water or topical application (Table 5). Table 5. Influence of the mode of application on the susceptibility of Chrysoperla carnea young adults to spinosad, tebufenozide and azadirachtin.
Chrysoperla carnea Adult treatments
RESIDUAL Insecticides Concentrations
mg a.i./ l % Mortality
at 48 h a Eggs/female,
days b %
Eclosion c IOBC class
Control 0 0a 42.0±1.5a 79.5±3.6a – Spinosad 80 – – – – 96 3.1±3.1a (*) (*) 1 400 87.5±5.1b (*) (*) 3 Tebufenozide 180 0a 38.2±4.3a 80.5±3.8a 1 Azadirachtin 48 0a 39.6±3.1a 78.3±5.4a 1
INGESTION* Insecticides Concentrations
mg a.i./ l % Mortality at 48 hours d
% Mortality at 6 days e
Eggs/female, days f
% Eclosion g
IOBC class
Control 0 0a 0 30.3±3.3a 90.0±3.8a – Spinosad 80 0a 66.0±10.4b (*) (*) 2 96 – – – – – 400 24.9±10.7b 100±0.0c (*) (*) 4 Tebufenozide 180 0a 0a 36.2±3.0a 88.2±2.7a 1 Azadirachtin 48 0a 0a 27.5±3.5a 84.5±5.4a 1
TOPICAL APPLICATION Insecticides Doses µg a.i./ g
adult (Concentrations
mg a.i./l)
% Mortality at 48 hours h
% Mortality at 6 days i
Eggs/female, days j
% Eclosion k
IOBC class
Control 0 0a 0a 24.4±3.5a 84.8±6.8a – Spinosad 5.40 (80) 0a 4.1±4.1a 34.1±2.9a 83.0±4.6a 1 27.02 (400) 4.1±4.1a 4.1±4.1a 37.1±2.2a 78.2±5.5a 1 Tebufenozide 12.16 (180) 0a 0a 28.3±1.4a 90.2±9.8a 1 Azadirachtin 3.24 (48) 0a 0a 30.4±3.6a 84.0±3.6a 1
Within the same column, data followed by the same letter do not differ significantly (P=0.05; a,b,c LSD mean separation. d,e,f,g,h,i,j,k Bonferroni mean separation) a F=210.27; df=4,15; P=0.000. b F=5.39; df=4,15; P=0.007. c F=79.75; df=4,15; P=0.000. d F=0.37; df=2,9; P=0.700. e F=1.80;df=2,9; P=0.221. f F= 0.06; df=2,9; P=0.938. g F=0.46; df=2,9; P=0.648. h F=1.00; df=4,15; P=0.438. i F=2.55; df=4,15; P=0.082. j F=2.59; df=4,13; P=0.086. k F=0.44; df=4,13; P=0.777. *Insecticides were supplied in water to adults during the preoviposition period (4 days). (*) Not measured.
Spinosad was harmful only at very high rates, a 87.5% mortality was recorded 48 h after treatment in residual tests, and a 24.9% mortality in ingestion ones. This naturalyte was fast acting via residual contact, but all predator adults also died in ingestion tests 6 days after
33
treatment. The insecticide had no effect on the enemy when topically applied. Normal reproductive parameters (fecundity and fertility) were recorded in survivors in every case.
Conclusions Azadirachtin was harmless for pupae and adults of C. carnea, pupae of H. didymator, and adults of P. maculiventris. However, adults of the braconid O. concolor were more sensitive, and the insecticide decreased their progeny size applied via residual contact, as well as the longevity, the progeny size and the number of attacked hosts when applied via ingestion. Tebufenozide was a safe insecticide for all the enemies studied, and only a slight decrease in fecundity was observed in P. maculiventris when the product was applied via residual contact at the maximum field recommended rate. Spinosad was very harmful for adults of O. concolor and P. maculiventris, and for pupae of H. didymator, but it was compatible with C. carnea pupae and adults applied at the maximum field rate. The insecticide was only slightly harmful to C. carnea adults applied at high rates via the drinking water. Acknowledgements This work was suported by the Spanish Ministry of Education and Culture (projects AGF98-0715 and AGF99-1135 to E. Viñuela). Mª P. Medina was recipient of a grant from the Autonomous Community of Madrid (Spain) and M. Schenider from CONICET (Argentina). References Albajes, R. & Santiago-Álvarez, C. 1980: Effects of larval density and food in the sex ratio of
Ceratitis capitata. Anales INIA/Serie Agrícola 13: 175-182. (In Spanish). Bahena, F., Budia, F., Adán, A., Del Estal, P. & Viñuela, E. 1999: Scanning electron
microscopy of Hyposoter didymator in host Mythimna umbrigera larvae. Annals ESA 92: 144-152.
Banken, J.A.O. & Stark, J. 1997: Stage and age influence on the susceptibility of Coccinella septempunctata after direct exposure to Neemex, a neem insecticide. Journal of Economic Entomology 90: 1102-1105.
De Clercq, P., Keppens, G., Anthonis, G. & Degheele, D. 1988: Laboratory rearing of the predatory stinkbug Podisus sagitta (F.). Medelingen Faculteit Landbouwkundige en Toegepaste Biologische Wetenschappen,Universiteit Gent 53: 1213-1217.
De Clercq, P., De Cock, A., Tirry, L., Viñuela, E. & Degheele, D. 1995: Toxicity of diflubenzuron and pyriproxyfen to the predatory bug Podisus maculiventris. Entomologia Experimentalis et Applicata 74: 17-22.
González, M. & Viñuela, E. 1997: Effects of two modern pesticides: azadirachtin and tebufenozide on the parasitoid Opius concolor. IOBC/wprs Bulletin 20(8): 233-240.
Greeve, L. 1984: Chrysopid distribution in northern latitudes. In: Biology of Chrysopidae. Canard, M., Séméria, Y. & New, T.R. (eds), Junk Publishers, The Hague: 180-186.
Harrington, S.A.; Hutchinson, P.; Ducth, M.E.; Lawrence P.J. & Michael, P.J. 1993: An efficient method of mass rearing two introduced parasitoids of noctuids. Journal Australian Enomological Society 32: 79-80.
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Jacas, J. & Viñuela, E. 1994. Analysis of a laboratory method to test the effects of pesticides on adult females of Opius concolor, a parasitoid of the olive fruit fly, Bactrocera oleae. Biocontrol Science & Technology 4: 147-154.
Salgado, V.L. 1997. The modes of action of spinosad and other insect control products. Down to Earth 52: 35-43.
Schneider, M., Budia, F., De Remes Lenicov, A. M. M., Gobbi, A. & Viñuela, E. 2000. Topic toxicity of tebufenozide, spinosad and azadirachtin on pupae of the parasitoid Hyposoter didymator. Boletín Sanidad Vegetal. Plagas 26: (in press). (In Spanish).
Schneider, M. & Viñuela, E. 1999. Evaluation of tebufenozide on immature stages of Hyposoter didymator, a parasitoid of noctuid larvae. Medelingen Faculteit Landbouwkundige en Toegepaste Biologische Wetenschappen, Universiteit Gent 64/3a: 287-295.
Smagghe, G., Viñuela, E., Budia, F. & Degheele, D. 1996. In vivo and in vitro effects of the non-steroidal ecdysteroid agonist tebufenozide on cuticle formation in Spodoptera exigua: an ultrastructural approach. Archives of Insect Biochemistry & Physiology 32: 121-134.
Smagghe, G., Viñuela, E., Budia, F. & Degheele, D. 1997. Tissue specific effects of the non-steroidal ecdysteroid mimic tebufenozide in the tomato looper Chrysodeixis chalcites: an ultrastructural analysis. Archives of Insect Biochemistry & Physiology 35: 179-190.
STSC, 1987. Statgraphics user’s guide, version 5.0. Graphic software system, STSC, Rockville, MD.
Thompson, W.T. 1992. A worldwide guide to beneficial animals used for pest control purposes. Thompson Publications. USA.
Viñuela, E., Adán, A., González, M., Budia, F., Smagghe, G. & Del Estal, P. 1998. Spinosad and azadirachtin: effects of two naturally derived pesticides against Podisus maculiventris (Say). Boletín Sanidad Vegetal. Plagas 24: 57-66. (In Spanish).
Viñuela, E., Adán, A., Smagghe, G., González, M., Medina, Mª. P., Budia, F., Vogt, H. & Del Estal, P. 2000. Laboratory effects of ingestion of azadirachtin by two pests (Ceratitis capitata and Spodoptera exigua) and three natural enemies (Chrysoperla carnea, Opius concolor and Podisus maculiventris). Biocontrol Science & Technology10 (2): 175-187.
Vogt, H., Bigler, F., Brown, K., Candolfi, M., Kemmeter, F., Kühner, C., Moll, M., Travis, A., Ufer, A., Viñuela, E., Waldburger, M. & Waltersdorfer, A. 2000: Laboratory method to test effects of plant protection products on larvae of Chrysoperla carnea (Neuroptera: Chrysopidae). In: Guidelines to evaluate side-effects of plant protection products to non-target arthropods. M.P. Candolfi, S. Blümel, R. Forster, F.M. Bakker, C. Grimm, S.A. Hassan, U. Heimbach, M.A. Mead-Briggs, B. Reber, R. Schmuck and H. Vogt (eds.): IOBC/WPRS, Gent, 27-44.
Pesticides and Beneficial Organisms IOBC/wprs Bulletin Vol. 24 (4) 2001
pp. 35 - 38
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Influence of the host density on the reproduction of Aleochara bilineata Gyll. (Coleoptera: Staphylinidae) K.M. Nienstedt & H.F. Galicia Springborn Laboratories (Europe) AG, Seestrasse 21, CH-9326 Horn, Switzerland Swiss Research Centre Abstract: The relation between the host density (Delia antiqua pupae) and the reproduction of Aleochara bilineata Gyll. (Coleoptera: Staphylinidae) was studied following a Moreth and Naton (1992) test design. The optimal host density was determined to be 2250 pupae (i.e. 3 supplies of 750 pupae each at weekly intervals). Lower as well as higher host densities resulted in lower reproduction. Keywords: Aleochara bilineata, functional response, reproduction, parasitation. Introduction Data on potential effects on non-target beneficial arthropods are to be provided for registration of new and existing plant protection products (PPP) in the European Union (European Directive 91/414/EEC). More precise guidance is given by the European Directive 96/12/EC (1996), Barrett et al. (1994) and a recent European Commission Working Document (July, 2000).
The parasitoid Aleochara bilineata Gyll. (Coleoptera: Staphylinidae) belongs to the beneficial arthropods recommended by Barrett et al. (1994). The 1st instar larvae of this species search for host puparia of cyclorrhapheous Diptera, which are found on soil substrates, and penetrate them. A summary of host species of Aleochara species is given in Maus et al (1998). Studies conducted to investigate the potential toxic effects of PPP’s usually follow the recommendations given by Moreth and Naton (1992).
In order to avoid competition between the test organisms, optimal experimental conditions for them should be provided (e.g. optimal environmental conditions, food and parasitation substrate ad libitum). Since previous results obtained in our laboratory indicated that the host density may need further consideration, the present study was carried out in order to determine if the reproduction of Aleochara bilineata in Moreth and Naton (1992) test designs is dependent on the host density provided during the test. Material and methods The test was performed following the method described by Moreth and Naton (1992). Glass vessels (approx. 14 cm diameter, 7.5 cm height) were filled with 800 g of dry, pure quartz sand (0.3 to 0.8 mm) moistened with deionized water (approximately 10% v/v). Ten males and ten females of adult A. bilineata (1 to 7 days old) were introduced into each vessel. Pupae of Delia antiqua (Diptera: Anthomyiidae) were used as host species for parasitation by A. bilineata.
Host pupae density was the variable tested in this experiment. On days 7, 14, and 21 of the experiment, 250, 500, 750, 1000, 1250 or 1500 pupae were added to the different
36
treatments, corresponding to total host densities of 750, 1500, 2250, 3000, 3750 or 4500 pupae, respectively. Three replicates per treatment were performed.
The test units were closed with a plastic lid with appropriate ventilation. During the test, A. bilineata were fed ad libitum with larvae of Chironomidae killed by freezing. The moisture in the sand substrate was held constant by controlling periodically the weights of the test vessels, and, if appropriate, by adding water to reach their initial weights. The test was performed at 20 ± 2°C, 60 to 90% relative humidity and a photoperiod of 16L:8D. Light intensity at the sand surface was 100 to 500 lux.
On day 28, surviving A. bilineata beetles were taken out of the test units. The sand substrate with the host pupae was refilled into the test vessels and was kept there for one week. Thereafter, the host pupae were recovered from the sand through sieving and the number of hatching beetles was quantified periodically (daily during the main hatching period) until no more beetles emerged. Hatched beetles were removed from the test units after each assessment. The total number of hatched A. bilineata was registered for each test vessel. Mean and standard deviations were calculated for each treatment. Results and discussion The mean number of hatched Aleochara bilineata adults varied with the amount of host pupae supplied during the experiment (Figure 1) and the observed pattern resembles that of a functional response. The number of hatched beetles increased with increasing host density until an apparent plateau value was reached at a total host density of 2250 pupae (i.e. 3 times 750 pupae). However, at host densities higher than 3750 (i.e. 3 times 1250 pupae), the total number of hatched beetles decreased (Figure 1). The percent of parasitation showed a slight increase at host densities lower than 2250 pupae and a continuous decrease at host densities higher than 2250 Delia pupae (Figure 2).
Total number of hosts (Delia pupae) provided 0 750 1500 2250 3000 3750 4500
No.
of h
atch
ed a
dult
Ale
ocha
ra
0
100
200
300
400
500
600
700
800
250 500 750 1000 1250 1500Hosts (Delia pupae) provided per week
Figure 1: Relation between the total number of hatched Aleochara bilineata beetles per test vessel (mean ± st.dev.) and the host density.
37
Figure 2. Percent parasitation (mean ± st.dev.) of Delia antiqua pupae by Aleochara bilineata in dependence of the host density.
The decrease in reproduction (number of hatched beetles) at host densities higher than 2250 pupae cannot be explained with this experimental design. It can only be suggested that the substrate conditions might have been influenced by the higher proportion of host pupae in relation to the amount of sand. As consequence, the A. bilineata larvae might have been exposed to different physical and/or biological conditions, which might have reduced their host finding capacity. This hypothesis, however, needs to be investigated.
In spite of this open question, the results clearly show that the host density has an influence on the reproduction of A. bilineata, i.e. on the total number of hatched beetles (offspring generation) recovered. The results indicate that under the conditions of this test the optimal host density is 2250 pupae (3 times 750 pupae).
Although at host densities lower or equal than 2250 pupae the percentage of parasitation did not vary substantially (25 to 30%), the mean total number of hatched A. bilineata adults varied by a factor 3.4 (191.7 to 654.7 hatched beetles). Consequently, since it is known that the survival of parasitoids can be related to the host density (Putman and Wratten, 1984), at host densities lower than 2250 pupae competition for host pupae between A. bilineata larvae cannot be neglected. Additionally, at these host densities potential negative effects of the chemical substances tested might not be detected appropriately. Further experiments would be useful in elucidating the extent and generalisation of these findings.
References Barrett, K.L., Grandy, N., Harrison, E.G., Hassan, S. & Oomen, P. (eds.) 1994: Guidance
document on regulatory testing procedures for pesticides with non-target arthropods. From the Workshop European Standard Characteristics on beneficial Regulatory Testing (ESCORT). Wageningen, 28-30 March. Setac: pp. 51.
European Council Directive 91/414/EEC 1991: Official Journal of the European Communities. No L 65: 26-27.
Total number of hosts (Delia pupae) provided0 750 1500 2250 3000 3750 4500
% P
aras
itatio
n
0
5
10
15
20
25
30
35
40
250 500 750 1000 1250 1500Hosts (Delia pupae) provided per week
38
European Commission Directive 96/12/EC. 1996: Official Journal of the European Communities. No L 65: 20-37.
European Commission. Directorate General for Agriculture. VI B II.1. 2021/VI/98 rev. 7 from 08.07.2000. Working Document. Guidance Document on Terrestrial Ecotoxico-logy. pp 15.
Maus, Ch., Mittmann, B. & Peschke, K. 1998: Host records of parasitoid Aleochara Gravenhorst species (Coleoptera, Staphilinidae) attacking puparia of cyclorrhapheous Diptera. Mitt. Mus. Naturkd. Berl., Dtsch. Entomol. Z. 45 (2): 231-254.
Moreth, L. & Naton, E. 1992: Richtlinie zur Prüfung der Nebenwirkung von Pflanzenschutz-mitteln auf Aleochara bilineata Gyll. (Col, Staphylinidae) (erweiterter Laborversuch). IOBC/WPRS Bulletin 15(3): 82-88.
Putman, R.J & Wratten, S.D. 1984: Principles of Ecology. University of California Press. Berkley and Los Angeles: 219-269.
Pesticides and Beneficial Organisms IOBC/wprs Bulletin Vol. 24 (4) 2001
pp. 39 - 46
39
Effects of Quassia products on predatory mite species Barbara Baier BBA, Institute for Ecotoxicology in Plant Protection, Stahnsdorfer Damm 81, D-14532 Kleinmachnow, Germany Abstract: The effects of three Quassia products on the predatory mite species Typhlodromus pyri, Amblyseius andersoni and Euseius finlandicus were investigated in laboratory. The tested application rates of the products were 1.2 l/ha for Quassin, 80.4 l/ha for Quassia solution and 120 l/ha for Quassetum. All treatments were applied in a spray volume of 200 l/ha. Investigations were conducted with protonymphs and females of all three mite species on hibiscus (Hibiscus rosa-sinensis) leaf disks. While the protonymphs were exposed to dried spray deposits on the leaf disks, the adult females were first placed onto the leaf disks before treatment with the test substances. Test criteria were cumulative mortality including repellent effect (in the tests with protonymphs and adult females) and cumulative mean number of eggs per female to day 14 (only in the tests with protonymphs). In addition, reduced application rates were investigated in the test with adult females for those products found to be harmful to the females. Investigations have shown that the effect of Quassin and Quassia solution on the protonymphs and females as well as the reproduction of A. andersoni was < 30 %. In comparison to A. andersoni the protonymphs and the reproduction of T. pyri showed stronger response to Quassin (61 % and 39 %) and Quassia solution (79 % and 39 %) whereas the effect on the females was < 30 % too. In the tests with protonymphs both products resulted in low mortality and showed signs of being repellent with T. pyri. The protonymphs and females of E. finlandicus responded very strongly to Quassin and Quassia solution (effect = 100 %). A large repellent effect was observed. Reduced application rates of 1.0 l/ha for Quassin and 60.3, 40.2 and 20.1 l/ha for Quassia solution resulted in the same outcome with females of E. finlandicus. A further decrease of the application rate of both test products led to a decrease of the effect with females of this species. The test substance Quassetum very strongly influenced all three predatory mite species. The effect on protonymphs was 95 % for T. pyri, 94 % for A. andersoni and 100 % for E. finlandicus. Strong signs of repellency were observed with E. finlandicus again, but not with T. pyri and A. andersoni in the protonymph test. The effect of Quassetum on the females of all three species was 100 %. Reduced application rates of 100 l and 80 l/ha for T. pyri and A. andersoni and 100 l, 80 l, 60 l and 40 l/ha for E. finlandicus resulted in the same effect. Only a further reduction of the application rate of Quassetum led to a decrease of the effect with the females of all three species. Key words: predatory mites, Typhlodromus pyri, Amblyseius andersoni, Euseius finlandicus, Quassia, side-effects, laboratory experiments Introduction Quassia products, which are based on Quassia amara, can be used as insecticides for agricultural, forestry and horticultural purposes in accordance with the German Plant Protection Act - List of substances and preparations for the preparation of plant protection products for use within one's own undertaking according to article 6a paragraph 4 sentence 1 no. 3b). Substances and preparations which are to be included or retained in that list must not have any negative effects on beneficial organisms (Kühne and Jahn, 1999). Quassia formulations are recommended in organic fruit production to control the apple sawfly, Hoplocampa testudinea (Klug) (Symphyta: Tenthredinidae). As there was insufficient
40
information on the effect of Quassia on predatory mites, laboratory investigations were carried out with the three Quassia products and the predatory mite species Typhlodromus pyri Scheuten, Amblyseius andersoni (Chant) and Euseius finlandcicus (Oudemans). Materials and methods The test products and their characteristics as well as the recommended application rates for orchards and the tested application rates (highest recommended application rate for orchards corrected by factor of 0.4 for foliage dwelling predators according to Barrett et al. (1994)) are shown in Table 1.
In addition, reduced application rates were investigated in the test with adult females for those products found to be harmful (effect > 99 %) to the females. Table 1: Test products and application rates
Product Active agent Extraction Producer
Highest recom-mended
application rate for
orchards
Tested application
rate according to Barrett et al.
(1994) Quassin Quassia extract
430 g/l Alcohol extract
Siegfried Agro AG CH-4800 Zofingen
3.0 l/ha 1.2 l/ha
Quassia solution
Quassia extract of 250 g dry
Quassia wood + 2 l water
(steeped for 24 h and cooked for 0,5 h)
Water extract
According to recipe by Kreuter (1995)
201.0 l/ha 80.4 l/ha
Quassetum Quassia extract 42 g/l
+ potash soap 100 g/l
+ Equisetum arvense + Artemisia vulgaris
Alcohol extract
Grieder Natur-
produkte CH-4497
Rünnenberg
300.0 l/ha 120.0 l/ha
The investigations with the three mite species were conducted on hibiscus leaf disks (Hibiscus rosa-sinensis). First, young protonymphs, less than one day old, were tested. Secondly, females at the stage of oviposition were tested. All mites were cultured in the laboratory prior to testing. T. pyri was reared with pollen of Betula and Pinus spp. (McMurtry and Scriven, 1965; Overmeer, 1981). A. andersoni and E. finlandicus were reared on hibiscus leaves in glass dishes with Tetranychus urticae and Pinus spp. pollen as food (Baier and Schenke, 1997).
41
Testing arenas comprised 5 cm diameter disks cut from hibiscus leaves. Each leaf disk was placed on a round wet piece of filter paper in a petri dish and surrounded by a glue barrier to contain the mites. Treatments were applied with a Potter Tower at a rate of 2 mg of wet deposit per cm2. When the spray film had dried, the leaf disks with the glue barrier were removed from the wet filter paper and placed on a cotton pad in a petri dish filled with water. Protonymphs were exposed to dried spray deposit on the leaf disks, adult females were placed on the leaf disks first and then sprayed with the Quassia substances. In all tests the control was treated with deionised water. Twenty test animals were placed on each leaf disk. Five replicates were included. T. pyri was fed with pollen, and A. andersoni and E. finlandicus were fed with pollen and spider mites, as in the rearing. The petri dishes with the test animals were kept in a controlled environment room (temperature 24 to 25 °C, relative humidity 70 to 88 %, and exposure to light for 16 h). Living and dead mites and those which had escaped and been trapped in the glue barrier were counted 1 day, 3 days and 7 days after application. The dead and glue-trapped test animals were removed. Mortality rates for the seventh day after application in the test substance arenas were corrected for control mortality (Abbott, 1925). In the tests with protonymphs, the cumulative mean number of eggs per female up to day 14 was also determined. The percentage reduction in egg production per female was calculated for the test substances as compared to the control. Results Effect on protonymphs including reproduction The results of the investigations with protonymphs are shown in Table 2 (mortality rates) and Table 3 (influence on reproduction). Quassin and Quassia solution caused a mortality rate of 61 % and 79 % with T. pyri, respectively. The majority of test animals had escaped into the glue barrier with both Quassia products on day 7. This suggests that both these treatments had a repellent effect. A. andersoni was not effected by Quassin. With 4 % mortality, Quassia solution had also only a weak effect on this species. In contrast to Quassin and Quassia solution, Quassetum resulted in 95 % mortality rate of T. pyri and 94 % mortality rate of A. andersoni, respectively. Only one day after treatment more than 70 % of the test animals of both predatory mite species were found dead on the leaf disks. Quassetum showed no repellent effects as Quassin and Quassia solution with the species T. pyri. All three Quassia substances showed a highly repellent effect in the test with protonymphs of E. finlandicus. Three days after application, no living test animals were left in either the Quassin and Quassia solution or Quassetum treatments. The majority of the predatory mites had escaped into the glue barrier. As a result of the high mortality rate of all three Quassia products, a determination of reproduction with the species E. finlandicus was not possible. Quassin and Quassia solution reduced the number of eggs per female of T. pyri by 39 %. An assessment of Quassetum was not possible, because there were results from one T. pyri female only. The surviving female produced 12 eggs showing that reproduction was not completely inhibited by application of Quassetum.
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In comparison to T. pyri the reproduction of A. andersoni was weakly influenced by Quassin and Quassia solution (Table 3). In the Quassetum treatment, only females of A. andersoni survived, so that reproduction was not possible.
Table 2: Mortality including repellent effect of Quassin, Quassia solution and Quassetum on protonymphs of Typhlodromus pyri, Amblyseius andersoni and Euseius finlandicus Test- Treatment Application Number of living (l), dead (d) and Mortality species rate escaped (e) animals 7 days after treatment rate (Ab- in l/ha l d e bott) in % (mean ± Std) (mean ± Std) (mean ± Std) on day 7 T. pyri Control - 16.4 ± 1.5 0.2 ± 0.5 3.4 ± 1.5 – Quassin 1.2 6.4 ± 1.5 0.4 ± 0.6 13.2 ± 1.9 61 Quassia solution 80.4 3.4 ± 1.5 0.6 ± 0.9 16.0 ± 2.0 79 Quassetum 120.0 0.8 ± 1.2 16.8 ± 1.9 2.4 ± 1.1 95 A. ander- Control – 18.2 ± 1.3 0.8 ± 0.8 1.0 ± 0.7 – soni Quassin 1.2 18.2 ± 0.5 0.4 ± 0.6 1.4 ± 0.6 0 Control – 19.2 ± 0.8 0.2 ± 0.5 0.6 ± 0.6 – Quassia solution 80.4 18.4 ± 1.1 0.4 ± 0.6 1.2 ± 0.8 4 Quassetum 120.0 1.2 ± 1.3 16.0 ± 1.9 2.8 ± 2.4 94 E. finlan- Control – 19.2 ± 1.1 0 0.8 ± 1.1 – dicus Quassin 1.2 0 0.2 ± 0.5 19.8 ± 0.5 100 Quassia solution 80.4 0 1.0 ± 1.2 19.0 ± 1.2 100 Quassetum 120.0 0 3.8 ± 2.2 16.2 ± 2.2 100 Table 3: Influence of Quassin, Quassia solution and Quassetum on reproduction of Typhlodromus pyri and Amblyseius andersoni Test- Treatment Application Number of eggs/female Reduction of species rate from day 7 – 14 number of (mean ± Std) eggs/female in % T. pyri Control – 11.84 ± 0.72 – Quassin 1.2 7.28 ± 1.19 39 Quassia solution 80.4 7.17 ± 1.89 39 Quassetum 120.0 12* – A. andersoni Control – 13.58 ± 1.39 – Quassin 1.2 11.98 ± 1.80 12 Control – 15.72 ± 1.92 – Quassia solution 80.4 11.48 ± 1.16 27 Quassetum 120.0 ** –
* result from one female only ** only females survived, so that reproduction was not possible
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Effect on females The results of the tests with females of all three species are shown in Table 4. The effect of Quassin and Quassia solution on the T. pyri females was weaker than the effect on the protonymphs, with a mortality rate of 29 % and 19 %, respectively. Again, more dead females were found in the glue barrier than on the leaf disk. This suggests a slight repellent effect of both treatments. With a mortality rate of 6 % and 8 %, the effect of Quassin and Quassia solution on females of A. andersoni was similarly weak as on the protonymphs of these species. In comparison to the females of T. pyri and A. andersoni the females of E. finlandicus showed a high repellent effect following a treatment by Quassin and Quassia solution. One day after treatment the survivors in these both treatments were observed to have impaired mobility. On day 7 after treatment the majority of E. finlandicus females were found dead in the glue and the rest of females were found dead on the leaves (Table 4). As for protonymphs Quassetum again showed the strongest effect of all three test substances with the females of T. pyri, A. andersoni and E. finlandicus too. One day after treatment, no living animals were left on the leaf disks with the females of all three predatory mite species. The majority of the females were found dead on the leaf disks, while only ≤ 5 % had escaped into the glue barrier. Table 4: Mortality including repellent effect of Quassin, Quassia solution and Quassetum on females of Typhlodromus pyri, Amblyseius andersoni and Euseius finlandicus Test- Treatment Application Number of living (l), dead (d) and Mortality species rate escaped (e) animals 7 days after treatment rate (Ab- in l/ha l d e bott) in % (mean ± Std) (mean ± Std) (mean ± Std) on day 7 T. pyri Control – 16.6 ± 0.6 0.8 ± 0.8 2.6 ± 0.6 – Quassin 1.2 11.8 ± 2.1 1.0 ± 1.4 7.2 ± 2.2 29 Quassia solution 80.4 13.4 ± 2.2 0.8 ± 1.8 5.8 ± 1.8 19 Quassetum 120.0 0 19.0 ± 0.7 1.0 ± 0.7 100 A. ander- Control – 18.6 ± 0.9 0.2 ± 0.5 1.2 ± 1.1 – soni Quassin 1.2 17.4 ± 1.8 0.8 ± 1.3 1.8 ± 1.8 6 Quassia solution 80.4 17.2 ± 1.3 0.4 ± 0.6 2.4 ± 0.9 8 Quassetum 120.0 0 19.2 ± 0.8 0.8 ± 0.8 100 E. finlan- Control – 17.8 ± 1.3 0.2 ± 0.5 2.0 ± 1.2 – dicus Quassin 1.2 0 1.4 ± 1.7 18.6 ± 1.7 100 Quassia solution 80.4 0 1.2 ± 1.6 18.8 ± 1.6 100 Quassetum 120.0 0 19.8 ± 0.5 0.2 ± 0.5 100 A reduction of the application rate of Quassetum to 100 l and 80 l/ha with T. pyri and A. andersoni produced the same results as an application rate of 120 l/ha in the female test (Table 5). The first surviving test animals of these two species were noticed only after the application rate had been lowered to 60 l/ha, yet the mortality rate was still very high with 94 % and 98 %.
44
With females of A. andersoni, a further reduction of the application rate to 50 l, 40 l, 30 l and 20 l/ha showed that rates of 30 l/ha or lower resulted in mortality rates of less than 50 %. In all experiments with reduced application rates of Quassetum and the test species T. pyri and A. andersoni, the number of dead females found on the leaf disks was higher or nearly the same than the number of females which had escaped into the glue. With females of E. finlandicus a reduction of the application rate of Quassetum to 100 l, 80 l, 60 l and 40 l/ha led to the same results as an application rate of 120 l/ha (Table 5). The first surviving females of this species were observed only after the application rate had been lowered to 30 l/ha, but the mortality rate was still very high with 99 %. Only an application rate of 5 l Quassetum/ha resulted in a mortality rate of less than 50 %. In the tests with E. finlandicus females, a decrease of the Quassetum application rate from 100 l to 10 l/ha caused a decrease of the number of the dead animals on the leaf disks and an increase of the number of animals that had escaped into the glue barrier. Signs of repellency with the E. finlandicus females were observed in the four lowest reduced Quassetum application rates. Table 5: Mortality including repellent effect of reduced application rates of Quassetum on females of Typhlodromus pyri, Amblyseius andersoni and Euseius finlandicus Test- Treatment Application Number of living (l), dead (d) and Mortality species rate escaped (e) animals 7 days after treatment rate (Ab- in l/ha l d e bott) in % (mean ± Std) (mean ± Std) (mean ± Std) on day 7 T. pyri Control – 16.2 ± 0.5 0.8 ± 0.5 3.0 ± 0.0 – Quassetum 100 0 19.2 ± 0.8 0.8 ± 0.8 100 Quassetum 80 0 18.8 ± 0.8 1.2 ± 0.8 100 Quassetum 60 1.0 ± 2.2 16.4 ± 2.7 2.6 ± 2.0 94 A. ander- Control – 18.4 ± 1.1 0.2 ± 0.5 1.4 ± 0.9 – soni Quassetum 100 0 18.0 ± 1.2 2.0 ± 1.2 100 Quassetum 80 0 17.6 ± 0.9 2.4 ± 0.9 100 Control – 18.0 ± 1.0 0 2.0 ± 1.0 – Quassetum 60 0.4 ± 0.6 17.0 ± 1.9 2.6 ± 1.8 98 Quassetum 50 3.2 ± 1.9 14.2 ± 2.3 2.6 ± 1.1 82 Quassetum 40 7.4 ± 2.3 9.2 ± 1.8 3.4 ± 2.0 59 Quassetum 30 9.8 ± 3.7 6.8 ± 3.2 3.4 ± 0.9 46 Quassetum 20 16.2 ± 2.2 2.0 ± 1.4 1.8 ± 1.5 10 E. finlan- Control – 17.2 ± 0.5 0.6 ± 0.6 2.2 ± 0.8 – dicus Quassetum 100 0 19.4 ± 0.9 0.6 ± 0.9 100 Quassetum 80 0 19.4 ± 0.9 0.6 ± 0.9 100 Quassetum 60 0 18.8 ± 0.5 1.2 ± 0.5 100 Quassetum 40 0 18.8 ± 0.8 1.2 ± 0.8 100 Control – 18.0 ± 1.6 0.2 ± 0.5 1.8 ± 1.3 – Quassetum 30 0.2 ± 0.5 11.0 ± 1.4 8.8 ± 1.3 99 Quassetum 20 0.2 ± 0.5 7.2 ± 4.2 12.6 ± 3.8 99 Quassetum 10 3.6 ± 2.7 1.0 ± 1.0 15.4 ± 3.2 80 Quassetum 5 10.6 ± 3.6 0.8 ± 1.1 8.6 ± 3.9 41
45
In comparison to Quassetum, all tested reduced application rates of Quassin and Quassia solution produced repellent effects with the females of E. finlandicus (Table 6). The majority of females had escaped into the glue barrier. The reduced application rates of 1.0 l/ha with Quassin and 60.3 l, 40.2 l and 20.1 l/ha with Quassia solution led to the same mortality rate as 1.2 l Quassin/ha and 80,4 l Quassia solution/ha, respectively. A further reduction of the application rate to 0.8 l to 0.1 l/ha with Quassin caused a decrease of the repellent effect with the E. finlandicus females, but a mortality rate of less than 50 % was reached only at 0.1 l Quassin/ha. With Quassia solution an application rate of 5 l/ha resulted still in an effect of more than 50 %. Table 6: Mortality including repellent effect of reduced application rates of Quassin and Quassia solution on females of Euseius finlandicus
Number of living (l), dead (d) and escaped (e) animals 7 days after treatment
l d e Treatment Application
rate in l/ha
(mean ± Std) (mean ± Std) (mean ± Std)
Mortality rate (Abbott)
in % on day 7
Control – 18.0 ± 1.4 0.4 ± 0.6 1.6 ± 1.1 – Quassin 1.0 0 1.4 ± 1.3 18.6 ± 1.3 100 Quassin 0.8 0.2 ± 0.5 0.8 ± 0.8 19.0 ± 1.0 99 Quassin 0.5 0.4 ± 0.6 1.0 ± 2.2 18.6 ± 2.6 98 Control – 17.0 ± 1.2 0.2 ± 0.5 2.8 ± 1.3 – Quassin 0.4 0.4 ± 0.6 1.6 ± 1.1 18.0 ±1.2 98 Quassin 0.3 0.8 ± 0.8 1.6 ±1.1 17.6 ± 1.1 95 Quassin 0.2 3.0 ± 2.5 1.4 ± 1.5 15.6 ± 2.2 82 Quassin 0.1 8.8 ± 2.4 0.6 ± 0.6 10.6 ± 2.1 48 Control – 17.6 ± 1.1 0.2 ± 0.5 2.2 ± 0.8 – Quassia solution 60.3 0 0.4 ± 0.9 19.6 ± 0.9 100 Quassia solution 40.2 0 1.6 ± 1.5 18.4 ± 1.5 100 Quassia solution 20.1 0 0.8 ± 0.8 19.2 ± 0.8 100 Control – 18.0 ± 0.7 0.6 ± 0.6 1.4 ± 0.6 – Quassia solution 10.0 1.0 ± 0.7 1.0 ± 0.0 18.0 ± 0.7 94 Quassia solution 5.0 7.4 ± 2.7 0.4 ± 0.6 12.2 ± 2.6 59 Discussion These results show that the three predatory mite species react very differently to the Quassia products Quassin and Quassia solution. Both test products can be classifed as harmless (effect < 30 %) according to IOBC categories (Hassan, 1992) with regard to protonymphs including reproduction and females of A. andersoni and females of T. pyri, respectively. Protonymphs of T. pyri were more affected by Quassin and Quassia solution than the females. Therefore these two substances are classified as slightly harmful (effect 30 - 79 %) to the juvenile stages of T. pyri. Quassin and Quassia solution led to very high repellent effects with the protonymphs as well as with the females of E. finlandicus. This results in the classification of Quassin and Quassia
46
solution as harmful (effect > 99 %) with regard to all tested developmental stages of E. finlandicus. The test product Quassetum was appreciably more harmful than Quassin and Quassia solution to all three predatory mites species. Quassetum is classified as moderately harmful (effect 80 - 99 %) to protonymphs of A. andersoni and T. pyri and as harmful (effect > 99 %) to females of all three species and protonymphs of E. finlandicus. The higher effect of Quassetum on the females of A. andersoni and T. pyri, which are a less susceptible life stage than the protonymphs of the two species may be attributed to the test method. The protonymphs were exposed to dried spray deposit on the leaf disks, the females were sprayed directly on the leaf disks. The strong effect of Quassetum, specifically in the test with females, is probably due to the 10 % potash soap and not to the Quassia extract. Investigations with Neudosan (active agent: potash soap) showed similar results on Phytoseiulus persimilis when the product was sprayed directly on this predatory mite species (Blümel et al., 1995). Finally it can not be said what the observed high repellency regarding the species E. finlandicus for this predatory mite in the field means. For example, would it be possible that the mites are repelled and seek out untreated parts of the plants as refugia or will they leave the plant altogether and be of no use for pest control? This question can only be answered by investigations in the field. Acknowledgements The author thanks Kevin Brown, Tavistock, United Kingdom, for helpful comments and suggestions on the manuscript. References Abbott, W.S. 1925: A method of computing the effectiveness of an insecticide. J. Econ.
Entomol.18: 265-267. Baier, B. & Schenke, D. 1997: Laboruntersuchungen zu den Auswirkungen von direkt
applizierten Insektiziden auf Weibchen von Euseius finlandicus (Oudemans) sowie Männchen und Weibchen von Typhlodromus pyri Scheuten (Acari: Phytoseiidae). Mitt. Biol. Bundesanst. Land- u. Forstwirtsch. Berlin-Dahlem (333): 36-51.
Barrett, K.L., Grandy, N., Harrison, E.G., Hassan, S.A. & Oomen, P.A. (eds.) 1994: Guidance document on testing procedures for testing pesticides and non-target arthropods. SETAC-Europe, 51 pp.
Blümel, S., Hausdorf, H. & Stolz, M. 1995: Wie wirken biologische Pflanzenschutzmittel auf Raubmilben? Der Pflanzenarzt 7-8: 6-8.
Hassan, S.A. 1992: Meeting of the Working Group ”Pesticides and Benefical Organisms”, University of Southampton, UK, September 1991. IOBC wprs Bulletin 15 (3): 1-3.
Kreuter, M.L. 1995: Pflanzenschutz im Biogarten. BLV, München Wien Zürich, 249 pp. Kühne, S. & Jahn, M. 1999: Liste der Stoffe und Zubereitungen für die Herstellung von
Pflanzenschutzmitteln zur Anwendung im eigenen Betrieb. Nachrichtenbl. Deut. Pflanzenschutzd. 51: 214-215.
McMurtry, J.A. & Scriven, G.T. 1965: Insectary production of phytoseiid mites. J. econ. Ent. 58: 282-284.
Overmeer, W.P.J. 1981: Notes on breeding phytoseiid mites from orchards (Acarina: Phytoseiidae) in laboratory. Meded. Fac. Landbouwwet. Rijksuniv. Gent 46: 503-509.
Pesticides and Beneficial Organisms IOBC/wprs Bulletin Vol. 24 (4) 2001
pp. 47 - 52
47
Effects of Quassia products on Chrysoperla carnea (Stephens) (Neuroptera, Chrysopidae) Vogt, Heidrun Federal Biological Research Centre for Agriculture and Forestry (BBA), Institute for Plant Protection in Fruit Crops, Schwabenheimerstr. 101, D-69221, Dossenheim, Germany Abstract: Quassia preparations have insecticidal activies and are used in organic fruit growing to control the apple sawfly, Hoplocampa testudinea (Klug) (Symphyta: Tenthredinidae). In laboratory tests the effects of three Quassia preparations on Chrysoperla carnea larvae and adults were tested by residual contact and by direct spraying. The tested application rates of the products were 0.4, 0.8 and 1.2 l/ha for Quassin, 26.8, 53.6 and 80.4 l/ha for Quassia solution and 40, 80 and 120 l/ha for Quassetum. Residual toxicity was evaluated by exposure of larvae and old adults on fresh, dry residue on glass. Larvae were held individually in arenas, the adults in groups of 10 in small, ventilated cages. For direct spraying larvae and adults, after anaesthetization with CO2, were sprayed with a Potter Tower. Controls were treated with water, the toxic reference was Perfekthion (EC, a.i. 400 g/l dimethoate). Preadult mortality and reproduction of surviving insects were recorded in the larval tests, mortality within 7 days in the adult tests. All preparations were tested with larvae, whereas only Quassin was tested with adults. The Quassia preparations were harmless to C. carnea by residual contact as well as by direct spraying. Preadult mortalites (Abbott corrected) were only slightly increased in the larval tests (3.3 - 33.1 %) and there were no effects on reproduction. In the adult tests with Quassin no mortality occurred. Keywords: Quassia, side-effects, Chrysoperla carnea, laboratory tests, residual uptake, direct spraying Introduction Quassia products, which are based on Quassia amara, can be used as insecticides for agricultural, forestry and horticultural purposes in accordance with the German Plant Protection Act - List of substances and preparations for the preparation of plant protection products for use within one's own undertaking (Pflanzenschutzgesetz, § 6a, paragraph 4, sentence 1 letter 3b; Anonymous 1998). Substances and preparations which are included in that list must not have any negative effects on beneficial organisms (Kühne & Jahn, 1999).
Quassia formulations are applied in organic fruit production to control the apple sawfly, Hoplocampa testudinea (Klug) (Symphyta: Tenthredinidae). As there was insufficient information on the effect of Quassia on beneficial arthropods, laboratory investigations were carried out with three Quassia products and the green lacewing, Chrysoperla carnea (Stephens) (Neuroptera, Chrysopidae). Routes of exposure for both, larvae and adults, were residual uptake and direct spraying. Material and methods The following Quassia preparations were used:
Quassia solution: Water extract according to Kreuter (1995), made from 250 g dry Quassia amara wood in 2 l water (steeped for 24 h, coked for 0.5 h). Recommended concentration for application: 13.42 % .
48
Quassin: Alcoholic extract of Quassia amara. Content: 30 % Quassia extract (430 g/l ). Recommended concentration for application: 0.2 %
Quassetum: Contains alcoholic extracts of Quassia amara, Equisetum arvense and Artemisia vulgaris plus potash soap. Content: 42 g/l Quassia extract und 100 g/l potash soap. Recommended concentration for application: 20 %.
The tested application rates were calculated for basic water amounts of 500, 1000 und 1500 l/ha and with a correction factor of 0.4 for 3dimensional crops to obtain the Predicted Initial Environmental Concentration (PIEC) (Barrett et al. 1994). The application rates are indicated in Table 1. Table 1. Application rates
Water amount per ha 500 1000 1500 Quassia solution (l/ha) 26.8 53.6 80.4 Quassin (l/ha) 0.4 0.8 1.2 Quassetum (l/ha) 40.0 80.0 120.0
Residual toxicity was evaluated by exposure of larvae (L1 at start of the test) and adults (2-3 days old at start of the test), respectively, on fresh, dry residue on glass. Larvae were held individually in arenas (6.3 cm in diameter), positioned on glass plates of 44 x 51 cm. 30 larvae were exposed per treatment. The larval test was conducted according to Vogt et al. (2000).
The adults were held in groups of 10 in ventilated cages. The cages were constructed similar to the model used in parasitoid tests by Jacas & Viñuela (1994). They consist of an acrylic glass ring (10 cm in diameter, 3 cm high) holding apart two square glass plates (12 x 12 cm), with their their treated surfaces to the inner of the cage. The ring and the glass plates are held together with elastic bands. The acrylic glass ring has 8 holes (0,8 cm in diameter): 5 covered by mesh for ventilation, one connected to a rubber bulb filled with drinking water, one used for insect manipulation and closed by a cork stopper and the last one, holding a hypodermic needle connected to a rubber providing a continous flow of air by an aquarium pump. Four replicates per treatment as well as for the control were used, i.e. 40 adults per treatment (20 females, 20 males).
All studies included a water control. In the adult tests Perfekthion (EC, a.i. 400 g/l dimethoate) was used as toxic reference at a rate of 20 ml/ha. Treatments were applied using a laboratory sprayer (Potter Tower, Burkhard, U.K.) with the adult residual test and with direct spraying of larvae and adults. Glass plates for the larval residual test were sprayed with a glass micro atomizer (Desaga) resulting in a deposit of 1 mg/cm2. The Potter Tower was calibrated to deliver a deposit of 1.5 mg/cm2 (direct spraying of larvae) and 2 mg/cm2 (adult tests), respectively. For direct spraying larvae and adults were anaesthetized with CO2. The insecticide solutions were prepared in the concentration needed to result in the rates given in Table 1.
Preadult mortality and reproduction of surviving insects were recorded in the larval tests. For assessment of fecundity and fertility 4 egg samples were taken within 2 weeks, each covering an egglaying period of 24 hours. In the adult tests mortality was recorded within 7 days. All preparations were tested on larvae, whereas only Quassin was tested on adults. Due
49
to technical reasons it was not possible to test the highest rate of Quassetum (PIEC 1500) in the larval residual test.
The significance of differences in mortality between the control and treated groups was analysed with a Fisher’s Exact Test under SAS, version 6.12. Results Residual toxicity – larvae The Quassia products resulted in mortalities (corrected according to Abbott, 1925) between 3.3 and 23.3 % (Figure 1). In the control no mortality occurred and 30 healthy adults hatched. In the different Quassia treatments the maximum number of dead individuals was 1 larva and 5 pupae; 1 adult had deformed wings. The number of healthy adults was between 23 and 29. Dose dependent effects were observed with Quassia solution and Quassetum. Significant differences between Quassia products and the control were found for Quassia solution and Quassetum at the highest tested rates (Fisher‘s Exact Test, p<0.05).
Figure 1: Mortality (Abbott) of Chrysoperla carnea after exposure of larvae (L1, n=30) on dry residues of Quassia products on glass (Control mortality: 0 %).
Concerning reproduction, the mean number of eggs per female per day in the different treatments ranged from 33 to 39 and the hatching rate of the eggs was above 86 % (Table 2). These values exceeded by far the threshold values indicating a normal reproductive performance. Based on a historical data base for C. carnea these threshold values have been agreed on a mean number of at least 15 eggs per female per day and a mean hatching rate of at least 70 % (Vogt et al. 2000). Thus, no sublethal effects on reproduction were observed after exposure of C. carnea to Quassia residues. Direct spraying – larvae With the exception of one treatment (Quassia solution, 26,8 l/ha) mortalities were very low and did not exceed 15 % (Abbott corrected) (Fig. 2). After direct spraying with Quassia solution at the rate of 26.8 l/ha a mortality (Abbott) of 33.1 % was recorded. This mortality differed significantly from the control (Fisher’s Exact Test, p< 0.05). However, with higher rates of the same Quassia product mortalities were much lower, i.e. below 15 %. In all treatments, mortalities mainly occurred in the pupal stage. This was also the case in the control, where 3 insects died as pupae and thus control mortality amounted to 10 %. In all treatments, reproduction was not affected (Table 3).
0
20
40
60
80
100
26,8 53,6 80,4 0,8 1,2 40 80
Mor
talit
y (A
bbot
t) %
Quassia solution Quassin Quassetum
l/ha
Mor
talit
y (A
bbot
t) %
50
Table 2: Reproduction of C. carnea after exposure on Quassia residues during larval development
Treatment Application rate in l/ha
No. of {{ }}
Fecundity a) Fertility % b)
Control - 17 11 33.3 ± 3.4 89.4 ± 5.1 Quassia 26.8 18 11 35.6 ± 1.3 88.4 ± 4.3 solution 53.6 15 13 36.1 ± 3.9 89.7 ± 2.9 80.4 16 9 32.5 ± 3.2 93.6 ±1.6 Quassin 0.8 15 11 36.8 ± 2.6 89.9 ± 3.6 1.2 12 14 34.7 ± 3.4 89.0 ± 1.7 Quassetum 40.0 13 12 39.0 ± 5.2 86.1 ± 4.4 80.0 15 8 36.8 ± 1.0 93.8 ± 2.7
a) mean no. of eggs eggs per female per day b) mean hatching rate of the eggs
Figure 2. Mortality of C. carnea after direct spraying of larvae (L1, n=30) (Control mortality: 10 %). Residual toxicity of Quassin to adults Like in the control no mortalities occurred in the Quassin treatments (0.4, 0.8 and 1.2 l/ha) within 7 days of exposure. With Perfekthion 97.5 % of the adults died. Direct spraying – adults All adults survived after being directly sprayed with Quassin at rates of 0.4, 0.8 and 1.2 l/ha. Also in the control no mortality occcured. It was remarkable, that also with Perfekthion all adults survived this type of exposure.
0
20
40
60
80
100
26,8 53,6 80,4 0,4 0,8 1,2 40 80 120
Mor
talit
y (A
bbot
t) %
Quassia solution Quassin Quassetum
l/ha
51
Table 3: Reproduction of C. carnea after direct spraying of the larvae with Quassia-products
Treatment Application rate in l/ha
No. of {{ }}
Fecundity a) Fertility % b)
Control – 7 19 33.3 ± 5.2 92.6 ± 1.6 Quassia 26.8 9 9 37.1 ± 3.8 91.0 ± 1.1 solution 53.6 12 14 31.7 ± 2.9 92.1 ± 2.8 80.4 10 13 32.7 ± 3.0 93.8 ± 2.0 Quassin 0.4 11 11 32.8 ± 4.6 89.3 ± 1.3 0.8 13 13 32.8 ± 2.3 92.2 ± 1.4 1.2 11 16 26.0 ± 2.2 91.2 ± 3.2 Quassetum 40.0 13 14 32.7 ± 2.7 94.6 ± 0.6 80.0 14 11 34.4 ± 4.0 90.6 ± 0.8 120.0 13 13 32.7 ± 1.7 91.9 ± 2.0
a) mean no. of eggs eggs per female per day b) mean hatching rate of the eggs Discussion and conclusions All Quassia products were of low toxicity to C. carnea exposed by residual uptake or by direct spraying. Mortalities in the larval tests were below 30 %, with the exception of the treatment with Quassia solution at the lowest rate and direct spraying of the larvae. This higher mortality however seems to be more of coincidental nature as higher rates of the same product resulted in lower mortalites. No sublethal effects with regard to reproduction were observed. Fecundity and fertility of the adults were excellent for all treatments. A question mark remains for the highest rate of Quassetum in the residual test (120 l/ha or 1,2 mg/cm2). The technical equipment did not allow to apply such a high rate: the maximum deposit, which can be achieved with the glass micro atomizer, and which fulfils the criteria of small droplets and even distribution, is limited to 1 mg/cm2. As a dose-dependent effect was observed with Quassetum, it cannot be excluded that this highest rate again would have resulted in a further increase of the mortality. In the tests with adult lacewings with Quassin no mortalities at all occurred.
According to the IOBC/WPRS working group „Pesticides and beneficial organisms“ (Hassan 1994) the tested Quassia products and rates can be classified as harmless for C. carnea (effect 0 - 30%).
A remarkable result was obtained in the adult tests with Perfekthion: wheras 97.5 % of the adults died with residual uptake, there was no mortality at all after direct spraying. The continous uptake of the insecticide by tarsal contact with the residue was obviously much more severe than direct contamination of the adults by small droplets, especially as the soft abdomen is well protected by the wings. Although the adults showed body-cleaning activities after waking up from the anaesthization, there was insufficient uptake of the insecticide to result in any mortality. Pesticide uptake by residual contact as one of the most important routes of exposure is also stated by Croft (1990).
52
References Abbott, W.S. 1925: A method of computing the effectiveness of an insecticide. J. Econ.
Entomol. 18: 265-267. Anonymous 1998: Gesetz zum Schutz der Kulturpflanzen (Pflanzenschutzgesetz – PflSchG)
in der Neufassung vom 14. Mai 1998. Bundesgesetzblatt Teil I: 971 ff, 1527ff, 3512 ff). Barrett, K.L., Grandy, N., Harrison, E.G., Hassan, S.A. & Oomen, P.A. (eds.) 1994: Guidance
document on testing procedures for testing pesticides and non-target arthropods. Society of environmental toxicology and chemistry. SETAC - Europe, 51 pp.
Croft, B.A. (ed.) 1990: Arthropod biological control agents and pesticides. John Wiley & Sons, New York, 723 pp.
Kühne, S. & Jahn, M. 1999: Liste der Stoffe und Zubereitungen für die Herstellung von Pflanzenschutzmitteln zur Anwendung im eigenen Betrieb. Nachrichtenbl. Deut. Pflanzenschutzd. 51: 214-215.
Hassan, S.A. 1994: Activities of the IOBC/WPRS Working group "Pesticides and Beneficial Organisms". IOBC/WPRS Bulletin 17(10): 1-5.
Jacas, J.A. & Viñuela, E. 1994: Analysis of a laboratory method to test the effect of pesticides on adult females of Opius concolor (Hym. Braconidae), a parasitoid of the olive fruit fly, Bactrocera oleae (Dip., Tephritidae). Biocontrol Science and Technology 4: 147-154.
Kreuter, M.L. 1995: Pflanzenschutz im Bio-Garten. BLV, München Wien Zürich, 249 S. SAS Institute Inc. 1989: SAS/STAT User’s Guide, Version 6, Fourth Edition, Volume I. Vogt, H., Bigler, F., Brown, K., Candolfi, M.P., Kemmeter, F., Kühner, Ch., Moll, M., Travis,
A., Ufer ,A., Viñuela, E., Waldburger, M. & Waltersdorfer, A. 2000: Laboratory method to test effects of plant protection products on larvae of Chrysoperla carnea (Neuroptera: Chrysopidae). In: M.P. Candolfi, S. Blümel, R. Forster, F.M. Bakker, C. Grimm, S.A. Hassan, U. Heimbach, M.A. Mead-Briggs, B. Reber, R. Schmuck & H. Vogt (eds.): Guidelines to evaluate side-effects of plant protection products to non-target arthropods. IOBC/WPRS, Gent: 27-44.
Pesticides and Beneficial Organisms IOBC/wprs Bulletin Vol. 24 (4) 2001
pp. 53 - 60
53
Extended laboratory methods to determine effects of plant protection products on two strains of Amblyseius andersoni Chant and their resistance level Gino Angeli, Diego Forti & Sergio Finato Istituto Agrario San Michele a/Adige, 38010 S.Michele a/A Trento, Italy Abstract: Extended laboratory trials were carried out in order to test the effects of four dithiocarbamate on eggs, protonymphs and adult females of two strains of Amblyseius andersoni. One strain (S) was collected in an apple orchard and reared for four years in laboratory. The second strain (R) was collected in the same orchard after treatments with dithiocarbamates for four years. The results of short-term effects showed that only the S strain was affected by the dithiocarbamates mancozeb, metiram and propineb. Ziram had also a slight effect on the S strain. On the long-term, the results showed that only mancozeb affected the fertility of the S strain whereas none of the four fungicides tested affected the fertility of the R strain. A resistance factor (LC50 R strain: LC50 S strain) of six for mancozeb was estimated. Mancozeb resistance was an inheritable trait primarily associated with the adult female.
Key words: Amblyseius andersoni, dithiocarbamate, susceptibility, resistance level, fungicide, phytoseiidae. Introduction Several field and laboratory experiments have shown that the use of certain dithiocarbamates may result in an increase in phytophagous mites (Ivancich Gambaro, 1972; Mori, 1985). In 1958, Mathys demonstrated for the first time that some dithiocarbamates were toxic to Typhlodromus species in the laboratory. Baynon and Penman (1987) conducted laboratory experiments on Typhlodromus pyri in wich metiram and mancozeb were observed to reduce egg hatching. The same authors reported a low mortality rate for adult females and nymphs directly exposed to the two products. Ioriatti et al. (1992) working with A. andersoni have shown the negative effect of mancozeb to be related to a reduction in female fecundity. Other field studies suggest that A. andersoni has developed resistance to mancozeb (Strapazzon & Rensi, 1989; Girolami et al., 1992; Mattioda et al., 1999).
In this report, we describe the susceptibility of a dithiocarbamate-susceptible and a dithiocarbamate-resistant strain of Amblyseius andersoni Chant in laboratory. The susceptible strain (S) was collected in an orchard in S. Michele (Tn-Italy) and reared for four years in the laboratory. The resistant strain (R) was collected in the same orchard after treatments with dithiocarbamates for four years. The experiment was carried out by comparing three bidithiocarbamates, mancozeb, metiram and propineb, generally considered to be harmful to A. andersoni (Ivancich Gambaro, 1972; Mori, 1985; Ivancich Gambaro, 1991; Ioriatti et al., 1992; Angeli et al., 1996), with one reference product, ziram, assumed to be harmless (Ivancich Gambaro, 1988).
The purpose was also to determine which parameters had changed in the R strain and whether these parameters varied in the same manner for the three bidithiocarbamates. A study of the susceptibility of the offspring (F1), obtained by crossbreeding the two strains, was also conducted in order to determine if resistance was heritable trait.
54
Materials and methods Insect rearing The two A. andersoni strains, S and R, used in the laboratory trials were reared in a climatic chamber at 25 ± 1°C, 75 ± 10 % relative humidity and a photoperiod of 17L:7D. Mites were reared on black plastic tiles (90 x 90 mm), bordered by strips of filter paper soaked in water (Overmer, 1985). Cohorts of 20 to 25 eggs in a single mass were placed on each arena. After hatching mites were fed with pollen of Quercus ilex L. (Fagaceae) and eggs of Tetranychus urticae C.L. Koch (Acari: Tetranychidae). Test units were maintained in a climatic chamber at 25 ± 0.5°C, 75 ±10 %RH and a photoperiod of 17L:7D.
Pesticides Commercial preparations of ziram (Pomarsol, 90%WP; 200 g l-1), mancozeb (M70, 80% WP; 200 g l-1), metiram (Polyram c. 80% WG; 200 g l-1) and propineb (Antracol 70% WP; 200 g l-1) were used. Formulated fungicides were dissolved in distilled water to produce concentrations equivalent to field applications rates of 15 hl ha-1.
Direct toxicity on eggs Series of 40 A. andersoni eggs, previously placed on leaf disks of apple (Golden delicious), were treated under a Potter spray tower (Burgerjon, 1956) on the first (i.e. 0-24 h old) and second day (i.e. 24-48 h old) after oviposition. Hatching rate was evaluated two and three days after treatment, respectively. Eggs were treated with one of the four selected dithiocarbamates or with distilled water as control. Four replicates were considered for each test.
Direct toxicity on protonymphs Pairs of 4-5 day old coeval protonymphs were deposited on leaf disks of apple tree. Series of 20 individuals were treated under a Potter spray tower for 1 minute with 8 ml of the water solution of each chemical tested. The amount of solution deposited on each disk was 1.7-1.8 mg cm-2, as recommended by the IOBC guideline (Hassan, 1985). Four replicates, each consisting of 20 predators on ten leaf disks, were used for each fungicide treatment, including a control treated with distilled water. Treated disks were allowed to dry before Q. ilex pollen and T. urticae eggs were placed on each leaf disk.
Post spray evaluations were made 15 minutes, 1, 4 and 7 days after the treatment by observing nymphal mortality. The effect of the fungicides was expressed as: E= 100% - (100% - M), where E was the percentage of toxicity and M was the percentage of mortality calculated according to Abbott (1925).
Direct toxicity on adult females Pairs of 10-12 day old coeval females were deposited on leaf disks of apple tree. Series of 20 females were treated under a Potter spray tower for 1 minute with 8 ml of the water solution of each chemical tested. The amount of fluid deposited on each disk was 1.7-1.8 mg cm2. Three replicates were considered for each fungicide tested including a distilled water test as a control. Post spray evaluations were made 15 minutes, 1, 4, 7 and 10 days after treatment by observing female mortality and fertility, as well as egg fertility. The effect of the fungicides was expressed as: E= 100% - (100% - M) x R1 x R2, where E was the percentage of toxicity, M was the percentage of mortality calculated according to Abbott, R1 was the fecundity (ratio of the number of eggs deposited in the treated sample and in the control) and R2 was the percentage of hatched eggs.
55
Long term effects The long term effects of the selected fungicides on the offspring obtained from females exposed to direct toxicity test was evaluated. Adult females offspring were obtained from youngs born from females exposed to direct toxicity. Youngs offspring, at age of 3-5 days, were placed and reared on glass plates treated with dithiocarbamates the same day of the short term effect trial. The gravid 10-12 day old females offspring obtained were placed in pairs on ten leaf disks of apple tree and the eggs deposited counted and removed each day for seven consecutive days. The experiment was replicated three times for each fungicide as well as for the water treated control.
Resistance level of the two strains S and R Females (10-12 day old) of each strain were treated under the Potter spray tower. Five concentrations of mancozeb were used. Ten leaf disks (with 2 females each) were treated with each concentration of mancozeb and each dose was replicated 3 times. Mortality was assessed 7 days after the treatment. The LC50 was estimated for each strain using the Probit analysis (Finney 1971).
Susceptibility level of the offspring F1, SxR and RxS The S and R strains were crossed in order to obtain both the S x R hybrid (i.e., with S mothers) and the R x S hybrid (i.e., with R mothers). Cross breeding was made by bringing together the corresponding R or S males and females on rearing arenas at the nymphal stage. Hybrid eggs were regularly collected and placed on separated rearing arenas. Hybrid females obtained were subsequently transferred onto leaf disks of apple tree and treated with mancozeb at the field dose (200 g l-1). Three series of 20 females each were treated under a Potter spray tower, including a water treated control. The effect of mancozeb was quantified following the same procedure used to determine short term effects and expressed as effect E.
Statistical analysis Results were subjected to one-way analysis of variance (ANOVA), and Duncan’s multiple range test was used for mean separation (SAS Institute, 1985). Results Direct toxicity on eggs The hatch rate of eggs of both S and R strains, was significantly reduced (P<0.01) when treated with mancozeb and metiram during the first 24 h after deposition (Table 1). When eggs were 24-48 h old, only mancozeb significantly affected the hatch rate (Table 1) (P<0.05, for S strain and P<0.01, for R strain;). Ziram and propineb showed no effect on the egg hatching.
Direct toxicity on protonymphs The effect (E) on both S and R strains was close to 100 % for mancozeb (100 % - S; 100 % - R), metiram (100 % - S; 94.4 % - R) and propineb (100 % - S; 100 % - R) and was much lower for ziram (74.0 % - S; 53.8 % - R). Death occurred during the protonymphal and deutonymphal stages.
Direct toxicity on adult females The effect (E) on both the S and R strains was maximal for mancozeb (71.7 % - S; 25.8 % - R), metiram (56.8 % - S; 19.4 % - R) and propineb (43.3 % - S; 27.3 % - R) and significantly lower (P<0.01 - S; P<0.05 - R) for ziram (1.9 % - S; 4.5 % - R) (Table 2 and 3).
56
Table 1. Amblyseius andersoni egg hatching rate (%) when treated with different dithiocarbamates one and two days after oviposition.
S strain R strain Fungicide 0-24 h 24-48 h 0-24 h 24-48 h Control Ziram Mancozeb Metiram Propineb d.f. Pr>f
95.10 A 90.65 A 15.53 B 34.10 B 90.90 A
4 0.0015
95.45 a 93.30 a 46.35 b 81.65 a 84.15 a
4 0.0423
97.90 A 96.65 A 23.75 B 22.83 B 95.00 A
4 0.0030
96.15 A 96.15 A 42.23 B 76.07 A 91.47 A
4 0.0027
Mean values in a column followed by the same letter are not significantly different (upper case, P<0.01; lower case, P<0.05). Table 2. Toxicity (E) of five fungicides based on A. andersoni mortality (M), fecundity (R1) and unhatched eggs (R2) of the S strain.
Fungicide % female mortality
(M)
Female fecundity
(R1)
% unhatched eggs (R2)
% toxicity (E)
Ziram Mancozeb Metiram Propineb d.f. Pr>f
1.76 b 35.22 a 34.05 a 29.89 a
3 0.0170
1.00 A 0.49 D 0.71 C 0.81 B
3 0.0001
0.14 B 10.67 A 7.64 A 0.07 B
3 0.0001
1.9 C 71.7 A
56.8 AB 43.3 B
3 0.0001
Within each column, values followed by the same letter are not significantly different, ANOVA (upper case letters, P<0.01; lower case letters, P<0.05). Table 3. Toxicity (E) of five fungicides based on A.andersoni mortality (M), fertility (R1) and unhatched eggs (R2) of the R strain.
Fungicide % females mortality
(M)
Females fertility
(R1)
% unhatched eggs (R2)
% toxicity (E)
Ziram Mancozeb Metiram Propineb d.f. Pr>f
0.51 -1.96 1.09 -0.63
3 0.8900
0.96 A 0.77 BC
0.84 B 0.74 C
3 0.0050
0 3.59 3.01 1.75
3 0.1400
4.5 c 25.8 ab 19.4 bc 27.3 a
3 0.0154
Within each column, values followed by the same letter are not significantly different, ANOVA (upper case letters, P<0.01; lower case letters, P<0.05).
57
For the S strain, these results were mainly due to female mortality (ranging from 30 to 35 %) and to a reduction of the fecundity of the survivors, which was 51 % for mancozeb, 29 % for metiram and 19 % for propineb. The effects on egg hatch were 10.67 % for mancozeb, 7.64 % for metiram and were completely negligible for propineb.
The toxicity on the R strain was primarily due to reduced female fecundity which was 23 % for mancozeb, 16 % for metiram and 26 % for propineb. Fungicides had no significant effect on both survival and fertility of the R-strain females.
Long term effects on the fertility of phytoseids offspring Only mancozeb affected the fecundity of the females belonging to the S strain (P<0.01) (Table 4). None of the four dithiocarbamates fungicides tested affected the fecundity of the R strain offspring, not even for mancozeb. Table 4. Average daily egg production of the S strain of A. andersoni during the first seven days after treatment with fungicide.
Fungicide
1st day
2nd day
3rd day
4th day
5th day
6th day
7th day
Control Ziram Mancozeb Metiram Propineb d.f. Pr>f
2.71 AB 2.86 A 0.23 D 1.98 C 2.18 BC 4 0.001
2.45 A 2.48 A 0.13 B 2.07 A 2.28 A 4 0.0001
2.67 A 2.66 A 0.28 B 2.43 A 2.54 A 4 0.0008
2.60 A 2.60 A 0.78 B 2.23 A 3.05 A 4 0.0098
2.56 A 2.59 A 1.40 B 2.33 A 2.66 A 4 0.0009
2.47 A 2.45 A 1.20 B 2.25 A 2.39 A 4 0.0002
2.49 A 2.15 A 0.80 B 2.20 A 1.88 A 4 0.0001
Within each column, values followed by the same letter are not significantly different, ANOVA (upper case letters, P<0.01). Resistance level of the two strains The LC50 of mancozeb for the S strain was 296 g l-1 (95 % confidence interval, 169 - 496) whereas the LC50 on the R strain was 1809 g l-1 (95 % confidence interval, 999 - 4625), resulting in a resistance factor of R strain of 6.13 (Table 5). Table 5. Slope of the linear relationship between mortality and log-dose and LC50 of mancozeb for the R and S strain of the A. andersoni.
Strain Slope (Standard
Error)
LC50 (Confidence
Interval)
Resistance Factor
LC50 R/LC50 S S 1.487
(0.484) 296
(159 - 496) –
R 0.370 (0.553)
1809 (999 - 4625)
6.13
58
Level of susceptibility of the offspring F1 The toxicity (E) of mancozeb on both R x S and S x R hybrids was in between values obtained for the S and R strains (Table 6). The effect on R x S hybrid (44.7 %) was significantly lower (P<0.01) than the effect on the S x R hybrid (61.7 %). This difference was due to the female mortality rates (18.67 % for the R x S strain and 41.69 % for the S x R strain). Non significant differences were observed for the other parameters determining E (Table 6). Table 6. Mancozeb toxicity (E) for the R x S and S x R hybrids of A. andersoni based on mortality (M), fertility (R1) and unhatched eggs (R2).
Hybrids % females mortality
(M)
Females fertility
(R1)
% unhatched eggs (R2)
% toxicity (E)
R x S S x R d.f. Pr>f
18.67 b 41.69 a
1 0.0224
0.72 0.73
1 0.9025
5.60 9.96
1 0.0775
44.7 B 61.7 A
1 0.0058
Values followed by the same letter are not significantly different; ANOVA (upper case letters, P<0.01; lower case, P<0.05). Discussion Mancozeb and metiram affected the hatching of 1-day old eggs in both R and S strains, whereas only mancozeb appeared highly toxic for 2-day old eggs.
The short term effects of mancozeb, metiram and propineb on both protonymphs and adult females were stronger for the S strain than for the R strain. Treated protonymphs suffered higher mortalities during the protonymphal and deutonymphal stages than control ones. On adults, the effect was due to both direct mortality and reduced fecundity, especially for the S strain. Ziram was completely selective for adult females and had a little effect on protonymphs of both R and S strains.
Mancozeb was the only product exhibiting a marked long term effect on the S strain It consistently reduced female fecundity. This effect could not be observed on the R strain. No long term toxic effects were reported for the other fungicides for both the R and S strains. The hypothesis that S and R were two strains with different susceptibility to dithiocarbamates and particularly to mancozeb, was confirmed by a comparison of the LC50 values. The R strain resulted in a resistance factor of 6.13. This feature was inherited and both the S x R and the R x S hybrids exhibited an intermediate susceptibility between those of the two parent strains. This confirmed the role of genetic factors in determining resistance. Moreover, the level of resistance differed depending on the sex of the resistant parent. This may be explained by the haplo-diploid sex determination system of phytoseiids (Schulten, 1985). It therefore appeared that the male genotype played a minor role in the inheritance of resistance, because the following pattern of toxicity of mancozeb was observed: R < R x S < S ~ S x R.
59
Conclusions There are numerous reports of phytoseid resistance to phosphoric esters, carbamates and even pyrethroids (Hoyt, 1972; Hoy, 1981; Overmeer & Van Zon, 1983; Ivancich Gambaro, 1985; Van de Baan et al., 1985). The present study has demonstrated the existence of dithiocarbamate resistance in A. andersoni. Dithiocarbamates are widely used in apple against scab caused by Spilocea pomi Fr. References Abbott, W. 1925: A method of computing the effectiveness of an insecticides. J. Econ. Entomol.
18: 265-267. Angeli, G., Forti, D., & Maines, R. 1996. Toxicity of a number of pesticides on mortality and
reproduction of the predatory mite Amblyseius andersoni Chant (Acarina: Phytoseiidae). Proc., New Studies in Ecotoxicology, Cardiff: 1-4.
Baynon, G.T. & Penman, D.R. 1987: The effects of pesticides and other spraying material on the predacious mite Typhlodromus pyri. Proc. New Zealand Weed and Pest Control Conference, 1: 104-107.
Burgerjon, A. 1956: Pulverisation et poudrage au laboratoire par des prèparations pathogènes insecticides. Ann. Epiphyt. 4: 675-684.
Finney, D.J. 1971: Statistical methods in biological assay. Griffin, London. Girolami, V., Greguoldo, M. & Saltarin, A. 1992. Controllo biologico degli acari del melo con
popolazioni di Amblyseius andersoni (Chant) tolleranti il mancozeb. Informatore Agrario 36: 55-58.
Hassan, S.A. 1985: Standard method to test the side-effect of pesticides on natural enemies of insects and mites developed by IOBC/WPRS Working Group "Pesticides and benificial Organisms". Bull. OEPP/EPPO 15 (1): 214-255.
Hoy, M.A.& Knop, N.F. 1981: Selection for genetic analysis of permethrin resistance in Metaseiulus occidentalis: genetic improvement of a biological control agent. Ent. Exp. Appl. 30: 10-18.
Hoyt, S.C. 1972: Resistance to Azinphosmetil of Typhlodromus pyri from New Zealand. N.Z.I. Sci. 15: 16-21.
Ioriatti, C., Pasqualini, E. & Toniolli, A. 1992: Effects of the fungicides mancozeb and dithianon on mortality and reproduction of the predatory mite Amblyseius andersoni. Exp. and Appl. Acarol. 15: 109-116.
Ivancich Gambaro, P. 1972: I trattamenti fungicidi e gli acari della vite. Informatore Agrario 8: 8141-8143.
Ivancich Gambaro, P. 1985: L'Amblyseius andersoni Chant (Acarina Phytoseiidae) biologia, ecologia, selezione di popolazioni resistenti agli esteri fosforici. Proceeding: Influenza degli antiparassitari sulla fauna utile in frutticoltura. Ed. C.C.I.A, Verona: 11-22.
Ivancich Gambaro, P. 1988: I trattamenti con ziram alla pianta ospite possono interessare la fecondita. Informatore Agrario 36: 57-58.
Ivancich Gambaro, P. 1991: Confronto fra la dinamica delle popolazioni di Amblyseius andersoni in meleti condotti con programmi di difesa a "lotta integrata". Informatore Agrario 29: 73-75.
Mathys, G. 1958: The control of phytophagous mites in Swiss vineyards by Typhlodromus species. Proc. 10° Int. Congr. Ent. 4: 607-610.
60
Mattioda, H., Auger, P. & Kreiter, S. 1999: Le mancozèbe et Typhlodromus pyri. La Dèfense des Végétaux 513: 44-47.
Mori, P. 1985: Effetto di alcuni fungicidi usati per la ticchiolatura del melo sugli acari predatori del ragno rosso. Proc., Influenza degli antiparassitari sulla fauna utile in frutticoltura, Ed. C.C.I.A., Verona: 65-73.
Overmeer, W.P.J. 1985: Rearing and handling. In: Spider mites their biology, natural enemies and control. Helle W., Sabelis M.W. (eds.), Elsevier, vol. 1B: 161-170.
SAS Institute 1985: SAS user’s guide: statistics. SAS Institute, Cary, N.C. Schulten, G.G.M. 1985: Pseudo-Arrhenotoky. In: Spider mites their biology, natural enemies and
control. Helle W., Sabelis M.W. (eds.), Elsevier, vol. 1B: 67-71. Strapazzon, A. & Rensi, F. 1989: Prospettive di controllo biologico di Panonychus ulmi (Koch)
su melo nel Veneto. Informatore Agrario XLV 12: 117-126. Van de Baan, H.E., Kuypers, L.A.M., Overmeer, W.P.J. & Oppenoorth, F.J. 1985: Organo-
phosphorous and carbamate resistance in the predacious mite Typhlodromus pyri due to insensitive acetyl cholinesterase. Exp. Appl. Acarol. 1: 3-10.
Pesticides and Beneficial Organisms IOBC/wprs Bulletin Vol. 24 (4) 2001
pp 61 - 66
61
Development of a laboratory test method to determine the duration of pesticide-effects on predatory mites M. Van de Veire, W. Cornelis & L. Tirry Laboratory of Agrozoology, Faculty of Agricultural and Applied Biological Sciences University of Ghent, Coupure Links 653, B-9000 Ghent E-mail: [email protected]; [email protected]; [email protected] Abstract: A reliable, simple and user-friendly apparatus was developed to study the duration of harmful activity of plant protection products on the predatory mite Amblyseius spp.. The device is based on the principle of the Munger cell, but has a number of advantages compared to the latter. The evaluation of the pesticide-effects (either direct contact toxicity or systemic toxicity) can be done easily under a stereomicroscope. There are no escapees and potential repellent activity of a pesticide can be recognised. Fifteen pesticides, amongst which a number of acaricides, were tested, using Amblyseius californicus as test organism. Data are presented and discussed in accordance with the classification of the IOBC/wprs Working Group “Pesticides and Beneficial Organisms”. Key words: laboratory persistence test; Amblyseius californicus; new test apparatus; side-effects of pesticides Introduction The duration of the harmful activity (persistence) of a pesticide determines when beneficial organisms can be safely introduced in the crop. Pirimicarb and dichlorvos are 2 examples of very toxic chemicals to parasitic wasps but as they are short-lived (< 1 week), beneficials can be introduced in the greenhouse crop again, one week after the treatment.
The usual procedure to study the persistence of plant protection products to predatory mites in the laboratory is to expose the mites to a treated leaf, which is put on wet cotton wool in a petri dish (Oomen, 1989). The main disadvantages of this method are that predatory mites often escape from the leaf surface, either to the wet cotton or to the underside of the leaf and that the quality of the leaf is not guaranteed for a period of 7 to 10 days.
To overcome these problems, we developed a device which is closely related to the “Munger” cage or cell (Oomen, 1989). We also used Amblyseius californicus (McGregor) (Acari: Phytoseiidae) eggs instead of nymphs, to ensure that all juvenile stages were exposed to the pesticide residue on the leaf.
In this paper we have studied the duration of the effect of 15 pesticides on the predatory mite, A. californicus, using the newly developed device. Materials and methods Description of the test cage The testing device, hereafter refered to as the test cage, consists of 6 parts: 1 plexiglass plate (10x5x0.25cm) in the center of which a round hole was made (Ø 3.5 cm); 1 plexiglass plate (10x5x0.25cm) without opening; 1 piece of cloth (10x5x0.2 cm) with a round hole in the center (Ø 3.5 cm); the bottom part of a tissue culture dish (1 cm height; Ø 3.5 cm, Nunc) and
62
2 plastic clamps (Fig. 1a, b, c). In the sides of the tissue culture dish 2 ventilation holes are made (Ø: 6 mm), covered with gauze (width: 140 µm) and one hole to introduce the test organisms. This hole can be connected with a pump to remove the potential pesticide vapours in the cage (Fig. 1d).
After introducing plant leaves in the cages, they can be placed in a holder, which in turn can be put in a reservoir, so that the petiole of the leaf hangs in the water. One reservoir can hold 10 cages (Fig. 1 e, f). Conduct of the test and mode of assessment To assemble the cage, the adapted tissue culture dish is first inserted into the opening of the plexiglass plate. Then the different parts are put together in the following order: the plexiglass plate (without opening), a sweet pepper leaf (Capsicum annuum), the cloth (with opening located at the leaf, and the second plexiglass plate, carrying the tissue culture dish. The different layers are clicked held together with plastic clamps.
Subsequently, 10 eggs of A. californicus are put in the cage via the opening at the side of the tissue culture dish, using a fine needle. The eggs originate from a continuous culture of A. californicus reared on sweet pepper plants, using the twospotted spider mite, Tetranychus urticae Koch (Acari: Tetranychidae) as prey. Then, 15 gravid T. urticae females from a culture on sweet peppers, are also put in the cage. These females soon lay eggs, which can be consumed by the younger stages of the predatory mites. Feeding spider mites to the A. californicus nymphs is a necessity, since feeding on pollen alone results in adults with a strongly reduced fecundity (Van de Veire, pers. comm.). The cages are then put in a climatic chamber (temperature: 24°C; RH: 80%; L/D: 16/8).
In controls, the introduction of T. urticae has to be repeated after 5 and 7 days; the A. californicus nymphs grow very rapidly (developmental time from egg to adult is approximately 1 week) and are able to consume T. urticae adults 5 days after hatching.
In treatments, the spider mites might be killed by the residue on the leaf surface. This can already be assessed after one day. In this case, the spider mite females are introduced every 2 days, until day 7 after the starting of the test.
The leaves used in the treatments were sweet pepper leaves containing aged residues (5, 15, 30 days) of the pesticides mentioned in Table 1. The leaves were taken from plants grown in a commercial greenhouse which were treated with a hand driven spray apparatus (300 ml) until run-off.
Four replicates (a total of 40 eggs) are used per treatment. In our tests, no forced ventilation was used, in order to simulate more realistic conditions for the sweet pepper leaves, because in the greenhouse, pesticide vapours can be also present.
Three to 4 days after the introduction of the A. californicus eggs in the cage, the number of living nymphs was counted to check the hatching rate. After 7 days, the number of developed A. californicus adults was counted.
The % mortality is then calculated. Mt is the % mortality (unhatched eggs and dead larval or nymphal stages) of the treated group. Mc is the % mortality (mainly unhatched eggs) of the control group. Mortality percentages were corrected for control mortality according to Abbott (1925). The actual mortality due to the pesticide effect (Ma) was:
Ma = (Mt – Mc)/(100 –Mc) * 100 To evaluate the effect of the pesticide residues on the fecundity and fertility of females which have developed on the residue, the number of living females and the number of eggs in each test container is counted on day 7. At the same time, the eggs are removed, and 2 days later (day 9), the number of eggs and the number of living females in the container is counted
63
again. At least one male A. californicus has to be present in every cage, in order to trigger oviposition by copulation. The average egg production per female in 4 days (day 7 to 11) was calculated (Rc for the control group, Rt for the treated group.
A B
C D
E F
Fig. 1. Test cage and outfit used in residual toxicity test on nymphs of Amblyseius californicus.
A = part of the test cage B = assembly of the cage C = assembled cage D = pump-connected tube E = detailed view of the test cage with leaf and water reservoir F = general view of the test cage and outfit
64
Finally, the total effect (E) of the pesticide on the beneficial capacity of the predatory mite was assessed by the following formula:
E = 100% - (100% - Mt)/(100% - Mc) * Rt/Rc
The pesticides are classified according to the 4 evaluation categories for laboratory persistence tests, developed by the IOBC Working Group “Pesticides and Beneficial Organisms”: Class A: shortlived (toxic effect < 5 days); Class B: slightly persistent (5 days <toxic effect < 15 days); Class C: moderately persistent (15 days < toxic effect < 30 days); Class D: persistent (toxic effect > 30 days) (Sterk et al. 1999). The toxic effects of the pesticides are classified according to the laboratory A test: toxicity < 30%: harmless; 30% < toxicity < 79%: slightly harmful; 80% < toxicity < 99%: moderately harmful; toxicity > 99%: harmful. Spray deposits of pesticides are thus tested further if more than 30% toxicity is found on leaves with spray deposits of 5 days old. Results and discussion The reproduction parameters Rt and Rc were calculated for all pesticide treatments and com-pared. This revealed that there were no significant differences between these parameters (t-test; P: 0.05) except in the case of amitraz. Thus the ratio Rt/Rc was 1 in all cases except with amitraz. The mean number of deposited eggs per female in a 4 day period ranged from 4 to 7.
The duration of the harmfulness of the pesticides on A. californicus is shown in table 1. The current data revealed that the fungicide sulphur, the insecticides spinosad, acetamiprid and novaluron, and the acaricides clofentezin, chlorfenapyr, fenpyroximate and tebufenpyrad were short-lived (toxic effect < 5 days). These products can be used in IPM to reduce pest populations to a low level, whereafter one week, A. californicus predatory mites can be introduced.
Endosulfan, imidacloprid, flufenoxuron and pyridaben were slightly persistent. The use of these compounds in IPM is more difficult, as one has to wait 2 weeks, before the predatory mites can be introduced.
The only acaricide with long lasting detrimental effect was amitraz. The compound was still harmful to the predatory mite A. californicus, 30 days after treatment of the leaves. It was not only toxic to the mite nymphs, but spray deposits of 15 days old also reduced the egg production of surviving mites. This compound is not suitable for use in IPM systems where A. californicus predatory mites have to be used.
The bacterial fermentation product avermectin, when used at the recommended rate for practical use in Belgium (10 ppm AI), was still slightly persistent as spray deposit of 15 days old. Using half of the recommended dosage resulted in lower persistence, in this case the toxic effect lasted only 5 days, and it can be used in IPM for spider mite control. The method itself showed some important features, as compared to other methods for persistence studies, e.g. the laboratory test methods for testing the side-effects of pesticides on Typhlodromus pyri Scheuten (Acari: Phytoseiidae) and Phytoseiulus persimilis Henriot (Acari: Phytoseiidae). There were no escapes of predatory mites during the experiment; the sweet pepper leaf remained healthy for a relevant period, i.e. more than 14 days. Since the plastic tissue culture dish is renewed for every new test, contaminations, e.g. by vapours of pesticides, are excluded. The “Munger” cell which is used for studying the persistence of pesticides (Oomen, 1989) has to be re-used again and again, and contamination with pesticide vapours can happen.
The apparatus is user-friendly, as the test unit consists only of 6 parts, which are easy to assemble.
Ta
ble
1. P
estic
ides
and
con
cent
ratio
ns te
sted
on
the
pred
ator
y m
ite A
. cal
iforn
icus
. Nym
phal
mor
talit
y pe
rcen
tage
s of
15
pest
icid
es a
fter
7 da
ys o
f exp
osur
e to
pes
ticid
e sp
ray
depo
sits
of 5
, 15
and
30 d
ays
old.
Tot
al e
ffec
t (m
orta
lity
+ ov
ipos
ition
eff
ect)
and
toxi
city
cla
ss a
ccor
ding
to
the
IOB
C c
lass
ifica
tion.
Act
ive
ingr
edie
nt
Tra
de
nam
e D
ose
(ppm
AI)
%
mor
t. (S
DP
5day
)*R
t/Rc
%
eff
ect
(E)
% m
ort.
(SD
P 15
day)
*R
t/Rc
% e
ffec
t (E
) %
mor
t. (S
DP
30da
y)*
Rt/R
c %
eff
ect
(E)
Tox
icity
cl
ass
Ace
tam
iprid
M
ospi
lan
80
16
.6
1 1
6.6
A
Am
itraz
M
itac
400
81
.5
0 10
0.0
67.7
0.
3 91
.0
64.7
1
64.7
D
A
verm
ectin
V
ertim
ec
5
58
.9
1 5
8.9
20.0
1
20.0
2
.5
1 2
.5
B
Ave
rmec
tin
Ver
timec
10
61.1
1
61.
1 55
.0
1 55
.0
5.0
1
5.0
Chl
orfe
napy
r In
trepi
d 2
40
23.9
1
23.
9
A
C
lofe
ntez
in
Apo
llo
200
25
.0
1 2
5.0
A
Endo
sulfa
n Th
ioda
n 5
00
40.0
1
40.
0
B
Fe
npyr
oxim
ate
Naj
a
40
8.6
1
8
.6
A
Fluf
enox
uron
C
asca
de
11
49
.0
1 4
9.0
35.0
1
35.0
B
Imid
aclo
prid
C
onfid
or
100
50
.9
1 5
0.9
10.0
1
10.0
B
Nov
alur
on
Rim
on 1
0 EC
50
26.0
1
26.
0
A
Pyra
zoph
os
Afu
gan
220
32
.6
1 3
2.6
B
Pyrid
aben
Sa
nmite
7
50
62.9
1
62.
9 2.
1 1
2.1
B
Sp
inos
ad
Trac
er
250
23
.9
1 2
3.9
A
Sulp
hur
Cum
ulus
50
00
14.8
1
14.
8
A
Te
bufe
npyr
ad
Mas
aï
30
0
.0
1
0.0
A
* : S
DP
= sp
ray
depo
sit
65
66
Acknowledgement The authors wish to thank Hendrik Van Caenegem for the critical reading of this manuscript. References Abbott, W.S. 1925: A method of computing the effectiveness of an insecticide. J. Econ.
Entomol. 18: 265-267. Oomen, P. 1989: Guideline for the evaluation of side-effects of plant protection products.
Encarsia formosa. Bulletin OEPP/EPPO Bulletin 19: 355-372. Sterk, G., Hassan, S., 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., Rovesti, L., Samsoe-Petersen, L., Sauphanor, B., Schaub, L., Stäubli, A., Tuset, J.J., Vainio, A., Van de Veire, M., Viggiani, G., Vinuela, E. and 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.
Pesticides and Beneficial Organisms IOBC/wprs Bulletin Vol. 24 (4) 2001
pp 67 - 70
67
A semi-field test for evaluating the side-effects of plant protection products on the aphid parasitoid Aphidius rhopalosiphi (DeStefani-Perez) (Hymenoptera, Braconidae) – First results M. Moll1 & M. Schuld2
1 IBACON GmbH, Arheilger Weg 17, D-64380 Roßdorf, Germany; 2 GAB-Biotechnologie, Eutingerstr. 24, D-75223 Niefern-Öschelbronn, Germany Abstract: A semi-field test with A. rhopalosiphi was carried out at two independent testing facilities (GAB-Biotechnologie and IBACON) under outdoor climatic conditions as a first validation test. Pots with barley planted in the central area were sprayed with 5 rates of Perfekthion (a.i. dimethoate 400 g a.i./l) and a tap water control. Each treatment group comprised 3 replicates. 15 female parasitoids per replicate were introduced into the exposure units. After 24, 48 and 72 hours of exposure potted barley seedlings infested with aphids (Rhopalosiphum padi) were added (5 pots per replicate). All pots were replaced by fresh ones after 24 and 48 hours. Ten to twelve days after the parasitization period the developed mummies on the barley were counted for each replicate separately. A dose response relationship was found. Key words: higher tier study, A. rhopalosiphi, semi-field, dose response relationship Introduction Higher tier studies will be required more often for A. rhopalosiphi since it is one of the most sensitive test organisms and one of the two indicator species for side-effect testing. Therefore, a method was developed to test the side-effects under outdoor climatic conditions in a semi field test as the following step after an extended laboratory test which showed adverse effects. The test design was based on the extended laboratory method developed by Mead-Briggs (1997). The study was carried out twice at IBACON and GAB, two independent testing facilities, working on side-effect testing of plant protection products (PPP), at the same time with test organisms from the same origin. Material and methods To evaluate the side-effects of PPP on the aphid parasitoid Aphidius rhopalosiphi under semi-field conditions a test with five different rates of Perfekthion and a water treated control was performed. The exposure units were placed under a UV permeable roof, in order to protect them against rain. Air temperature and relative humidity were measured with a data logger during the exposure phase. Approximately 50 - 60 barley seedlings were planted in pots of 32 cm in diameter in the central area of the pots in an area of approximately 20 cm. The seedlings were sprayed with 5 rates of Perfekthion and water. The application rates were 5, 10, 20, 30 and 40 ml per ha in a water volume of 400 l per ha. In addition, a tap water treated control was prepared with the same water volume. Each treatment group consisted of 3 replicates. Before application the plants were sprayed with a 25 % fructose solution, to increase the attractiveness of the treated plants for the parasitoids. Within one hour after the sprayed solutions were dried, the pots were covered with a fine mesh gauze. Then 15 female
68
parasitoids (obtained by PK Nützlingszuchten, Dr. Peter Katz, Industriestr. 38, D-73642 Welzheim) per replicate, less than 48 hours old, were introduced into the exposure units. After 24 hours of exposure 5 reproduction units with potted barley seedlings, infested with Rhopalosiphum padi, were introduced into each replicate and placed around the treated plants. These reproduction units were replaced by new ones after 24 hours and this procedure was repeated once. In total, 15 reproduction units per replicate were obtained. The reproduction units were transferred into the laboratory in a climatic chamber with a temperature of 20 ± 2°C and relative humidity of 70 ± 20 %, under long day conditions (16 h light, 8 h dark, with more than 2000 lux light intensity). Before the test units were transferred to the laboratory, the plants were checked for female parasitoids. Detected parasitoids were transferred back to the exposure units. Ten to twelve days after the parasitation period the developed mummies on the barley seedlings were counted for each replicate separately. Some aphids migrated from the reproduction units onto the treated plants during the parasitization phases. Since they could have been parasitised as well, the exposure units were checked for mummies as well. Statistical analysis was performed with SAS 6.2. Results and discussion The average air temperature and relative humidity during the exposure and the parasitization phase of the semi-field study differed not very much comparing IBACON in Darmstadt, which is approximately in the middle west of Germany, and GAB in Pforzheim, which is located in the south of Germany (Figure 1 and Figure 2). The differences in the average temperature and relative humidity were at maximal 1.5 °C and 23 %.
0
2
4
6
8
10
12
14
16
18
20
17.07.2000 18.07.2000 19.07.2000 20.07.2000 21.07.2000
Date
Tem
pera
ture
[°C
]
IBACON
GAB
Figure1. Average air temperature during exposure and parasitization phase of the semi-field study
69
0
10
20
30
40
50
60
70
80
90
100
17.07.2000 18.07.2000 19.07.2000 20.07.2000 21.07.2000
Date
IBACON
GAB
Rel
ativ
e H
umid
ity [%
]
Figure 2. Average relative humidity during exposure and parasitation phase of the semi-field study In both studies a clear dose response relationship was found. The ER50 was 10.5 ml Perfekthion/ha at GAB and 2.4 ml Perfekthion/ha at IBACON. As shown in Figure 3 the reduction of the parasitization rate differed between GAB and IBACON by the factor 5, which is not unusual for such a kind of study (discussion in the ring testing group).
0
10
20
30
40
50
60
70
80
5 ml/ha 10 ml/ha 20 ml/ha 30 ml/ha 40 ml/ha
Perfekthion
IBACONGAB
Para
sita
tion
Rat
e [%
]
Figure 3. Parasitation rate relative to the control
70
To discuss these differences first the similarities are summarised in the following: − both studies were conducted at the same time from July the 17th to July the 21st − the climatic conditions differed only slightly − the test organisms originated from the same breeder and had the same age − the same food was taken (barley seedlings were sprayed before treatment with a 25 %
fructose solution until the point of run off) − the exposure units were absolutely similar: same size, number of plants, age of plants,
same mesh gauze type and size − the reproduction units were absolutely similar as well: same size, number of plants,
number of host aphids − same handling of the reproduction units during their change (controlling of wasps under a
glass cage).
One reason for the observed differences could be a higher exposure of the females to the spray residues at IBACON, so that more females died. Much more mummies on the plants were observed at GAB than at IBACON supporting this assumption. In the control groups a mean of 16.7 mummies per female were observed at GAB compared to 10.8 mummies per female at IBACON.
Another reason could be the 1.5 degree difference in the average temperature during the study (temperature was slightly higher at GAB in Pforzheim) (Bigler, personal communication).
The main difference between the two studies was the origin of the host aphids (R. padi). The aphids used at GAB were bred in a mass rearing at the testing facility and the aphids used at IBACON were delivered by PK Nützlingszuchten. According to Messing & Rabasse (1995) the aphid host lines are of fundamental importance in parasitization efficacy.
One more explanation for the different parasitization rates could be the different host strains used in the studies. A different host colony structure of size probably occurred, which might also be responsible for different numbers of parasitised aphids (Messing & Rabasse 1995, Weisser 1995). Acknowledgements The authors thank the technical personnel who were mostly responsible for the successful practical performance of the studies. References Mead-Briggs, M. 1997: A standard ‘extended laboratory’ test to evaluate the effects of plant
protection products on adults of the parasitoid, Aphidius rhopalosiphi (Hymenoptera, Braconidae), (unpubl.).
Messing, R.H. & Rabasse J.M. 1995: Oviposition behaviour of the polyphagous aphid parasitoid Aphidius colemani Viereck (Hymenoptera: Aphidiidae). Agric. Ecosyst. Environm. 52: 13-17.
Weisser, W.W. 1995: Within-patch foraging behaviour of the aphid parasitoid Aphidius funebris: plant architecture, host behaviour and individual variation. Ent. Exp. Appl. 76: 133-141.
Pesticides and Beneficial Organisms IOBC/wprs Bulletin Vol. 24 (4) 2001
pp. 71 - 81
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A sequential testing program to assess the side effects of pesticides on Trichogramma cacoeciae Marchal (Hym., Trichogrammatidae) Sherif Hassan & Hayder Abdelgader Federal Biological Research Centre for Agriculture and Forestry (BBA), Institute for Biological Control, Heinrichstr. 243, D-64287 Darmstadt, Germany
Abstract: A sequential program for testing the side effects of pesticides on Trichogramma cacoeciae Marchal (Hym., Trichogrammatidae) is presented. The program includes the initial toxicity test (most susceptible life stage of the parasitoid), pupae initial toxicity test (less susceptible life stage-pupae within the host eggs), persistence test (duration of harmful activity), extended laboratory test (exposure of adults to fresh dried residues) and field test. Keywords: Trichogramma, pesticides, side effects, sequential testing. Introduction Parasitoids of the genus Trichogramma are distributed world-wide and play an important role as natural enemies of lepidopterous pests on a wide range of agricultural crops. The use of Trichogramma in biological control has gained widespread interest in many countries. At the moment, about 18 species of Trichogramma have been used in more than 23 countries. Approximately 10 different Trichogramma spp. are being mass-reared to control pests on corn, sugarcane, tomato, rice, cotton, sugar beet, apple, plum, vineyard, pasture, cabbage, chestnut, sweet pepper, pomegranate, paddy and forests in at least 23 countries.
The formulated plant protection products, tested during the sequential testing program, are of a known batch, code and technical data. They are usually tested at the highest recommended field rate. A diluted spray fluid of the formulated product is prepared
The sequential testing program presented here describes five different types of methods to test the side effects of pesticides on Trichogramma as follows: I. Adult initial toxicity test is carried out by exposing the adult parasitoids (the most susceptible life stage) to fresh dry pesticide film applied on glass plates at recommended rate. The method was described by Hassan (1974, 1977, 1988 and 1992), a guideline was drafted for use by German registration authorities (Hassan 1989) and an actualised and validated guideline was published recently (Hassan et al. 2000). This worst case test allows the classification of products as harmless. But if the test product shows harmful effects over the threshold value, further testing is recommended depending on the attended use of the product; II. Pupae initial toxicity test, less susceptible life stage, spraying of product on parasitoid pupae within the host eggs (Hassan 1992); III. Persistence test, duration of harmful activity, exposure of adults to pesticide residues applied on plants at intervals after treatment (Hassan 1980, 1992). About 70 chemicals were tested for persistence using this method and the results were published and compared with those of the initial laboratory tests (Hassan 1994); IV. Extended laboratory test, exposure of adults to pesticide film applied on plant leaves under controlled laboratory conditions;
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V. Field test, relevant crops are directly sprayed with the product after laboratory reared organisms are released. Experiments are conducted at the appropriate time of season for the chemical. BASIC INFORMATION A - Rearing of test insects The parasitoids can be cultured in cylindrical clear glass cages (about 25 cm long and 10 cm in diameter). Food and host eggs are replaced three times a week. One third of the parasitised host eggs are returned to each cage to allow continuous rearing. Under laboratory conditions (26 ± 2°C; 75 ± 20% RH; 16 h light, about 500 Lux and 8 h darkness.) the adult longevity is about 10 days, the development time is about 13 days and egg production is about 30 per female B - Preparation of the host During the sequential testing program Trichogramma is offered host eggs for parasitism. To facilitate handling and counting, the S. cerealella eggs are glued on strips of paper. The Traganth glue is applied evenly on paper using a paint brush or roller and a plastic stencil with circular holes. The stencil is carefully lifted and the host eggs, without moth fragments, are scattered onto the glued areas, a fine sieve can be used (0.5 mm mesh) to ensure even distribution. Fresh eggs are used for the experiment, the use of older reddish eggs or clusters should be avoided. In order to maintain fresh eggs in a suitable condition they should be stored at about 5-7 °C for not longer than 2 weeks. Companies producing Trichogramma for use in Biological Control are also usually able to provide the parasitoid, the host as well as food for the test, all of which are generally of suitable quality. C - The test unit A basic test unit is used to expose adults to the tested pesticides in three of the above 5 mentioned methods (Adult initial toxicity test, Persistence test and Extended laboratory test).
The exposure cage is an aluminium frame (13 cm x 13 cm, with walls 1.5 cm high and 1 cm wide, see Figure 1). The upper and lower surfaces of which are covered by foam material (such as Tesamoll®) to provide pads for the two glass plates (2 mm, 13 x 13 cm). Two glass plates are fitted onto the square aluminium frame forming the floor and ceiling of the test unit (treated surfaces of the glass facing inwards). Each one of three sides of the aluminium frame has six holes for ventilation (Ø approximately 1 cm). The inside surface of the frame is coated with black, tightly stretched, porous material (such as Helancabatist®) to cover the ventilation holes preventing wasp escape. The fourth side of the aluminium frame has two different holes. The smaller hole (Ø approximately 1 cm) is used to introduce the starting population of the parasitoid and the second bigger hole (approximately 3.5 cm long x 1 cm high) is used to introduce the host eggs to be parasitized and the food (honey/gelatine) to the insects. The holes on this fourth side of the frame are closed from the outside with adhesive tape, which can be removed. The sticky inside surface area of this tape with which the wasps may come into contact is covered by additional tape thus preventing adhesion of the insects. The large hole is essentially a door that can be opened and closed when necessary. In order to deter the parasitoids from remaining on the untreated sides of the frame, the exterior edges of the glass plates can be covered with black card. A central square (ca. 7 x 7 cm) having been cut out from the black card, thus establishing the contact area of the insects to the spray residues within the illuminated area. The cage is held together with clamps and/or strong elastic bands (At least 2). The insects in the cage stay active on the surface of the treated glass exposed to the light, thus maximising exposure to the spray residues and ensuring “worst-case” exposure
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conditions. A paper strip (ca. 1 cm x 3 cm) with a few thin strips of food (honey/gelatine), is introduced during the assembly of each test unit. The tests are carried out in a controlled environment chamber or room under the same temperature and relative humidity conditions as for the rearing of Trichogramma. Therefore no acclimatisation period should be required between rearing and the toxicity test. The cages are connected to a vacuum pump via a tube system to prevent possible accumulation of plant protection product vapours. The pumps are adjusted so that the entire air in the cages is removed by suction about every 1-2 min. The use of this exposure cage is recommended, however any unit that provides the same conditions of exposure on glass, adequate ventilation and the addition of food and host may be considered.
Figure 1. A diagrammatic representation of a suitable test unit with which to determine the toxicity of plant protection products under “worst case” laboratory conditions. Glass plates (13 cm x 13 cm x 2mm) are secured to the upper and lower surfaces of the unit. D-Preparing the parasitoids ”emerging tubes” To facilitate handling of the hosts and parasitoids, the use of Traganth glue which is completely harmless to Trichogramma spp. is recommended. (Hassan et al 2000) Parasitoids of uniform age are prepared for the experiments. Host eggs (Sitotroga cerealella) are parasitized by Trichogramma and are left until the parasitoids emerge. Fresh host eggs, cleaned from moth fragments as much as possible, are glued with Traganth in circles on strips of white paper. To facilitate counting, graph paper with millimetre markings may also be used (e.g. graph paper 80 g/m2). Each disc is about 0.6 cm in diameter and contains about 100 ± 50 eggs . As far as practically possible, similar numbers should be introduced into each tube. In order to obtain adult parasitoids of uniform age, the egg-cards are placed in the Trichogramma rearing units and taken out 24 h later. To prevent further parasitism after this time, adults remaining on the egg-strips should be driven away.
The parasitized eggs are then kept in a controlled environment chamber to allow the parasites to develop. The parasitoid reaches adulthood in about 10 days. As a parasitoid develops within the moth egg, the egg turns from being translucent/white to progressively darker in colour. Two days before emergence, the black eggs containing pupae of the
Inside surface covered by black netting
Aluminium frame
Upper and lower surfaces covered by foam
Ventilation hole
Wasp introduction hole Host/food introduction hole
13 cm
13 cm
1,5 cm
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parasites are transferred to ‘emergence tubes’ for use in the experiments (see Figure 2). Each emergence tube comprises a transparent glass unit 120 mm in length x 20 mm Ø at one end and 7 mm Ø at the other. A single circle with parasitized eggs is placed into each such tube. A few thin lines of food (honey/gelatine) are applied with a syringe on a thin strip of paper that is then placed inside the tube or it can be directly applied on the internal tube surface. The honey/gelatine should be applied thin and condensed, so that Trichogramma do not get stuck on it. The tubes are closed from both ends with cloth of sufficiently small mesh size tightly held in place with rubber bands or rings. The adult parasitoids emerge and are left in the tube until they are 24 h old. At the beginning of the test, the adult parasitoids should be ca. 24 hours old, active and still have food in excess.
Optional: The number of parasitoids in the emerging tube can be increased by using a larger egg disc of about 1.2 cm diameter that includes ca. 400 ± 100 eggs. With these higher numbers, that were previously used, the number of progeny to be counted for monitoring is unnecessarily high. The higher numbers may be used i.e. for reasons of comparison with older results.
Figure 2. Diagrammatic representation of a suitable emergence tube design. Both the larger 20 mm and the smaller 7 mm holes at either end are sealed using cloth and elastic bands/rubber rings to prevent emerged wasps from escaping. The narrower end of the tube is used for introduction of the test organisms to the test units. 1. Part I: Initial toxicity test (most susceptible life stage of the parasitoids)
This part was validated and published in cooperation with a number of Research Institutions (Hassan et al. 2000): Huntingdon Life Sciences Limited, Cambridgeshire, United Kingdom; Novartis Crop Protection AG, Basel, Switzerland. GAB, Biotechnologie GmbH, Nierfern-Oschelbronn, Germany; IBACON GmbH, Rossdorf,Germany; Mitox Consultants, Amsterdam, The Netherlands; ECT Oekotoxikologie GmbH, Bad Soden, Flörsheim, Germany; Agricultural Research Centre, Alexanderia, Egypt. In this test adults of Trichogramma (the stage of the life cycle that is most exposed to pesticides) are exposed to a fresh dried film of a test substance applied on glass plates (Figure 1). The reduction in parasitism with Trichogramma within one week compared to untreated controls is used to assess the side-effect of the product. The egg laying capacity of the parasitoid in the control cages is used as a validity criteria. The detailed description of the method is given in Hassan et al. (2000).
Emergence tube
parasitised egg circlelines of honey/gelatine 7 mm 20 mm
120 mm
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2. Part 2: Pupae initial toxicity test (less susceptible life stage, pupae within the host eggs)
2.1 Experimental conditions 2.1.1 Principle of the trial
Mature pupae of T. cacoeciae within S. cerealella-eggs (the stage of the life cycle which is least exposed to plant protection products) are directly sprayed with the product. Mortality or capacity of parasitism is compared to those of water treated control.
2.1.2 Trial conditions About 1 day old Sitotroga- eggs are glued in discs ca. 0.5 cm in diameter (about
100 ± 50 eggs) on strips of paper and parasitized by Trichogramma for 24 hours (see 1.4.2 and 1.4.3). The parasitized eggs are left for 7 days to develop at: 26 ± 2°C; 75 ± 20% RH.
2.1.3 Design and lay-out of the trial Treatment: test product (s) and control treated with water.
Replicate: one egg-disc. Number of replicates: four (discs).
2.2 Application of test compounds The treatment is carried out at the 7th day by spraying four egg-discs (black eggs
containing pupae) with each pesticide to be tested. In each experiment, 3 discs are sprayed with water for control. Calibration is carried out by spraying glass plates. The untreated control is sprayed with tap water.
2.3 Conduct of trial and mode of assessment After spraying, the treated eggs are left for 3 hours to dry, transferred to new
containers (i.e. glass tube 14 cm long and 2 cm diameter) and the parasitoid is left to develop under the standard experimental conditions. Two days before emergence, the sprayed black eggs containing Trichogramma pupae are transferred into “emergence tubes” (Figure 2).
If the initial toxicity test on the adults has been performed and parasitism tested, only the “mortality” immobility, and not the capacity of parasitism, is assessed in the present test. The rate of immobilisation is determined 24 h after the beginning of exposure by counting the number of mobile or immobile individuals, whichever is more practical. The glass tube is examined by placing it over white paper without opening it and the adults are counted through the glass. Immobilisation is reached when an adult is not able to move within 3 seconds after gently knocking on the glass. The total number of adults is counted after killing the adults by exposing the glass tube to about 70 °C for ca.30 min.
If the initial toxicity test on the adults has not been performed or if no parasitism occurred, the capacity of parasitism is determined in the present test. The same experimental cage as described in figure 1 is normally used. Assessment of parasitism is then carried out.
2.4 Results Products causing effect below the accepted threshold combined with persistence of
less than 15 days (see persistence test) are considered to have acceptable hazard to the parasitoids for single treatment. Products causing effect above the accepted threshold are further tested.
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3. Part 3: Persistence test (duration of harmful activity, exposure of adults to pesticide residues applied on plants at intervals after treatment)
3.1 Experimental conditions 3.1.1 Principle of the trial To assess the duration of harmful activity, the product is applied to potted vine
plants. After drying and ageing of the residue under field or field simulated conditions for different periods of time, adults of T. cacoeciae are exposed to the treated leaves in test cages and the capacity of parasitism is assessed (Hassan 1980, 1992). About 70 chemicals were tested for persistence using this method and the results were published and compared with those of the initial laboratory tests (Hassan 1994).
3.1.2 Trial conditions For ageing of residue, the treated plants should be kept in the field under
transparent polyethylene rain cover about 50 to 100 cm high. To expose the plants to direct sun, the rain cover is removed for 3 hours in a sunny day once every week. Research experiments showed that the ageing can also be done by placing treated plants in a greenhouse or in a climatic chamber under field simulated summer day conditions. This is particularly useful for pesticides intended to be used in greenhouse. In a controlled environment chamber, the following conditions are suitable for simulating field conditions: 13 h at 27 ± 2°C, 65 ± 20 % RH – 11 h at 17 ±2°C, 95 ±20 % RH and 15 h light – 9 h darkness. The light in the climatic chamber may be supplied by i.e. 7 OSRAM- L tubes (2 White-Universal 30 W/25-2, 2 Fluora 30 W/77-2, 2 Warmtone de Luxe 30 W/32-2 and Fluorescence-Black-Light 20 W/73), that provide a total of 2000 Lux at a distance of 30 cm from the lamps with a large spectrum of light wave length. In addition to ventilators that circulate the air inside the chamber, a ventilator is used to renew the air such as Helios R 10 air pump (Schwenningen, GFR) with the capacity of 95 m3/h. These conditions can also be simulated in a suitable greenhouse. The use of artificial weathering conditions allow to conduct experiments in all seasons of the year.
3.1.3 Design and lay-out of the trial Treatments: test product (s) and control treated with water.
Replicates: one cage with treated leaves (Figure 1). Number of replicates: at least 3.
3.2 Application of chemicals/water Vine plants (e.g. variety Müller-Thurgau) are grown in pots of about 15 cm in
diameter and 16 cm in height under field or greenhouse conditions. The plants should be about 30-40 cm high with about 10 leaves. After sprouting, the plants need at least 6 weeks to reach the height of 40 cm, higher plants are cut back. Each plant is used only once for testing. The product is applied to the test plants until the point of incipient run off, with any suitable currently used equipment. It is usually tested at the highest concentration registered. After spraying, the plants are left for at least 3 hours in a well ventilated area to dry.
3.3 Conduct of trial and mode of assessment Exposure tests are carried out at intervals after treatment of the vine plants, possibly after 3, 10, 17, 24 and 31 days. Leaves with residue are picked and spread inside the exposure cage (Figure 1) to cover the entire lower surface. Trichogramma is introduced into these exposure cages using emergence tubes
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(Figure 2) and the capacity of parasitism is assessed as given in the first laboratory test. Determination of the initial population: The number of wasps that entered the test unit is calculated by examining the emergence tube. Because some parasites emerge in the tube after the beginning of the experiment, the emergence tube should be examined not earlier than 4 days after the beginning of the test. All the parasitised host eggs (black in colour) on the egg-disc are counted and the number of adult Trichogramma stuck on the food strips or remaining in the tube is subtracted. The use of graph paper with a millimetre scale may facilitate counting. Assessment of parasitisation of host eggs: The numbers of host eggs parasitised during the course of the experiment are counted on the egg-strips not earlier than 10 days after the introduction of the particular egg-strip to the exposure cage. After this time, parasitised eggs turn black and are easily recognised. If the parasitised eggs are not to be counted until after the 10th day after their introduction to the test units, the eggs should be heated to 70°C for about 30 min or frozen for one day. This will stop Trichogramma from emerging without affecting the black colouration of the parasitised eggs. The egg-strips can then be stored at room temperature or at 5-70C for counting later.
3.4 Results The number of parasitized eggs is counted for each cage. This is divided by the
number of parasitoid adult females (starting population) that entered the same cage at the beginning of the experiment. For each test product and time interval, the mean % reduction in parasitism compared to the control is calculated as follows:
Reduction in parasitism (%) = Eggs/female in Control - Eggs/female in Treatment x 100 Eggs/female in Control
The reduction in the capacity of parasitism for each product and interval is plotted on probit scale against time. The “duration of harmful activity” is the time required for the pesticide residue to loose effectiveness so that a reduction in parasitism of less than 50% is reached, compared with the control. If 15-day old residues on leaves cause an effect above the accepted threshold on T. cacoeciae adults, further testing is recommended. Products causing an effect below the accepted threshold for less than 15 days (approximate development time of Trichogramma) are considered to have acceptable hazard to the parasitoids for single treatment. The period during which a product remains harmful may be mentioned on the label, valuable information.
4. Part 4: Extended laboratory method, exposure of adults to fresh dried pesticide film applied on plant leaves under controlled laboratory conditions
4.1 Experimental conditions 4.1.1 Principle of the trial The product is sprayed to plant leaves. After drying, adults of T. cacoeciae are
exposed to the treated leaves in test cages and the capacity of parasitism is assessed.
4.1.2 Trial conditions The use of vine leaves (variety Müller-Thurgau or similar) is recommended but
other leaves if relevant to the use of the pesticide may be used. Leaves that have a
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thick wax layer, e.g. cabbage, if not relevant, should be avoided. An even film of the product is sprayed on the leaves. After spraying, the drying time should be about 3 hours. The exposure cage described under 1.4.1 my be used.
4.1.3 Design and lay-out of the trial Treatments: test product (s) and control treated with water.
Replicates: one cage with treated leaves. Number of replicates: at least 4.
4.2 Application of chemical/water A stationary sprayer similar to that described under 1.6 may be used. 4.3 Conduct of trial and mode of assessment Exposure tests are carried out on about 3 hours treated leaves. Leaves with residue
are spread inside the exposure cage to cover the entire lower surface. Trichogramma is introduced into these exposure cages using emergence tubes (Figure 2) and the capacity of parasitism is assessed (see 3.3).
4.4 Results The number of parasitized eggs is counted for each cage. This is divided by the
number of parasitoids adult females (starting population) that entered the same cage at the beginning of the experiment. For each test product, the mean % reduction in parasitism compared to the control is calculated (see 3.4). Products causing effects below the accepted threshold are considered to have acceptable hazard to the parasitoids for single application. Products causing effect above the accepted threshold may be further tested.
5 Part 5: Field test
5.1 Experimental conditions All products not classified according to the earlier tests should be subjected to field test.
5.1.1 Selection of crop and cultivar, test organisms Trials can be carried out on any fruit (i.e. apple, prune) or field crop (i.e. wheat).
Any cultivar may be used. Test organism: Any Trichogramma sp. relevant to the crop can be used i.e. T. cacoeciae in fruit orchards and Trichogramma evanescens or Trichogramma brassicae for field crops.
5.1.2 Trial conditions The experiments are carried out in fruit orchards or in fields that have not recently
been treated with any plant protection product. Trichogramma is released in the field by distributing cardboard devices that contain parasitized Sitotroga eggs, shortly before emergence. Growth and cultural conditions, temperature and humidity should be as homogeneous as possible for all plots.
5.1.3 Design and lay-out of the trial Treatment: Test products(s), reference product(s) and water treated control(s),
arranged where possible in a randomised design. Plot size (net): At least 3 large apple trees, or the equivalent of pillar trees i.e. 9 trees or ca. 100 m2 of a field crop. The distance between the plots should be about 3 large trees, 9 pillar or ca. 10 m. Parasitoid release should be carried out at the rate of about 18000 adults per large tree, 3000 per pillar tree, releasing cards used of biological control may be used, usually with about 3000 individuals per cared. Replicates: At least 3.
5.2 Application of products
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5.2.1 Test product(s) The named usually formulated product under investigation. 5.2.2 Reference product(s) Use products registered for use on fruit or field crops and which have proved
satisfactory in practice. At least one should be recognised to be harmless and another recognised to be harmful to T. cacoeciae. In general, formulation type and mode of action should be close to those of the test product.
5.2.3 Mode of application Applications should comply with good agricultural practice. 5.2.3.1 Type of application According to the instructions of the (proposed) label. Normally a spray. 5.2.3.2 Type of equipment used Application with currently used equipment which should provide an even
distribution of product on the whole plot. Factors which may affect action on T. cacoeciae (such as operating pressure, nozzle type) should be recorded together with any deviations in dosage of more than 10%. Special attention should be paid to avoid drift.
5.2.3.3 Time of application The treatment should be carried out one day after the main wave of Trichogramma
has emerged. This is usually two days after the beginning of emergence. 5.2.3.4 Doses and volumes used The product should normally be applied at the highest dosage recommended on the
(proposed) label for use in the relevant crop. This will normally be expressed in kg (or litre) of formulated products per ha. It may also be useful to record the dose in g of active ingredient per ha. For sprays, data on concentration (%) and volume (liter per ha) should also be given.
5.2.3.5 Data on chemicals used against other pests Interference with other chemicals should be avoided. 5.3 Mode of assessment, recording and measurements 5.3.1 Meterorological data Records of temperature and humidity should be made during the whole trial
period, thermohygrograph may be used. 5.3.2 Type, time and frequency of assessment 5.3.2.1 Type Assessment is carried out by randomly distributing baiting cards (Sitotroga eggs
glued on small strips of possibly green paper) in the plots. About 18 egg cards per large tree, 6 per group of spindle bush trees each with about 400 Sitotroga eggs, are fastened preferably to the under surface of the leaves, clamps may be used.
5.3.2.2 Time and frequency The first baiting cards are distributed about 24 hours after the application of the
plant protection product. After 1 or 2 days, the cards are collected, replaced by new cards and the old ones taken to the laboratory of examination. Egg cards are usually distributed four times preferably on day 1, 2, 4 and 6 after the treatment. After collection, the egg cards are kept at about 25 °C until they are examined. The number of parasitized host eggs (which have turned black) are counted at least 5 days after the exposure in the field. The reduction in parasitism, as compared with control (treated with water) is used to measure the effect of the chemical.
5.3.2.3 Disturbance by weather and by predators or egg parasitoids in the field Rain can reduce the concentration of the product. Rain can also damage the baiting
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cards. The experiments should therefore be stopped if it rains during the first two days of the experiment. Cold weather reduces the activity of Trichogramma in the plots resulting in lower parasitism, making assessment difficult. If so, monitoring could be extended for few days i.e. one week. Predators may feed on the baiting cards and disturb assessment. These losses can be met by increasing the number of baiting cards and changing them at shorter intervals. To reduce the interference by predators, trees could be searched and or protected by adhesive rings before the experiment. If high level of natural egg parasitism is expected i.e. presence of Lepidoptera eggs, this may be assessed by distributing baiting cards one week before the experiment.
5.3.3 Direct effects on the crop Direct effects on the crop should already have been evaluated in the trials on
product efficacy (see relevant guidelines), but any particular effects observed should be recorded.
5.3.4 Repeated treatments Preparations that are treated more than once are tested by repeating the points 1, 2
and 3 for each treatment. 5.4 Results Effects on T. cacoeciae comparable to those caused by the harmful reference
product are classified as harmful; effects consistently comparable to those caused by the harmless reference product are classified as harmless. Intermediate results are classified as harmless in special conditions.
6.0 Sequential Scheme – Presentation of all test results At each stage of the sequential scheme, the results should be reported in a
systematic form and the report should include an analysis and evaluation. Original (raw) data should be available. Statistical analysis should be used, where appropriate, by methods which should be indicated. Interpretation of all test results – Adults initial toxicity test: Effect below 30%– harmless. If harmful continue; – Pupae initial toxicity test and adult persistent: Effect below 50%, combined
with less than 15 days persistence – harmless. If harmful continue; – Extended laboratory test: Effect below 50% - harmless. If harmful continue. – Field test: Compared to reference products – harmless or harmful.
References Hassan, S.A. 1974: Eine Methode zur Prüfung der Einwirkung von Pflanzenschutzmitteln auf
Eiparasiten der Gattung Trichogramma (Hymenoptera: Trichogrammatidae) – Ergebnisse einer Versuchsreihe mit Fungiziden. Z. angew. Entomol. 76: 120-134.
Hassan, S.A. 1977: Standardized techniques for testing side-effects of pesticides on beneficial arthropods in the laboratory. Z. Pflanzenkrankh., Pflanzensch. 84: 158-163.
Hassan, S.A. 1980: Reproduzierbare Laborverfahren zur Prüfung der Schadwirkungsdauer von Pflanzenschutzmitteln auf Eiparasiten der Gattung Trichogramma (Hymenoptera, Trichogrammadidae). Z. angew. Entomol. 89: 281-289.
Hassan, S.A. 1989: Auswirkungen von Pflanzenschutzmitteln auf Trichogramma cacoeciae Marchal als Vertreter der Mikrohymenopteren im Laboratorium. Richtlinie f. d. Prüf. v.
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Pflanzenschutzmitteln im Zulassungsverfahren. Biolog. Bundesanst. Land-Forstwirtsch., (Teil 6, 23-2.1.1), 21 S..
Hassan, S.A. (ed.) 1988: Guidelines for testing the effects of pesticides on beneficial organisms. Short description of test methods. IOBC/WPRS Bulletin 11 (4).
Hassan, S.A. 1992: Guideline for the evaluation of side effects of plant protection product on Trichogramma cacoeciae, sequential testing programme. In: Guidelines for testing the effects of pesticides on beneficial organisms: description of test methods. IOBC/WPRS Bulletin 15 (3): 18-39.
Hassan, S.A. 1994: Comparison of three different laboratory methods and one semi-field test method to assess the side effects of pesticides on Trichogramma cacoeciae. IOBC/WPRS Bulletin 17 (10): 133-141.
Hassan, S.A.; Halsall, N.; Gray, A.P.; Kühner, C.; Moll, M.; Bakker, F.M.; Roemke, J.; Yousif, A.; Nasr, F. & Abdelgader, H. 2000: A laboratory method to evaluate the side effects of plant protection products on Trichogramma cacocciae Marchal (Hym., Trichogrammatidae). In: M.P. Candolfi, S. Blümel, R. Forster, F.M. Bakker, C. Grimm, S.A. Hassan, U. Heimbach, M.A. Mead-Briggs, B. Reber, R. Schmuck & H. Vogt (eds.) 2000: Guidelines to evaluate side-effects of plant protection products to non-target arthropods. IOBC/WPRS, Gent: 107-119.
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Pesticides and Beneficial Organisms IOBC/wprs Bulletin Vol. 24 (4) 2001
pp. 83 - 88
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Effects of Confidor 20 LS and Nemacur CS on bumblebees pollinating greenhouse tomatoes
P. Bielza1, J. Contreras1, M.M. Guerrero2, J. Izquierdo3, A. Lacasa2 & V. Mansanet3
1 Universidad Politécnica de Cartagena, E.T.S. Ingeniería Agronómica, Paseo Alfonso XIII, 52. E-30203 Cartagena (Murcia), Spain
2 Protección Vegetal, Centro de Investigación y Desarrollo Agroalimentario, Consejería de Agricultura, Agua y Medio Ambiente, C/ Mayor, s/n., E-30150 La Alberca (Murcia), Spain
3 Bayer Hispania, División Agro. Pau Claris, 196, E-08037 Barcelona, Spain Abstract: In the intensive vegetable-cultivation area of southeastern Spain, bumblebees are widely used for greenhouse-tomato pollination. Knowledge of the selectivity of insecticides towards bumblebees is a key point to develop plant-protection programs in this scenario. A large-scale greenhouse trial was performed to assess the effects on bumblebees of soil-applied Confidor 20 LS (imidacloprid SL 200) and Nemacur CS (fenamiphos CS 240). The experimental layout employed 8 greenhouses to set a total of 12 experimental plots of ca. 1000 m2 each. Confidor 20 LS was applied 38, 48, 58 and 68 days after transplanting of tomato plants at 0.05 ml/plant (0.75 l/ha). Nemacur CS was applied 9 and 23 days after transplant at 1.33 ml/plant (20 l/ha). Assessments of pollinating activities were performed 38, 44, 52, 59, 66, 73 and 80 days after transplant. Evaluations included percentages of flowers pollinated, aborted, closed/non-marked and marked, as well as bumblebee flight frequencies. Laboratory assessments of final hive status, which included up to 10 biological indicators, were performed at the end of the experiment. No significant differences could be detected between untreated control plots and treated plots for pollinating activities or flying frequencies. After laboratory evaluation of hives at the end of the experiment, no significant differences were detected between treatments for any of the parameters studied. Keywords: Bombus terrestris, imidacloprid, fenamiphos, drip irrigation, pollinating activity, flying frequencies, effect on hives.
Introduction
In the tomato cultivation area of Southeast Spain (Murcia, Almería), bumblebees are used to pollinate practically 100% of protected tomato crops. Due to the high incidence of Tomato Yellow Leaf Curl Virus (TYLCV), pesticide treatments are required to control Bemisia tabaci, especially during the crop first 3 months. Furthermore, where crop rotation is not feasible or helpful, nematicides are often applied before/at transplanting of tomatoes. Theoretically, soil- and foliar-applied systemic insecticides could reach the flower structures of tomato plants, and thus affect pollinating insects.
Confidor 20 LS (imidacloprid SL 200) is widely used for control of whiteflies in Spain. This active substance is also commonly used for seed dressing. Imidacloprid proved highly toxic to honeybees in feeding tests (Schmidt, 1996). Despite this toxicity and its systemic properties, residues in nectar and pollen of seed-treated sunflower were found too low to cause adverse effects on honeybees and bumblebees (Schmidt, 1996, Curé et al., 2000, Tasei et al., 2000). The use of Confidor 20 LS via drip irrigation improves its selectivity towards beneficial arthropods and pollinators in vegetable cultivation under plastic. Several trials were
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carried out with this product in drip irrigation showing no adverse effects on the activity of bumblebees pollinating tomato crops (Hernández et al., 1999).
Nevertheless, there is still certain controversy between pesticide manufactures, suppliers of bumblebees, researchers, consultants and growers, on the effects of imidacloprid on bumblebees. The aim of this work was to gather information on the effects of soil-applied systemic pesticides on bumblebees, using an experimental layout as close as possible to a commercial scenario, still with an appropriate statistical design. In particular, we assessed the effects of soil-applied Confidor (imidacloprid SL 200) and Nemacur CS (fenamiphos CS 240) on the activity of bumblebees pollinating greenhouse tomatoes.
Materials and methods
In autumn, 1999, a large-scale greenhouse trial was performed in Ramonete (Lorca), in the area of intensive vegetable-cultivation of Murcia, Southeast Spain. There were 3 treatments, each replicated 4 times and arranged in randomised blocks: Untreated control; Nemacur CS (fenamiphos 240 g a.i./L) at 1.33 ml/plant (20 l/ha, applied twice); and Confidor 20 LS (imidacloprid 200 g a.i./L) at 0.05 ml/plant (0.75 l/ha, 4 applications).
A total of 8 greenhouses were used to set 12 experimental plots of ca. 1000 m2 each, belonging to 3 different growers. Three parallel and consecutive greenhouses from the first grower (ca. 2000 m2 each) were half-divided with a fine mesh to prevent bumblebee movement, corresponding each half to block 1 and block 2. Block 3 used three parallel and consecutive greenhouses from the second grower (ca. 1000 m2 each). From the third grower, a 2000 m2 subdivided greenhouse, together with a ca. 1000 m2 greenhouse, accounted for block 4. Experimental treatments were assigned at random within each block. This allowed for testing the effects on bumblebees under different agronomic conditions, still controlling variability within each block.
Tomato plants were transplanted on September 25th, 1999. Cultivar Daniella was transplanted in blocks 1 and 2, and cultivar Gabriella in blocks 3 and 4. Plant density was 1.5 plants/m2, two stems per plant.
Commercially available bumblebee hives were selected to adjust the number of workers to plot size. Hives were very homogeneous regarding number and the age of workers. Hives were set at random 34 days after transplant, at the crop growth-stage of 2nd inflorescence (GS 62). In all plots, regardless of treatment, hives were closed on late afternoon of the day preceding each application, and opened again early in the morning the day after application.
Pesticide treatments were performed with the irrigation system on, to assure a thorough and even distribution of the chemicals in soil. The appropriate amounts of product were poured onto the drip zones aside each individual plant, using a volumetric dispenser. In the Nemacur plots, the product was applied undiluted (1.3 ml/plant), 9 and 23 days after transplant. In the Confidor plots, the compound was applied 38, 48, 58, and 68 days after transplant, diluted 1:40 in water. Each plant received 2 ml solution containing 0.05 ml Confidor 20 LS.
Weekly assessments on pollinating activities were performed 38, 44, 52, 59, 66, 73, and 80 days after transplant. In each experimental plot, 4 groups of 10 plants were randomly selected for evaluation. Assessments on these 40 plants per plot included: F1, number of pollinated flowers (set fruits and marked flowers); F2, aborted flowers; F3, closed/non-marked flowers; and M, marked flowers (bumblebees marks the flowers that visited, showing small brown areas). Average percentages of each parameter were calculated for each experimental plot.
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To control bumblebee flight-frequencies, at each assessment date the number of workers going into and out of the hives was counted for 30 minutes. Flight-frequency assessments were performed at the same time in the three experimental plots of each block.
At the end of the experiment, all hives were closed and transferred to the laboratory and stored in a freezer until retrieved for evaluation. Laboratory assessments of final hive status included queen survival; numbers of queen larvae, new queens, and males; percentage mortality of workers, larvae and pupae; number of provisions; weight of molasses; and egg setting.
Statistical analysis of raw data included two-way analysis of variance, using critical level P≤0.05 for declaring significant differences between treatments.
Results and discussion
Pollinating efficiency No significant differences could be detected between treatments for pollinating activities at any assessment date. This was true for the four parameters studied: percentages of flowers pollinated (F1), aborted (F2), closed/non-marked (F3), and marked (M). Means and standard errors are shown in Figure 1 and Table 1.
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Fig. 1. Mean percentage of pollinated flowers (F1) (error bars = standard errors). Evaluations 1-7 were made 38, 44, 52, 59, 66, 73 and 80 days after transplant of plants.
In all treatments, percentage pollinated flowers showed a fast increase from the introduction of the hives until 2 weeks later (Fig. 1). After a remarkably cold period of 2-3 days preceding week 4, a phase of steady increase followed, and a plateau was reached 5 weeks after hive introduction. Equilibrium was maintained until the end of the hives life span. For all treatments, maximum percentage of pollinated flowers was around 70%.
Percentage aborted flowers (F2) was very low for all treatments, showing a constant decrease from hive setting until the end of the experiment. Percentage closed/non marked flowers (F3) showed a mirror pattern of that of percentage pollinated flowers. Percentage
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marked flowers (M) was relatively low for all treatments, indicating that bumblebee densities were far below the saturation threshold. Thus, under these conditions chances of detection of negative effects due to treatments were enhanced.
Significant block effects were detected for most pollination parameters at several assessment dates, suggesting that crop conditions had much more impact on pollination efficiency than experimental treatments. Table 1. Mean ± Standard Error for each evaluation date of the parameters: aborted flowers (F2), closed/non marked flowers (F3) and marked flowers (M). Evaluations dates 1-7 were 38, 44, 52, 59, 66, 73 and 80 days after transplant of plants.
Evaluation Date 1 2 3 4 5 6 7 Treatment M ± SE M± SE M±SE M±SE M±SE M± SE M ± SE F2 Control 3.5 ± 4.0 3.6± 2.1 1.4±0.6 1.0±0.6 1.0±0.6 0.9± 0.4 1.0 ± 1.2 Nemacur 5.0 ± 4.1 3.2± 1.8 3.3±2.0 1.6±1.3 1.1±0.9 1.4± 0.6 0.6 ± 0.3 Confidor 3.9 ± 3.1 2.5± 1.6 1.2±1.1 1.2±0.7 1.2±1.1 1.8± 1.8 0.4 ± 0.3 F3 Control 66.3 ± 7.4 41.4± 9.3 33.1±8.7 33.5±7.1 29.9±5.5 28.9± 9.0 30.6 ± 12.2 Nemacur 61.5 ± 3.8 41.5± 7.9 30.8±4.7 34.3±4.4 30.1±5.4 29.0± 6.1 29.3 ± 6.3 Confidor 63.3 ± 2.4 44.6± 2.8 34.5±6.3 34.8±7.1 33.0±7.1 29.3± 5.1 32.1 ± 7.0 M Control 2.6 ± 2.9 10.3± 7.6 10.7±3.4 11.2±5.6 10.1±2.6 7.1± 4.1 5.3 ± 2.8 Nemacur 5.0 ± 4.5 9.4± 7.1 8.5±2.6 10.0±3.2 9.2±2.4 8.1± 4.2 5.7 ± 3.3 Confidor 3.9 ± 3.1 7.5± 5.6 9.9±3.5 8.9±4.1 8.8±1.7 6.9± 4.2 3.9 ± 2.5
M: Mean, SE: Standard Error
Flight frequency Similarly to pollinating activities, no significant differences could be detected between treatments regarding flight frequencies, at any evaluation date. Again, there were significant differences between blocks at several evaluations dates.
Flight frequencies increased from hive introduction until 4 weeks later, decreasing thereafter until the end of the experiment (Fig. 2). The above-mentioned unusually low temperatures registered just before the 4th evaluation, probably accounted for reduced hive life span. Flight frequencies at weeks 6 and 7 were very low, which coincided with limited percentage pollinated flowers on these dates. Data on flight frequencies showed high levels of variability. Influences of ethological and meteorological parameters probably make this parameter a poor indicator for hive activity.
Hive status After laboratory evaluation of hives at the end of the experiment, no significant differences were detected between treatments or blocks for any of the parameters studied (Figure 3). The only exception was for block differences in larvae mortality, which was significantly lower in block 4. This result agrees with a higher pollinating efficiency in this block. Elevated mortality in some hives can be explained by its ageing, given that assessments were performed 1.5 months after setting. Growers usually replace bumblebee hives after one month of use, which is the period providing high pollinating activity.
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0
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Fig. 2. Mean flight frequency (no. of worker bumblebees entering and leaving the hive/30 minutes). (Error bars = standard errors).
alivek
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Fig. 3. Final hive status (error bars = standard errors). To summarise, under the experimental conditions described, chances of detection of side
effects on bumblebees were enhanced, as indicated by the limited percentages of marked flowers registered. The finding of significant block effects suggest that agronomic conditions had a clear influence on pollinating activities. On the contrary, soil-applied Confidor 20 LS or Nemacur CS had no noticeable effect on bumblebees pollinating greenhouse tomatoes in a commercial scenario.
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References Curé, G., Schmidt, H.W. & Schmuck, R. 2000: Results of a comprehensive field research
programme with the systemic insecticide imidacloprid (Gaucho). Hazards of pesticides to bees, ICPBR 7th Bee Protection Symp. Avignon, Sept 99. IOBC wprs Bulletin 23 (3): 6.
Hernández, D., Mansanet, V. & Puiggrós, J.M. 1999: Use of Confidor 200 SL in vegetable cultivation in Spain. Pflanzenschutz-Nachrichten Bayer 52 (3): 364-375.
Schmidt, H.W. 1996: The reaction of bees under the influence of the insecticide imidacloprid. Proc 6th Int. Symp. on Hazards of pesticides to bees. Braunschweig. Appendix: 12.
Tasei, J-N., Lerin, J. & Ripault, G. 2000: Sub-lethal effects of imidacloprid on bumblebees, Bombus terrestris (Hymenoptera: Apidae), during a laboratory feeding test. Pest Manag. Sci. 56: 784-788.
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Pesticides and Beneficial OrganismsIOBC/wprs Bulletin Vol. 24 (4) 2001
pp. 89 - 95
Pre-sampling for Large Field Trials – a valuable instrument to chose the ‘perfect’ site? Silvio Knäbe1, Jon F.H. Cole2 & Anna Waltersdorfer3 1 GAB-Biotechnologie, Eutinger Strasse 24, 75223 Niefern-Öschelbronn, Germany 2 Syngenta Ltd, Jealott’s Hill International Research Centre, Bracknell, RG42 6ET, UK 3 Aventis CropScience GmbH, Ecotoxicology, 65926 Frankfurt am Main, Germany Abstract: The Joint Initiative on higher tier testing suggested that some pre-sampling of the study site is appropriate to ensure a sufficient abundance and diversity of arthropods. To ensure the selection of a high quality site an assessment of several sites (pilot study) can be a valuable tool to investigate the suitability of alternative field sites. The fauna diversity found in early sampling, however, is not always comparable to the fauna present later in the year. Nevertheless the pilot study does give valuable information about the population dynamics and other factors concerning the field site that might pose difficulties (physical and biological) during the main study. Additionally, the pilot study can be used as an invaluable tool for familiarisation of the site and the training of field staff in new methods etc. To demonstrate the usefulness of pilot studies, data from examples are presented. The designs of the studies as well as the results are discussed. Keywords: side-effects, large scale field trials, pre-sampling, arable crops, orchards
Introduction
The testing of a plant protection product on non-target arthropods (NTAs) may generate a large amount of laboratory data on the toxicity of the chemical. However, even with results available from laboratory studies, it is not always possible to estimate whether or not a pesticide possesses a high risk to the non-target arthropods in a natural system. In order to fully evaluate the potential risk to NTA’s, further investigations in the field can be undertaken to study effects at the population level. A system for evaluation developed over the last years includes large-scale field trials (Anonymous 1995 and Candolfi et al. 2000). The aim of a large field trial is to demonstrate in the agricultural environment the immediate effects of the PPP (plant protection product) and the duration of possible effects on non-target organisms.
For such studies certain requirements for the experimental site should be met. The main requirements are the adequate presence, abundance and diversity of the key arthropod fauna. To ensure these particular requirements, it is advisable to work in less intensive grown crops on a site that is managed extensively. For instance, avoiding ploughing or the non-usage of insecticides would be such a measure. Additionally the field should have relatively uniform structure and boundary to avoid further factors influencing the study - a requirement not easily met due to the required size of the experimental site. For instance a large field study in arable crops will necessitate a minimum size of 12 ha, and a study in orchards needs at least 1100 trees for a design with about 100 trees per plot (Brown 1998).
To guarantee all the above-mentioned requirements, it is possible to imagine a ‘perfect’ study site. The field should be rectangular, flat and extensively used, and preferably not more than a few minutes away from the field station. The arthropod fauna should have been sampled over several years and the main factors influencing the re-colonisation process should be known, since many of the factors i.e. size of the reservoir population, distance over which re-colonisation takes place, the rate of decline for PPPs tested, availability of food and
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weather pattern cannot be influenced during the study (Mead-Briggs 1998). To avoid complications or interference, the best set-up for a study would be on a field site maintained by the contracting lab/sponsor. At present very few contract laboratories possess enough property to run a large field trial, so a field managed by a local farmer has to be used. Additionally, even if there should be a large enough area available, due to the nature of the relationship between agrochemical industry and contract labs, it is very difficult to continue sampling and maintain a field site in the hope, that there might be some day a study taking place. It is a problem all contractors and the industry are aware of, and it has been suggested that some pre-sampling should be done prior to the start of the study to ensure a sufficient abundance and diversity of arthropods (Candolfi et al. (2000). To ensure the selection of a high quality site, an assessment of several sites (pilot study) can be a valuable tool to investigate the suitability of alternative field sites for the main study.
However, it is possible that the diversity seen in the field is not always comparable to the fauna present later in the year. Nevertheless the pilot studies from early sampling give valuable information about the population dynamics and other factors concerning the field site that might pose difficulties (physical and biological) during the main study. Additionally, the pilot study can be used as an invaluable tool for site familiarisation, and the training of field staff in new methods.
To demonstrate the usefulness of pilot studies, data from examples are presented. One example will be for a pilot study in arable crops, and one in a citrus orchard. The designs of the studies as well as the results are discussed. Material and methods All the studies took place not more than four weeks before the start of the main trial to record some arthropod activity (occurrence and abundance). For the pilot study in arable crops, the starting criteria was the soil temperature, and for the study in orchards the beginning of flowering. Pilot study in arable crops The pilot study was needed to assess the condition of two field sites before the study took place. The design was similar for both fields. The field number one was separated into three units by a path and a ditch. The separate units were used as subplots within the field. The second field was split into two parts because of its large size. The pilot study included 20 pitfall traps and one yellow water trap. Ten pitfall traps were randomly distributed on the edge of the field. The traps were set up directly on the field margin or not more than two meters inside cropped area. Ten pitfall traps and the yellow water trap were set up within the field at least 50 m away from the field margin. Pitfall traps were randomly distributed within the field. The two types of traps were sampled after four days and after eight days. The idea to separate the traps into outside and inside was based on the immigration of arthropods from the edge towards the middle (Thacker and Jepson 1993). After collection of the samples, specimens were identified to order and in some cases to family.
Additional assessments were visual observations of 100 stems and weed assessments with quadrants. Soil samples were taken from the different parts of the fields. The pesticide and the crop histories of the site were recorded. All the data from the identification results and the agricultural history were summarised in a report.
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Figure 1: Set-up for inventory sampling Pilot study in a citrus orchard The pilot study was designed as an inventory study of two orchards. Both fields were treated as one unit. Three trees were randomly chosen in each orchard. However, the trees had to be at similar growth stage and not situated at the edge of the field. The sampling was carried out as an inventory assessment. In such sampling the whole fauna of one tree is sampled – a method that produces less variable data than sampling methods like D-vac or beating (Brown 1989).
For an inventory assessment, two collection sheets were set underneath the selected tree (Figure 1) and dichlorvos was applied to the tree with a motorised backpack mist blower. All specimens that fell into the sheets were collected three hours after the application. The sampled material was washed from the collection sheets with water into large photo dishes. From there they were transferred into collecting pots. All trees were sampled and the insects kept separately. Samples were transferred to the lab and identified to order within one week after the sample was taken. There also was an assessment of phytophagous insects, made by sampling 50 leaves at random on each tree.
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Results
Arable crops The results for a study in southern Europe (Italy) are presented. Additionally there were pilot studies in central Europe and northern Europe at a similar developmental stage of the crop. Table 1: Sum of arthropods caught in pitfall traps
Field 1 edge Field 1 centre Field 2 edge Field 2 centre Sampling 1 Three days after setting Carabidae large 13 2 12 6 Carabidae small 3 0 3 1 Lycosidae 124 127 66 93 Araneae other 44 36 20 12 Staphylinidae 14 5 35 7 Collembola 73 75 14 18 Acari (mites) 7 0 1 5 Carabidae larvae 0 0 0 0 Sampling 2 Seven days after setting Carabidae large 32 6 37 8 Carabidae small 10 2 22 5 Lycosidae 81 78 87 94 Araneae other 51 48 38 49 Staphylinidae 6 4 47 90 Collembola 349 1633 250 32 Acari (mites) 2 1 0 0 Carabidae larvae 2 1 2 0
Ten pitfall traps were distributed on the edge and in the centre of each field. For the second collection only nine traps were emptied on the edge of field one and two.
In the study in southern Italy two samplings with pitfall and yellow water traps were made. The traps were set up on the 21st March and sampled three and seven days after setting. The results for the pitfall trap sampling are given in Table 1. Generally the number of individuals increased for nearly all groups in the second sampling. However, the increase was more pronounced on the edge than in the centre of the field with exception of the Collembola. There was no statistical difference between the fields for the two samplings.
The number of arthropods caught in yellow water traps was too low for any analysis. There were no aphids or other phytophagous insects found during visual checks of 100 plants in each field.
The soil types of the two fields were very similar. In contrast to Field 2, Field 1 was situated much closer to the facility. Additionally, the maintenance of the surrounding land was less intensive for the first field. The crop and pesticide history was favourable for the experiment for both field sites, i.e. no pesticides had been used for the last two years, the farmer was cooperative and reliable, and the fields had secure surroundings (they were on private property, and separated from public areas with fences and ditches).
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At the end of the pilot study the decision was taken to start the trial on Field 1. The main reasons for this were, because of the diversity of the surrounding environment, the extensive maintenance of the field margins and the closer distance to the field station. Orchards The pilot study was conducted in two citrus orchards in Spain. The orchards were about 1 km apart. The inventory sampling took part at the starting point of flowering. The sampling in the two orchards was three days apart from each other due to bad weather. The results for the sampling are given in Table 2. There were more individuals of the orders Sternorrhyncha, Diptera, Hymenoptera and Araneae in Orchard 1. However, the individuals of Sternorrhyncha and Hymenoptera in Orchard 1 were mainly from one tree that was heavily infested by aphids. There was no statistical difference between the numbers of all insects in the two orchards sampled. Three different aphid species were found in the orchards.
No aphid specimen was parasitised. The distribution of the developmental stages of the aphids indicated the beginning of an infestation. No difference in the number of aphids was found between the two orchards.
Table 2: Arthropods caught with inventory sampling of three trees in each orchard
Field 1 Tree 1 Tree 2 Tree 3 Sum Caelifera 0 0 0 0 Psocoptera 0 0 6 6 Thysanoptera 2 0 0 2 Sternorrhyncha 5 1651 38 1694 Heteroptera 0 0 2 2 Coleoptera 5 8 5 18 Hymenoptera 13 102 8 123 Lepidoptera 0 0 0 0 Diptera 103 23 24 150 Araneae 24 0 29 54 Acari 0 4 0 4 Field 2 Tree 1 Tree 2 Tree 3 Sum Caelifera 0 1 0 1 Psocoptera 0 9 0 9 Thysanoptera 0 0 0 0 Stenorrhyncha 22 34 24 80 Heteroptera 1 0 2 3 Coleoptera 1 3 12 16 Hymenoptera 2 1 6 9 Lepidoptera 1 0 0 1 Diptera 5 21 21 47 Araneae 1 2 3 6 Acari 0 0 0 0
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Orchard 1 was managed more extensively than Orchard 2. There was no pruning in Orchard 1 for the last two years. Additionally, the pesticide usage for Orchard 1 was lower and the orchard was closer to the facility. In Orchard 2 the application of insecticides started in May and ended in October the previous year, while in Orchard 1 the last application was in September and the first application of an insecticide in June. The shape of Orchard 1 was more regular than Orchard 2. While Orchard 1 had a rectangular shape (180 m x 160 m) that of Orchard 2 was a pentagram, separated into 5 parts on different terraces. The surroundings were very diverse for both orchards. At the end of the pilot study the decision was taken to run the study in Orchard 1. The decision was based on the start of a heavy infestation of aphids, the shape and the maintenance of the orchard, and the smaller distance to the test facility. Discussion The pilot studies were confirmed as a valuable tool for both the contract facility and the sponsor. The pilot study gave more background information on the field site than a short visit to the sites. This included factors that were considered for the later randomisation of the plots, for instance the shape of the fields and the soil type. These factors (arthropod fauna in the field, field history, surroundings and contact with the farmer) were described in much more detail than normally possible. Also, the potential for recovery from the surrounding habitats as well as the taxa already present in the field gave some indication of the suitability of the site for the later field study. The results of the sampling from the pilot study in arable crops does not always give information about the development of the populations later in the season The pilot studies in arable crops were carried out in several localities at similar crop stages and period of the year. This gave indications of differences in species composition in the fields before the main arthropod activity began (presence of important groups). For the orchard study, the sampling of the chosen field was used as a pre-sampling before the first application.
For the sponsor, the pilot study has the advantage of providing data prior to a final decision about the field study. Since the pilot studies were run shortly before the projected start of the large-scale main study, the application dates could be scheduled with more precision. A further advantage was the introduction of the personnel involved in the main study to the sampling techniques, the field site and to any study-specific problems that might be encountered. It also was an opportunity for a rough introduction into the basic taxonomy of the sampled organisms for the technical personal involved in a later study.
A short summary about the advantages of a pilot study: • A collection of needed data within a short period • Sponsor has an influence on the final decision about field site • Reduces the risk of failure (cost of study) • Can be used as training for technical personnel • Collection can be used as first samples before the application Conclusion The pilot study will not exclude all the risks that a large-scale study possesses in terms of the populations needed for a large field study and the financial involvement. However, it can give in a very short period a good overview on the potential of different field sites. From the experiments of the last year criteria were underlined, that are as important as the arthropod
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fauna of the field. These are the surroundings of the field site and the cooperation with the farmer or owner of the field. References Anonymous 1995: Guideline to Study the Within-Season Effects of Insecticides on Non-
Target Terrestrial Arthropods in Cereals in Summer. Part three, A3, Appendix 2 (formerly working document 7/7) of data requirements on pesticides. Ministry of Agriculture Fisheries and Food, London.
Brown, K.C. 1989: The designs of experiments to assess the effects of pesticides on beneficial arthropods in orchards: replication versus plot size. In: Pesticides and Non-target Invertebrates. P.C. Jepson (ed.). London, Intercept, Dorset: 71-80.
Brown, K.C. 1998: Field studies with pesticides and non-target arthropods in orchards. In: Ecotoxicology: pesticides and beneficial organisms. Haskell, P.T. & McEwen (eds.). London, Chapman & Hall: 139-147.
Candolfi, M., Bigler, F.,Campbell, P., Heimbach, U., Schmuck, R., Angeli, G., Bakker, F., Brown, K., Carli, G., Dinter, A., Forti, D., Forster, R., Gathmann, A., Hassan, S., Mead-Briggs, M., Melandri, M., Neumann, P., Pasqualini, E., Powell, W., Reboulet, J.-N., Romijn, K., Sechser, B., Thieme, Th., Ufer, A., Vergnet, Ch. & Vogt, H., 2000: Principles for regulatory testing and interpretation of semi-field and field studies with non-target arthropods. Anzeiger für Schädlingskunde 73: 141-147.
Mead-Briggs, M. 1998: The value of large-scale field trials for determining the effects of pesticides on the non-target arthropod fauna of cereal crops. In: Ecotoxicology: Pesticides and beneficial organisms. Haskell, P.T. & McEwen (eds.), London, Chapman & Hall: 182-190.
Thacker, J.R.M. & Jepson, P.C. 1993: Pesticide risk assessment and non-target invertebrates: integrating population depletion, population recovery and experimental design. Bulletin Environmental Contamination and Toxicology 51: 357-368.
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Pesticides and Beneficial Organisms IOBC/wprs Bulletin Vol. 24 (4) 2001
pp. 97 - 101
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Side effects of some pesticides on predatory mites (Phytoseiidae) in citrus orchards
G. Viggiani1 & U. Bernardo2 1 Dipartimento di Entomologia e Zoologia Agraria, Università degli Studi di Napoli
“Federico II”, Via Università 100, I-80055 Portici (NA), Italia 2 Centro di Studio CNR sulle Tecniche di Lotta Biologica, Via Università 133, I-80055 Portici
(NA), Italia
Abstract: Several trials were carried out in Campania (Southern Italy) for the evaluation of the side effects of some pesticides on predatory mites in citrus orchards, where Amblyseius stipulatus A. H. is the dominant species. The tested products were the following: Biolid E (a.i.: mineral oil 80 %) at 1000 ml/hl, Biolid E (a.i.: mineral oil 80 %) + Folithion (a.i.: fenitrothion 48,5 %) at 1000 ml/hl + 150 ml/hl, Magister 200 SC (a.i.: fenazaquin 18,32%) at 35 ml/hl and Oikos (a.i.: azadirachtin A 3,2 %) at 150 ml/hl. In unsprayed citrus (lemon and orange) orchards, trees with a sufficient number of predatory mites were selected by sampling at random 20 leaves/tree, before treatment. The plants were treated by using a knapsack sprayer. Each product was tested at least in 4 replications, distributed at random and represented by a single tree or three trees/replication. The control was treated with water. After 1, 5 days and subsequently at 7-day intervals, the mobile population of predatory mites was recorded by sampling at random 20 leaves/tree. For the data evaluation the Henderson-Tilton formula was used. Results showed the good selectivity (OILB/IOBC category: 1) of Biolid E and Oikos. Folithion, mixed in rather low dosage as recommended to control mealybugs when needed, had toxic effect (OILB/IOBC category: 4), but the detrimental action persisted at maximum between 13 and 20 days after treatment. Finally, Magister showed in general the highest and longest (>30 days) toxic effect (OILB/IOBC categories: 4). Keywords: citrus orchards, predatory mites, Amblyseius stipulatus, side effects, field test, persistence Introduction Predatory mites are a very important factor in the natural control of phytophagous mites in citrus orchards. In Southern Italy, where the spider mites Tetranychus urticae Koch and Panonychus citri (McGregor) are present, the dominant predatory species is Amblyseius stipulatus A. H. (Viggiani, 1982). To select products that can preserve the complex of the predatory mites and other beneficials, several field trials were carried out. In this paper the obtained results are presented. Material and methods A first trial was carried out in Amalfi (Campania, Salerno province) on lemon (cv. Sfusato amalfitano), in a typical, terraced cultivation, each with at least ten plants irregularly disposed.
At least 10 plants in four plots were treated on 25.5.2000 with the pesticides reported in table 1 and a fifth plot (check) with water. In each plot five plants were considered replications.The density of predatory mites (mobile stages/leaf) was evaluted the day before
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the treatment by counting with a magnifying lens on 20 leaves/tree, sampled at random on each plant/plot.
All plants were treated by using a knapsack sprayer and the control was treated with water. In this trial, as in the others, an amount of 1000 l/ha was used.
After 1, 6 days and subsequently at 7-day intervals, the mobile population of predatory mites was recorded using method above-mentioned.
Table 1. Pesticides tested
Trade name Active ingredient Formulation %
Dosage (cc/hl)
Biolid E Mineral oil 80 1000 Biolid E+ Folithion
Mineral oil + Fenitrothion
80 + 48.5
1000+150
Magister Fenazaquin 18.32 35 Oikos Azadirachtin A 3.2 150
A second trial was carried out in Portici (Campania, Napoli province) in a familiar
orange orchard (cv. Washington navel), in a plantation design of 6 x 6 m. The experimental design was fully randomized with 4 replications (one plant for replication) per product.
The tested products were the same as in the first trial, except Biolid E. A third trial was carried out in a lemon orchard located in Nocera Inferiore (Campania:
Salerno province) where only Oikos was tested in randomized plots formed by three plants with four replications.
Results were calculated with the Henderson-Tilton formula (100 x (1-(K1/K2 x R2/R1)) and divided into four categories corresponding to the set standard for field methods of the IOBC Working group “Pesticide and Beneficial Organisms”. Results and discussion Amalfi trial Of all used products, the mixture Biolid E+Folithion affected the density of mobile phytoseiids/leaf more heavily, but the detrimental action persisted no longer than two weeks (Fig. 1).The effect of Magister was moderate, but moderately persistent (Table 2). No or very slight effect was shown by Biolid E and Oikos. Portici trial Data obtained in this trial confirmed those of Amalfi, except for Magister which was more toxic and long persistent (Fig. 2, Table 3). Nocera trial In this trial the harmless effect of Oikos on the predatory mite populations was confirmed (Fig. 3, Table 4).
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Fig. 1. Effects of pesticides on number of mobile phytoseiids/leaf. – Amalfi trial. Table 2. Reduction (%) of phytoseiid population according to Henderson-Tilton caused by the tested pesticides and evaluation categories (Cl) according to OILB/IOBC. – Amalfi trial.
Trade name T + 1 T + 6 T + 13 T + 20 T + 27 % Cl % Cl % Cl % Cl % Cl Biolid E 14,6 1 0 1 0 1 0 1 0 1 Biolid E + Folithion
76,5 4 59,5 3 20,3 1 0 1 0 1
Magister 36,0 2 7,3 1 41,5 2 40,7 2 30,0 2 Oikos 0 1 0 1 0,7 1 0 1 20,0 1
Table 3. Reduction (%) of phytoseiid population according to Henderson-Tilton caused by the tested pesticides and evaluation categories (Cl) according to OILB/IOBC. – Portici trial.
Trade name T + 1 T + 6 T + 13 T + 20 T + 27 T + 34 % Cl % Cl % Cl % Cl % Cl % Cl Biolid E + Folithion
89,6 4 75,9 4 49,9 2 12,8 1 4,6 1 0 1
Magister 73,5 3 84,3 4 80,6 4 73,6 3 76,0 4 52,1 3 Oikos 0 1 22,8 1 1,8 1 0 1 0 1 0 1
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Fig. 2. Effects of pesticides on number of mobile phytoseiids/leaf. – Portici trial. Fig. 3. Effects of pesticides on number of mobile phytoseiids/leaf. – Nocera trial. Table 4. Reduction (%) of phytoseiid population according to Henderson-Tilton caused by the tested pesticide and evaluation categories (Cl) according to OILB/IOBC. – Nocera trial.
Trade name T + 1 T + 6 T + 13 % Cl % Cl % Cl Oikos 18,6 1 23,5 1 19,0 1
In conclusion, in all trials Biolid E and Oikos revealed to be selective for predatory mites. Biolid E+Folithion showed high detrimental effect (OILB/IOBC categories: 4) until 6 days
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from treatment; later the mite predatory population was slightly affected. In the first trial Magister caused a slight toxic effect, but it persisted more than 27 days. On the contrary, in the second trial and also in another one not included in this paper, the product showed high and persistent detrimental effect (OILB/IOBC category: 4), as reported by other authors (Sterk et al., 1994; Espinha et al., 1999). According to the known data, the use of Magister in IPM programs should be restricted or not allowed. References Espinha, I.G., Torres, L. M., Avilla, J. & Carlos, C. 1999: Testing the side effects of
pesticides on phytoseiid mites (Acari, Phytoseiidae) in field trials. XIV International Plant Protection Congress, Jerusalem, Israel, July 25-30, 1999. Abstracts: 113.
Sterk, G., Creemers, P. & Merckx, K. 1994: Testing the side effects of pesticides on the predatory mite Typhlodromus pyri (Acari, Phytoseiidae) in field trials. IOBC wprs Bulletin 17 (10): 27-40.
Viggiani, G. 1982: Effetti collaterali di fitofarmaci su acari fitoseidi degli agrumi. Atti Giornate fitopatologiche 1982. Cooperativa Libraria Universitaria Editrice Bologna, 3: 171-179.
Viggiani, G. & Tranfaglia, A. 1978: A method for laboratory test of side effects of pesticides on Leptomastix dactylopii (How.) (Hym. Encyrtidae). Boll. Lab. Ent. agr. "Filippo Silvestri" 35: 8-15.
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Pesticides and Beneficial Organisms IOBC/wprs Bulletin Vol. 24 (4) 2001
pp. 103 - 112
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Side-effects of pesticides on selected natural enemies occurring in citrus in Spain Josep-Anton Jacas Miret1 & Ferran Garcia-Marí2 1 Departament de Ciències Experimentals, Universitat Jaume I, Campus de Riu Sec, E-12071
Castelló de la Plana (Spain), E-mail: [email protected] 2 Departament d’Ecosistemes Agroforestals, ETSE Agrònoms, Universitat Politècnica de
València, E-46022 València (Spain), E-mail: [email protected] Abstract: Spain is one of the largest producers of citrus for the fresh market worldwide (around 5.5 million tons in 1998), mainly oranges, tangerines and lemons. Many of our potential pests are kept under excellent (Icerya purchasi, Insulaspis gloverii) or satisfactory (Aleurothrixus floccosus, Panonychus citri, Chrysomphalus dyctiospermi, Coccus hesperidum, Ceroplastes sinensis, Plano-coccus citri, Saissetia oleae) natural control. Three scales are considered the key pests of citrus in Spain: Parlatoria pergandei, Cornuaspis beckii and Aonidiella aurantii. Besides, aphids (Aphis gossypii, A. spiraecola and Toxoptera aurantii), Tetranychus urticae and Phyllocnistis citrella may occasionally require especial attention from the grower, and, because of quarantine regulations, Ceratitis capitata is subjected to mandatory control by governmental agencies. This means pesticides applied against these pests should be as harmless as possible against natural enemies responsible for natural control of the former group of phytophages. Side-effect testing of pesticides with the most important natural enemies (Rodolia cardinalis, Cales noacki, Euseius stipulatus, Cryptolaemus montrouzieri, and Leptomastix dactylopii) has been routinely done in Spain for many years. Results of both laboratory and field assays are presented and discussed. Keywords: citrus, pesticide side-effects, Rodolia cardinalis, Cales noacki, Euseius stipulatus, Cryptolaemus montrouzieri, Leptomastix dactylopii, Lysiphlebus testaceipes. Introduction Implementation of integrated pest management (IPM) strategies requires the evaluation of the impact of pesticides on the natural enemies of pests. Selective pesticides that can be used to control pests without adversely disrupting the activity of relevant natural enemies are needed for modern pest management. Citrus IPM has since long recognised the need for such data. For many years, researchers working on citrus IPM in Spain (Grupo de Trabajo de Cítricos, GTC) have been routinely testing the side-effects of the most commonly used pesticides on the most relevant natural enemies. As a consequence, the GTC has produced a database containing around 270 records referred to 6 important citrus biocontrol agents and 80 different pesticides. These natural enemies are the following: Cales noacki (Hymenoptera, Aphelinidae), a parasitoid of the woolly whitefly Aleurothrixus floccosus (Homoptera, Aleurodidae), Cryptolaemus montrouzieri (Coleoptera, Coccinellidae), a predator of the citrus mealybug Planococcus citri (Homoptera, Pseudococcidae), Euseius stipulatus (Acarina, Phytoseiidae), a predator of the citrus brown mite Panonychus citri (Acarina, Tetranychidae), Leptomastix dactylopii (Hymenoptera, Encyrtidae), a parasitoid of the citrus mealybug, Lysiphlebus testaceipes (Hymenoptera, Braconidae), a parasitoid of aphids, and Rodolia cardinalis (Coleoptera, Coccinellidae), a predator of the cottony cushion scale Icerya purchasi (Homoptera, Margarodidae). These natural enemies are responsible of their
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prey/hosts being categorised as under either excellent or satisfactory biological control (Ripollés et al., 1995). Nevertheless, some citrus pests are not under good biological control yet. These include the chaff scale, Parlatoria pergandei (Homoptera, Diaspididae), the purple scale, Cornuaspis beckii (Homoptera, Diaspididae), and the red scale, Aonidiella aurantii (Homoptera, Diaspididae). Besides, aphids (Aphis gossypii, A. spiraecola and Toxoptera aurantii), the two-spotted spider mite Tetranychus urticae (Acarina, Tetranychidae), and the citrus leaf miner, Phyllocnistis citrella (Lepidoptera, Gracillariidae) may occasionally require especial attention from the grower. Because of quarantine regulations, the medfly, Ceratitis capitata (Diptera, Tephritidae) is subjected to mandatory control by governmental agencies. Chemical control is commonly used against these pests. Therefore, conservation tactics aimed at maximising the activity of natural enemies responsible for the biological control of the former group of phytophages should be implemented, and testing the side-effects of pesticides on these beneficials is necessary. Materials and methods The bulk of the data presented come from internal reports of the GTC. Some of these data have also been published elsewhere (Castañer et al., 1988; Costa-Comelles et al., 1994, 1997; Departamento de Producción Vegetal, 1995; Garrido, 1983, 1992, 1999; Garrido & Beitia, 1992; Garrido & del Busto, 1986; Garrido et al., 1986; Garrido & Ventura, 1993; Ripollés, 1986; Sterk et al., 1999). Methodology used for each species is given in Table 1. Effects were categorised in four classes (1: harmless, 2: slightly harmful, 3: moderately harmful, and 4: harmful) determined by preset threshold values according to the IOBC WG „Pesticides and Beneficial Organisms“ (Sterk et al., 1999). Pesticides were always applied at the highest recommended field rate, and although they are listed according to their active ingredient, commercial products were always used. Table 1. Test methods used in the assays considered. Species Method Exposure
type Exposure substrate
Stage Variable
Euseius stipulatus
Costa-Comelles et al., 1994
Field, Direct spray Citrus tree Mixed Initial toxicity
Rodolia cardinalis
Garrido & del Busto, 1986
Laboratory, Direct spray Citrus leaf Pupae Initial toxicity
Cryptolaemus montrouzieri
Ripollés, 1986 Laboratory, Direct spray Glass Pupae Initial toxicity
Leptomastix dactylopii
Ripollés, 1986 Laboratory, Dry residue Glass Adults Initial toxicity
Lysiphlebus testaceipes
Garrido et al., 1986
Laboratory, Direct spray Citrus leaf Parasitized
pupae Initial toxicity
Cales noacki
Garrido, 1992 Laboratory, Direct spray Citrus leaf Parasitized
pupae Initial toxicity
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Table 2. Effects of different plant protection compounds on the selected natural enemies. Shadowed: Active ingredients allowed under Integrated Production labelling (Government of the Comunitat Valenciana). Rating according to the 4 categories established by the IOBC WG “Pesticides and Beneficial Organisms”.
Euse
ius
stip
ulat
us
Cry
ptol
aem
us
mon
trou
zier
i
Rodo
lia
card
inal
is
Lept
omas
tix
dact
ylop
ii
Lysi
phle
bus
test
acei
pes
Cal
es n
oack
i
ORGANOCHLORINE Endosulfan (Thiodan) 2-3 3 1 3 1 1 Lindane (Agronexa-60) 2 3
ORGANOPHOSPATES Acephate (Orthene) 4 4 4 1-2 Carbofenthion (Spider Spray) 1 3 Chlorfenvinfos (Birlane) 2-3 4 3 2 1 2-3 Chlorpyrifos (Dursban) 2 2 1-2 3 3 2-3 Diazinon (Diazinon) 2 3 3 1-2 Dimethoate (Perfektion) 2-3 4 1 4 1-2 2 Ethion (Ethion Emuls.) 2-3 1 1 1 2 1 Ethylazinfos (Azifene) 1 3 Etrimfos (Ekamet) 1 2 2 3 Fenitrothion (1) 3 1-2 4 1-2 3 Fenthion (Lebaycid) 3 2 1 2 2 Fentoate (Cidalina) 4 1 3 3 Fosalone (Zolone) 2 1 3 Fosfamidon (Dimecron) 3-4 3 1 Fosmet (Imidan) 2-3 4 4 3 1 4 Malathion (Volktion) 2 4 2-3 4 3 3-4 Mecarbam (Murfotox) 1-2 4 Metamidofos (Tamaron) 3 1 2-3 Methidathion (Ultracid) 2-3 3 2 3-4 2 3 Methylazinfos (Gusathion) 2-3 3-4 3 3-4 1 1-2 Methylchlorpyriphos (Reldan) 3 1 1 3-4 3 1-2 Methyloxydemeton(Metasystox) 2 3 1 3 1 1-2 Methylparathion (Folidol) 1-2 2 4 Methylpyrimifos (Actellic) 1-2 1-2 4 3-4 Mevinfos (Fosdrin) 2-3 Ometoate (Folimat) 3-4 1 Pyridafenthion (Ofunack) 1 4 Quinalfos(Ekalux) 2-3 4 3 3-4 2 3 Tetraclorvinfos (Gardona) 1 1 3 Tiometon (Ekatin) 2 3 1 3 Triazofos (Hostathion) 2 3 Triclorfon (Dipterex) 1 1 2-3 1-2 3 2
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Euse
ius
stip
ulat
us
Cry
ptol
aem
us
mon
trou
zier
i
Rodo
lia
card
inal
is
Lept
omas
tix
dact
ylop
ii
Lysi
phle
bus
test
acei
pes
Cal
es n
oack
i
CARBAMATES Carbaryl (Sevin) 3-4 2 1 2-3 Carbosulfan (Marshal) 1-2 1-2 Etiofencarb (Croneton) 2 2 2 2 1 1 Mehtomyl (Lannate) 4 4 4 2 3-4 Pirimicarb (ZZ-Aphox) 1-2 2 1-2 1 1 1
PYRETHROIDS Bifentrin (Talstar) 3-4 3-4 1 Cihalotrin (Karate) 3 Cipermethrin (Ambush C) 4 4 4 1 3-4 Deltamethrin (Decis) 4 3-4 4 4 1 3 Fenvalerate (Sumicidin) 3-4 4 1 Flucitrinate (Cybolt) 2 3 Fluvalinate (Klartan) 3-4 3 4 3 1 2 Permethrin(Ambush) 4 4
JUVENILE HORMONE ANALOGUES Fenoxycarb (Insegar) 1-2 4 2 Pyriproxifen (Atominal) 1 4 4 2-3
INSECT GROWTH REGULATORS Buprofezin (Applaud) 1-2 3 1 Diflubenzuron (Dimilin) 1 Flufenoxuron (Cascade) 2-3 1-2 Hexaflumuron (Consult) 1 Lufenuron (Match) 1 1 4 1 Teflubenzuron (Nomolt) 1
MOULTING ACCELERATING COMPOUNDS Tebufenozide (Mimic) 1
NEONICOTINOIDS Imidacloprid (Confidor) 2-3 4 3
ACARICIDES Amitraz (Mitac) 4 1 1 2-3 Benzoximate (Artaban) 2 1-2 Bromopropilate (Neoron) 3-4 3 1 2-3 Clofentezine (Apollo) 1-2 1 Dicofol (Kelthane) 4 1 1 1 2 Fenazaquin (Magister) 4 3 4 Fenbutatin oxide (Torque) 2 1 1 1 1 Fenotiocarb (Panocon) 3 1-2
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Euse
ius
stip
ulat
us
Cry
ptol
aem
us
mon
trou
zier
i
Rodo
lia
card
inal
is
Lept
omas
tix
dact
ylop
ii
Lysi
phle
bus
test
acei
pes
Cal
es n
oack
i
Hexithiazox (César) 1 1 Pyridaben (Sanmite) 4 Propargite (Omite) 4 2-3 Tetradifon (Tedion) 1-2
FUNGICIDES Captan (1) 1-2 1 1 Copper Oxychloride (1) 1-2 1 1 Zineb (1) 1
OTHERS Avermectin (Vertimek) 2-3 Azadirachtin (1) 1 B. thuringiensis (Delfin) 1 1 1 1 1 Benfuracarb (Oncol) 1 Petroleum Spray Oil 1-2 1 1 2 2-3 Soap sprays 1 1 Butocarboxim (Darwin) 4 2 2 3-4 1 1
(1) Different commercial products were tested Results and discussion Results are shown in Table 2. By comparing average toxicities against the different beneficial species tested, the following pattern of sensitivity was obtained: L. testaceipes (average toxicity, a.t.: 1.5 ± 0.1; n = 43) > R. cardinalis (a.t.: 2.2 ± 0.2; n = 39) > C. noacki (a.t.: 2.3 ± 0.1; n = 62) > E. stipulatus (a.t.: 2.4 ± 0.1; n = 60) > C. montrouzieri (a.t.: 2.6 ± 0.2; n = 39) > L. dactylopii (a.t.: 2.7 ± 0.2; n = 30). Therefore, L. dactylopii appeared to be the most sensitive beneficial. This is not surprising because this was the only beneficial tested as adult (the sensitive stage), whereas the others were tested in their protected stages (Table 1). Unfortunately no assays on the most exposed stages of the other citrus beneficials have been routinely performed. In a recent comparison of sensitivity of different non-target arthropod species and laboratory test systems to insecticides included in the IOBC Joint Pesticide Testing Programmes (Vogt, 2000), L. dactylopii ranked fourth out of six adult parasitoids tested, and tenth out of 17 when including all beneficials considered. Nevertheless, it should be kept in mind that all parasitoids reacted very similarly against insecticides (Vogt, 2000). Therefore L. dactylopii can not be considered as a specially sensitive species to insecticides.
In general, and as expected, pyrethroids (average toxicity: 3.0 ± 0.2; n = 25), organo-phosphates (a.t.: 2.4 ± 0.2; n = 134) and organochlorine (a.t.: 2.1 ± 0.3; n = 8) exhibited the most harmful effects against the selected natural enemies. There were very few exceptions to that rule. In contrast, selective products could be sorted out from the other groups of pesticides. Nevertheless, very selective pesticides, such as pirimicarb or fenbutatin oxide are
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no longer recommended in citrus because resistance from target pest species (aphids and the two-spotted spider mite, respectively) has appeared (Viñuela, 1998).
Table 3. Toxicity of active ingredients allowed under Integrated Production (Government of the Comunitat Valenciana). Classification according to IOBC WG “Pesticides and Benficial Organisms” standards, both for pests and beneficials. For beneficials, results from the SELCTV databank (toxicity scale 1-5) are also provided.
Average toxicity against natural enemiesActive ingredient Pest (toxicity) GTC (n) SELCTV (n)
Chlorpyrifos Scales (4), Cacoecimorpha pronubana(4), Flower moth (4)
2.3 (6) 3.06 (34)
Dimethoate Aphids (2-4) 2.5 (6) 2.97 (209) Malathion Medfly (4) 3.2 (6) 3.12 (256) Methidathion Scales (3-4) 2.7 (6) 2.88 (42) Methylpyrimifos Scales (3-4) 2.6 (4) 3.83 (12) Carbosulfan Aphids (3-4) 1.5 (1) – Diflubenzuron Leafminer (4) 1.0 (1) 1.97 (77) Flufenoxuron Spider mites (3-4),
leafminer (4) 2.0 (2) –
Hexaflumuron Leafminer (4) 1.0 (1) – Lufenuron Woolly white fly (3-
4), leafminer (4) 1.8 (4) –
Imidacloprid Leafminer (4) 3.2 (3) – Bromopropilate Eryophyes sheldoni
(4) 2.5 (4) 1.63 (8)
Dicofol Spider mites (4) 1.8 (5) 1.59 (68) Hexithiazox Spider mites (3-4) 1.0 (2) – Copper Oxichloride Phytophthora spp. 1.3 (3) – Avermectin Leafminer (4) 2.5 (1) 2.95 (18) B. thuringiensis Cacoecimorpha
pronubana (4), Flower moth (4)
1.0 (5) 0.00 (2)
Benfuracarb Aphids (3-4), leafminer (4) 1.0 (1) –
Petroleum Spray Oil Scales (3-4), spider mites (3-4) 1.6 (5) –
In contrast to other crops, Juvenile Hormone Analogues (average toxicity: 2.7 ± 0.5; n =
7), which are quite selective for parasitoids, have a very limited use in citrus because of their tremendous effect on relevant predators such as lady beetles. Recently, pyriproxifen has been recommended against armoured scales (A. aurantii, for example), but its use is restricted to once per season and careful monitoring of scale population is required. Secondary pest outbreaks have been sometimes observed after its use (Category 4 both for C. montrouzieri
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and R. cardinalis). This applies to Insect Growth Regulators (IGR’s), as well. IGR’s were extensively used following arrival of the citrus leafminer in 1993. Since then, its use against the leafminer under Integrated Production has been granted, but reports on cottony cushion scale explosions following their use have not been uncommon. Usually deformed adult R. cardinalis were found in these cases.
Some of the products appearing on Table 2 are no longer permitted in citrus, and many of them are no longer being recommended. One of the reasons for not endorsing these products is their impact on beneficials. Products recommended for Integrated Production in the Valencian Community have been shadowed on Table 2. In Table 3, these products have been listed and their toxicity against target pests (using the same scale as used for beneficials), as well as average toxicity against beneficials, both from GTC data and from SELCTV database (http://www.ent3.orst.edu/Phosure/database/selctv/selctv.htm) are presented. All these pro-ducts are quite effective against target pests (toxicity ranging from 3 to 4) whereas their effects on beneficials are either low (B. thuringiensis, Benfuracarb, Petroleum Spray Oils, etc.) or not long-lasting (Chlorpyrifos). Although average toxicities are helpful, in some cases they can be misleading. For example, the acaricide Dicofol has an average toxicity of 1.8 (n = 5), but it actually is very harmful for E. stipulatus. Selective use of some of the most harmful products, such as imidacloprid or malathion is achieved by exploiting ecological selectivity (Croft, 1990): either trunk painting or drip irrigation for imidacloprid, or bait treatments with malathion. Other harmful products are only recommended in spring, when natural enemies are not present (Carbosulfan and Benfuracarb against aphids; Bromopropilate against Aceria sheldoni). In some cases, spring should be avoided. This is the case of imidacloprid, which is not recommended from April till July in order to protect immigrant R. cardinalis who invade orchards at that time. Nevertheless, where there is no choice, harmful long-lasting products are still recommended (metidathion against black scale, or dimethoate against aphids). Future changes in that list will certainly occur. New more selective active ingredients may appear, but well known selective products, such as petroleum spray oils, as well as other types of oil, will certainly play a crucial role in future citrus IPM. Acknowledgements To J.M. Llorens, convenor of the Grupo de Trabajo de Cítricos, on behalf of all testing members whose results were compiled in this study. References Castañer, M., A. Garrido & T. del Busto. 1988: Comportamiento del metil-oxidemetón sobre
Cryptolaemus montrouzieri Muls. Fruits 43 (5): 325-330. Costa-Comelles, J., A. Soto, A. Alonso, J.M. Martínez & F. García-Marí. 1994: El pulgón Aphis
gossypii Glover: eficacia de algunos plaguicidas en cítricos y su acción sobre el fitoseído Euseius stipulatus A.H. Levante Agrícola 328: 201-213.
Costa-Comelles, J., A. Santamaria, A. Alonso, J.M. Rodríguez, D. Troncho, D. Alonso, C. Marzal & F. García-Marí. 1997: Efectos secundarios sobre el fitoseído Euseius stipulatus A.H. de los plaguicidas utilizados en el control químico del minador de hojas de cítricos. Levante Agrícola 339: 140-144.
Croft, BA. 1990: Arthropod biological control agents and pesticides. John Wiley & Sons, New York, USA: 723 pp.
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Departamento de Producción Vegetal. 1995: Ensayo de campo de efecto del plaguicida Match (Lufenurón) sobre el ácaro fitoseído Euseius stipulatus A.H. en cítricos. Levante Agrícola 331: 134-135.
Garrido, A. 1999: Fauna útil en cítricos: control de plagas. Levante Agrícola 347: 153-169. Garrido, A. 1992: Pesticides toxicity over pupal of Cales noacki Howard (Hym.: Aphelinidae).
IOBC/WPRS Bull. 15 (3): 56-60. Garrido, A. 1983: Enemigos naturales de la mosca blanca de los cítricos (Aleurothrixus floccosus)
y métodos de control. Levante Agrícola 246: 77-83. Garrido A. & F. Beitia. 1992: Plaguicidas y pequeños animales útiles en la agricultura. Levante
Agrícola 319: 106-113. Garrido, A. & T. del Busto. 1986: Algunas cochinillas no protegidas que pueden originar daños en
los cítricos españoles, I: Icerya purchasi Mask. (Subfamilia: Margarodinae). Levante Agrícola 267-8: 63-71.
Garrido, A., T. del Busto & J. Tarancón. 1986: Toxicidad de algunos plaguicidas en laboratorio sobre el parásito de pulgones, Lysiphlebus testaceipes (Gresson) (Hym. Aphidiidae). Actas del II Congreso Nal. Soc. Esp. Cienc. Hortíc. II: 973-981.
Garrido, A., J. Tarancón & T. del Busto. 1982: Incidencia de algunos plaguicidas sobre estados ninfales de Cales noacki How., parásito de Aleurothrixus floccosus Mask. Anales INIA/ Ser. Prot. Veg. 18: 73-96.
Garrido, A. & JJ. Ventura. 1993: Plagas de los cítricos. Bases para el manejo integrado. Ministerio de Agricultura, Pesca y Alimentación, Madrid, Spain: 183 pp.
Ripollés JL. 1986. La lucha biológica: utilización de entomófagos en la citricultura española. Integrated Pest Management in Citrus. Parasitis 86. Genève, CH.
Ripollés, JL., M. Marsà & M. Martínez. 1995. Desarrollo de un programa de control integrado de las plagas de los cítricos de las comarcas del Baix Ebre-Montsià. Levante Agrícola 332: 232-248.
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. Lewis, L. Moreth, L. Polgar, L. Rovesti, L. Samsøe-Petersen, B. Sauphanor, L. Schaub, A. Stäubli, J.J. Tuset, A. Vainio, M. Van de Veire, G. Viggiani, E. Viñuela & H. Vogt. 1999: Results of the seventh joint pesticide testing programme carried out by the IOBC/WPRS-Working Group “Pesticides and Beneficial Organisms”. BioControl 44: 99-177.
Viñuela, E. 1998: Insecticide resistance in horticultural crops in Spain. In: Pesticide resitance in horticultural crops. F.I.A.P.A., Almería (Spain): 117 pp.
Vogt, H. 2000: Sensitivity of non-target arthropod 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. 24 (4) 2001
pp. 113 - 120
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Impact of pesticides on beneficial arthropod fauna of olive groves Ruano, F. 1, Lozano, C.1, Tinaut, A.2, Peña, A.1, Pascual, F.2, García, P.3 & Campos, M.1 1 Estación Experimental del Zaidín, CSIC, Profesor Albareda, 1, E-18008 Granada, Spain 2 Departamento de Biología Animal y Ecología, Universidad de Granada, E-18071 Granada,
Spain 3 Departamento de Estadística e I.O., Universidad de Granada, E-18071 Granada, Spain Abstract. Effects of pesticides on the beneficial arthropod fauna of two commercial olive groves in the province of Granada (Southern Spain), with different management policies (treated and organic), have been studied. Samples of the arthropod fauna were taken monthly from the foliage of 30 trees between March and October 1999. There were significant differences in the communities of predatory as well as parasitoid arthropods in the two olive groves studied, both concerning the abundance as well as the faunistic composition and seasonal fluctuations. The group of predatory arthropods which appeared to be affected by chemical management were: Heteroptera, Coleoptera and Araneids. Neuropteroidea were only slightly affected in abundance by treatments, but their diversity was higher in the organic olive grove. Thus, Chrysoperla carnea was much more abundant and constant in the treated grove. Parasitoid populations in the chemically treated grove declined by 75% compared to the organic grove. Key words: Olive groves, organic management, pesticides, beneficial insects. Introduction Olive groves are very widely extended in Andalusia (South of Spain), representing about 43% of the total cultivated surface area. Control measures have mostly relied for years on the use of pesticides, but in this agroecosystem, different environmental problems have been reported by several authors, related to the excessive use of agrochemicals (Heim 1984, Pastor 1990, Cirio 1997, Civantos 1999). Negative effects of pesticides on the crop dynamics are well known and include, for example, development of resistance (Van Driesche & Bellows 1996, Polaszek et al. 1999); negative effects on the beneficial arthropod fauna (Croft, 1990) and explosion of new invasive pests (Kerns & Gaylor 1993, Van Driesche & Bellows 1996). In olive groves the application of non-selective pesticides, continuously and with unappropiate timing, has contributed to the elimination of parasitoids, predators and alternative preys (Heim 1984). For instance, aerial treatments against Bactrocera oleae (Gmelin) (Diptera: Tephritidae) induced an increase in the populations of Saissetia oleae (Olivier) (Homoptera: Coccidae) and other coccids (Homoptera: Coccoidea) (Lichtensia viburni Signoret, Parlatoria oleae (Colvée) and Pollinia pollini Costa) due to their negative effects on the beneficial fauna (Kapatos & Flecher 1983, Crovetti 1996). Similarly, for Prays oleae (Bernard) (Lepidoptera: Plutellidae), it has been reported that insecticidal treatments against the fruit generation reduce the activity of one of its main predators, Chrysoperla carnea (Stephens) (Neuroptera: Chrysopidae) (Fimianni 1965, Ramos et al 1978). Nevertheless, it is difficult to estimate the overall impact of pesticides in agroecosystems, since the diversity and abundance of beneficial insects present in them remain largely unquantified. However, such determinations have become possible with the establishment and spreading of organically
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grown olive groves in Andalusia (Spain). In these organic groves the use of fertilizer, fungicides and insecticides is regulated by the Council Regulation (EC) Nº 2092/91 (June 1991). Other characteristics of organic olive groves are compiled in Alonso Mielgo & Guzmán Casado (1999).
The objective of this study was to evaluate the effects of pesticides at the community level, on the main groups of beneficial arthropods present in olive groves, by comparing two management crop policies: conventional and organic. Materials and methods In 1999, two commercial olive groves were chosen in the province of Granada (Southern Spain) for performing the assays. They were located at similar altitudes, rather close (25 Km apart), with similar environmental characteristics but two opposite management policies. These two plots had the same surface (14.400 m2) and number of trees (144 trees).
One grove was under conventional intensive management (which we shall call treated) and the other was organic. The treated olive grove (Colomera) was drip irrigated, frequently ploughed deeply, and received at least three annual treatments of different insecticides, fungicides and herbicides: 1. At the end of March or beginning of April, a dimethoate spray against the phylophagous
generation of P. oleae, and a foliar fertilizer and copper were applied. 2. At the beginning of June, an α-cypermethrine spray against the anthophagous generation of
P. oleae, a foliar fertilizer and a soil treatment with the herbicide simazine were done. 3. At the end of October, dimethoate against B. oleae, red copper and potassium, and a second
herbicide treatment with simazine were done. Meanwhile, the organic olive orchard (Deifontes) was drip irrigated, ploughed only to a
shallow depth (10 cm) from the end of May to the beginning of June and neither permitted insecticides nor Bacillus thuringensis Berliner were applied during our study.
Monthly from March to October 1999, in both groves, 30 trees were randomly chosen and sampled by beating 4 limbs per tree (one per orientation) over an insect net of 50 cm in diameter. The samples were frozen and afterwards arthropods were separated from vegetal and inorganic remains. Adults and juveniles were identified and both considered for the total number of each taxa.
Data are presented for the main groups of natural enemies: parasitoids (Hymenoptera) and predators (Araneidae, Neuropteroidea, Coleoptera and Heteroptera). Samples were classified up to the family level in most cases and up to the superfamily level in the case of the hymenopteran parasitoids. In Neuropteroidea classification was undertaken up to the family level, except for the more abundant genera of Chrysopidae: Chrysoperla carnea and Mallada, Navás (Neuroptera: Chrysopidae). The taxonomic study of Araneidae was not undertaken.
A two-ways t of Student test was applied to the monthly average number of individuals per sample in each group, after testing homogeneity of variances (Levene test). A χ2 test was applied in order to compare the faunistic composition of Neuropteroidea taxa. The diversity of the Neuropteroidea assemblage was calculated through the index of Shannon (Magurran 1988). Results By analysing first the seasonal fluctuation of parasitoids and predators considering the total number of individuals collected every month, we can see major quantitative differences
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between the treated and the organic groves (Figure 1). A higher number of parasitoids and predators were collected from May to October in the organic grove. In October, before the pesticide application was done in this month, the parasitoid and predatory community of the treated grove had a tendency towards a recovery after 4 months without insecticide applications.
Figure 1. Seasonal fluctuation of the total number of parasitoids (circles) and predators (squares) for the organic (bold) and the treated (open) groves.
A more detailed study by groups, taking into account the average number of individuals per sample in each month, was also done (Figures 2 and 3). Number of araneids fluctuated seasonally in a similar way in both zones, but significant differences were detected in June, July, August and September (t Student test p < 0.0005, p < 0.0005, p = 0.009, p = 0.004 respectively) as well as in March (p = 0.001) in the two groves. The taxonomy of this group needs to be studied in depth to find out whether there also were qualitative differences in the faunistic composition of the two groves.
The predatory Coleoptera collected belonged mainly to the coccinelid family (87.8%). This group is not frequent in either zone until June, when it sharply increases its frequency in the organic olive grove. These differences are significant in June, July, August and September. In October significant differences arise again, but in this case caused by the recuperation of this group in the treated olive grove, while an inverse trend appears in the organic orchard (t Student test, p < 0.0001 in all cases).
The predatory Heteroptera, consisting primarily of Mirids (51.3%) and Anthocorids (43.4%), were practically absent from the treated olive grove and heavily abundant in the organic one during the months of June and July. This group was apparently more influenced by seasonal environmental changes and showed no trend to recover as it was observed in other groups such as Coleoptera. Significant differences arose only in June and August (t Student test, p < 0.0001, p = 0.013 respectively), because of the high variability observed in the organic grove in July.
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Figure 2. Mean number of individuals by sample (mean ± SE) for: A) Araneids, B) Predatory Coleoptera and C) Predatory Heteroptera. (Treated zone in white and organic grove dark grey. Bars mean SE. Stars show significant differences between groves: * p < 0.05; ** p < 0.005; *** p < 0.0005).
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Figure 3. Mean number of individuals by sample (mean ± SE) for: A) Parasitoids Hymenoptera and B) Neuropteroidea. (Treated zone in white and organic grove in dark grey. Bars mean SE. Stars show significant differences between groves: * p < 0.05; ** p < 0.005; *** p < 0.0005).
Parasitoid Hymenoptera (Figure 3) belonged mostly to the superfamily Chalcidoidea (87.7%) and quantitatively, they presented strong significant differences between groves. They were very abundant in the organic grove in June and July (t Student test, p < 0.0001) and significant differences were maintained in August and September (t Student test, p < 0.0001 and p = 0.005 respectively). Populations of the treated olive grove recovered very weakly in autumn, before the third treatment.
For the supraorder Neuropteroidea significant differences between the groves were only found in March, May and October (t Student test, p = 0.04, p<0.0001, p = 0.001), perhaps due to the small seasonal variations. Thus pesticides did not quantitatively affect the Neuropteroidea community.
Nevertheless, when we analyse the faunistic composition and the frequency of the different groups of Neuropteroidea (Table 1), significant differences do arise between the two olive groves (χ2= 107.13; df = 4; p < 0.0001). Taxa richness was similar in the two groves, but evenness was very different. Thus, Chrysoperla carnea appeared in a higher frequency in the treated grove, while in the organic one the genus Mallada was present in large
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proportions, as well as other Neuropteroidea belonging to the family Coniopterygidae or order Raphidioptera. Thus, the Neuropteroidea supraorder was more diverse in the organic grove. Table 1. Abundance of the different groups belonging to the Supraorder Neuropteroidea found in the two groves
NEUROPTEROIDEA Treated Organic
individuals larvae (adults)
% individuals larvae (adults)
%
Order Neuroptera (s.s.) Family Chrysopidae C. carnea 139 (38) 80 64 (16) 38 Mallada sp. 3 1 51 (1) 25 Chrysopidae
indet. 21 9 11 5
Family Coniopterygidae (18) 8 (38) 18 Order Raphidioptera (5) 2 (29) 14 Total no. of individuals 224 100 210 100Diversity (Shannon Index) 0.7 1.4
Discussion Beneficial insects were strongly affected by the conventional management of olive groves. Most of the groups were seriously affected by treatments, nevertheless predatory Coleoptera appeared to maintain a short-term recuperation ability.
Hymenoptera parasitoids were seriously affected by most insecticides, as it was already known (Jacas et al 1992, Jacas & Viñuela 1994, Viñuela et al 2000) and showed less recovery capacity. Heteroptera predators exhibited differences in abundance in both groves in certain moments and appeared to be more influenced by seasonality.
The araneids proved to be affected quantitatively by pesticides, although this group needs a more detailed study.
Neuropteroidea presented marked qualitative differences, reflected in an appreciable decline in diversity in the treated zone. C. carnea appeared to be strongly favoured in the treated zone, while the genus Mallada, the family Coniopterygidae and the order Raphidioptera are unfavoured in the treated zone.
Several factors might have contributed to the fact that C. carnea was so abundant in the treated olive grove, masking the total effect of pesticides on the Neuropteroidea. Some of these factors are the ability of C. carnea to develope resistance (Zaki et al 1999), at least in some populations (Sterk et al 1999). C. carnea is especially resistant to pyrethroids (Ishaaya 1993, Jansen 2000), but also to other insecticides (Jansen 2000) at least in some of its instars (Viñuela et al 1996, Vogt et al 1998, Viñuela et al 2000). The great flight capacity of dispersion of this species (Duelli 1984) and its ability to survive during periods of prey scarcity (Limburg & Rosenheim 1998) also contribute to enhance its possibilities of surviving in unpredictable habitats such as treated agroecosystems.
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Other behavioural (oviposition places, position of the larvae on the tree), biological (number of generations per year, eggs’ peduncle length and egg-laying size, hatching period) or ecological factors (absence of natural enemies) might also have an influence on the abundance of C. carnea in the treated grove. These aspects will be examined in future studies. Acknowledgements CICYT (project number AMB 98-0946) has supported this work. Authors thank Saturno Recio, Manuel Recio Alcalde and Jose Luis Romero Benitez for permission for using their olive groves, and Maria Ortega and Herminia Barroso for field and laboratory assistance. References Alonso Mielgo, A.M. & Guzmán Casado, G.I. 1999: Cultivo del Olivar en Agricultura
Ecológica. Ed. Junta de Andalucía. Sevilla: 11 pp. Cirio, V. 1997: Productos agroquímicos e impacto ambiental en olivicultura. Olivae 65: 32-
39. Civantos, M. 1999: Control de Plagas y Enfermedades del Olivar. Ed. C.O.I. Madrid: 207 pp. Croft, B.A. 1990: Arthropod Biological Control Agents and Pesticides. John Wiley & Sons.
New York: 723 pp. Crovetti, A. 1996: La defensa fitosanitaria. Desarrollo de metodologías y salvaguarda de la
producción y del medio ambiente. In: Enciclopedia Mundial del Olivo. C.O.I. (ed.): 225-250.
Duelli, P. 1984: Dispersal and oviposition strategies in Chrysoperla carnea. In: Progress in World's Neuropterology. Aspöck & Hötzel (eds.): 133-145.
Fimiani, P. 1965: Effetti del "Sevin" sull' entomofauna dell' olivo e degli agrumi. Is. Wnt. Agr. Portici 30: 3-8.
Heim, G. 1984: Effect of insecticidal sprays on predators and different arthropods found on olive trees in the north of Lebanon. In: Integrated Pest Control in Olive Groves. Cavalloro & Crovetti (eds.): 456-465.
Ishaaya, I. 1993: Insect detoxifying enzymes: their importance in pesticide synergism and resistance. Arch. Insect Biochem. Physiol. 22: 263-276.
Jacas, J. & Viñuela, E. 1994: Side-effects of pesticides on Opius concolor Szepl. (Hymenoptera, Braconidae), a parasitoid of the olive fruit fly. IOBC/wprs Bull. 17 (10): 143-146.
Jacas, J., Viñuela, E., Adan A., Budia, F., del Estal, P. & Marco, V. 1992: Laboratory evaluation of selected pesticides against Opius concolor Szepl. (Hymenoptera, Braconidae). Ann. Appl. Biol., TAC 13: 40-41.
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.
Kapatos, E.T. & Fletcher, B.S. 1983: Development of a pest management system for Dacus oleae in Corfu by utilizing ecological criteria. In: Fruit-flies of economic importance. Cavalloro (ed.), CEC/OILB Int. Symp. Proc., Athenas: 593-602.
Kerns, D.L. & Gaylor, M.J. 1993: Induction of cotton aphid outbreaks by insecticides in cotton. Crop Protection 12: 387-393.
Limburg, D.D. & Rosenheim, J.A. 1998: The role of extrafloral nectar in the diet of the common green lacewing larva Chrysoperla carnea. National Cotton Council: 1311-1313.
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Magurran, A.E. 1989: La Diversidad Ecológica y Su Medición. Ed. Vedrá. Barcelona: 200 pp.
Pastor, M. 1990: El no laboreo y otros sistemas de laboreo reducido en el cultivo del olivar. Olivae 34: 18-30.
Polaszek, A., Riches, C. & Lenné, J. M. 1999: The effects of pest management strategies on biodiversity in agroecosystems. In: Agrobiodiversity: Characterization, Utilization and Management. Wood & Lenné (eds.): 273-303.
Ramos, P., Campos, M. & Ramos, J.M. 1978: Osservazioni biologiche sui trattamenti contro la tignola dell'olivo (Prays oleae Bern., Lep. Plutellidae). Boll. Ent. Agr. "Filippo Silvestri" 35: 16-24.
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. Lewis, L. Moreth, L. Polgar, L. Rovesti, L. Samsøe-Petersen, B. Sauphanor, L. Schaub, A. Stäubli, J.J. Tuset, A. Vainio, M. Van de Veire, G. Viggiani, E. Viñuela & H. Vogt. 1999: Results of the seventh joint pesticide testing programme carried out by the IOBC/WPRS-Working Group “Pesticides and Beneficials Organisms”. BioControl 44: 99-117.
Van Driesche, R.G. & Bellows, T.S., Jr. 1996: Biological Control. Chapman & Hall, NY: 539 pp.
Viñuela, E., Adán, A., Smagghe, G., González, M., Medina, Mª.P., Budia, F., Vogt, H. & del Estal, P. 2000: Laboratory effects of ingestion of Azadirachtin by two pests (Ceratitis capitata and Spodoptera exigua) and three natural enemies (Chrysoperla carnea, Opius concolor and Podisus maculiventris). Biocontrol Sci. & Technol. 10 (2): 165-177.
Viñuela, E., Händel, U. & Vogt, H. 1996: Evaluación en campo de los efectos secundarios de dos plaguicidas de origen botánico, una piretrina natural y un extracto de neem, sobre Chrysoperla carnea Steph. (Neuroptera: Chrysopidae). Bol. San. Veg. Plagas 22 (1): 97-106.
Vogt, H., González, M., Adán, A., Smagghe, G. & Viñuela, E., 1998: Efectos secundarios de la azadiractina via contacto residual en larvas jóvenes del depredador Chrysoperla carnea (Stephens) (Neuroptera, Chrysopidae). Bol. San. Veg. Plagas 24: 67-78.
Zaki, F.N., Farag, N.A. & Abdel-Aziz Shadia, E. 1999: Evaluation of tolerance of Chrysoperla carnea Steph. (Neuropt., Chrysopidae) to successive insecticidal treatments. J. Appl. Entomol. 123: 299-302.
Pesticides and Beneficial Organisms IOBC/wprs Bulletin Vol. 24 (4) 2001
pp. 121 - 137
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Selectivity of Lufenuron (Match ®), Profenofos and mixtures of both versus cotton predators Burkhard Sechser 1, Sharif Ayoub 2 & Naglaa Monuir 2 1 Consultant for Syngenta Crop Protection AG 2 Syngenta Crop Protection AG, Kaha Experimental Station, Egypt Abstract: The predator fauna was monitored following the application of lufenuron, profenofos or the mixture of both in a test on Egyptian cotton over a period of 20 (profenofos) or 30 (all other treatments) day period by a combined drop cloth/DVac method. In each treatment 90 m of cotton row was sampled at -3, 3, 10, 20 and 30 days after application (DAA).
The recovery of the moulting inhibition effect caused by lufenuron was measured by the separate evaluation of young (L1-2) and old nymphs (L3-5) of the minute pirate bug Orius albidipennis. The development to older nymphal stages was interpreted as loss of the moulting inhibition effect and can be used as the parameter to measure recovery. Lufenuron was safe at the tested rates between 10 and 100 g ai/ha to all adult stages of the tested predator species. It allowed normal egglaying and -hatch of O. albidipennis. This was proved by the continuous presence of first instar nymphs, which must have hatched from freshly laid eggs.
There was a reduction of older nymphal populations after the application of lufenuron, but the degree and duration of the suppression could be proved to be dependent on the dosage. The reappearance of older nymphal stages became visible in the 10 DAA evaluations at the 10 g ai/ha rate, at 50 g ai/ha in the 20 DAA counts and at 100 g ai/ha 30 DAA.
Lufenuron proved to be safe to all adult stages of all predator species (Campylomma verbasci, Scymnus spp., Coccinella undecimpunctata, Paederus alfierii and all spider families). The moulting inhibition effect versus nymphs of the predatory mirid bug C. verbasci was of the same type as with O. albidipennis, but the mirid species occurred only in low numbers. It exerted also some moulting inhibition of larvae of C. carnea, but 30 DAA the populations were again comparable to the one in the untreated control.
Profenofos caused a temporary moderate reduction of the adults, and a stronger one of the nymphs of both predatory bug species. It was safe to larvae of C. carnea, adults of P. alfierii and Scymnus spp., and all stages of spiders. It caused some suppression of populations of C. undecimpunctata. Key words: selectivity, cotton, predators, lufenuron, profenofos Introduction Cotton is a crop with a manifold complex of chewing and sucking pests. Sucking pests such as aphids, jassids and whiteflies have a broad spectrum of predators, which can be useful to suppress or delay the outbreak of damaging populations. It is therefore important to know the effect of pesticides on predator populations in order to make best use of them in the most selective way.
Many insecticides belonging to different chemical classes are used for pest control in cotton, amongst them the insect growth inhibitors belonging to the benzoylphenylureas. One of these is lufenuron (Match®), which is applied for the control of chewing lepidopterous pests. Lufenuron interferes with the moulting process from one immature stage (larva or nymph) to the next one, but does not affect adults (Buholzer et al. 1992). By being safe to adults and therefore selective, it offers already an advantage over some of the older
established compounds (e.g. organophosphates, carbamates, pyrethroids) which have a broader spectrum of activity. Lufenuron's selectivity may therefore be enhanced by either a split application of a high dose into two lower ones or by mixing at low rates with other products. Pest control might be comparable to a single high rate application whilst the effect on predators might be reduced. The degree of moulting inhibition of immature predator stages and the duration seems to be largely dose-dependent.
The testing of these possible uses of lufenuron and its mixture with the organophospate profenofos (Curyom®) on cotton predators was the aim of this study. Materials and methods The experiment was carried out at the Novartis Experimental Station in Kaha in the Nile Delta, 30 km north of Cairo, Egypt. This offered the advantage of non-interference from farmers’ side during the testing period. Furthermore, Egypt offers the advantage of having a cotton predator complex which is rich in numbers of individuals but with a limited number of species. The predator complex includes the minute pirate bug (mainly Orius albidipennis), a mirid species (Campylomma verbasci), the green lacewing (Chrysoperla carnea), the ladybird beetles Scymnus spp. and Coccinella undecimpunctata, the rove beetle Paederus alfierii and several families of spiders (Araneae).
The test focused on the evaluation of lufenuron, which is registered at the rate of 40-50 g ai/ha for the control of the tobacco budworm worm (Heliothis virescens) on cotton in the Americas. A new registration at 100 g ai/ ha is intended for use against the harder to kill cotton bollworm (Helicoverpa armigera and H. punctigera) in Australia. There is also hope for an extended residual activity with the higher rate. To reduce the potential risk to the predator fauna, the split application of 2 x 50 g ai/ha was also included in the study. Mixtures of lufenuron at the low rates of 10 and 30 g ai/ha in mixture with profenofos were also tested to obtain selectivity data for these registered rates against cotton leafworms (Alabama argillacea, Spodoptera frugiperda) (10) and H. virescens (30) in the Latin Americas. The commonly used cotton insecticide profenofos served as a positive control. Details of the rates, formulations and number of sprays are given in Table 1.
The test fields were distributed over the experimental area of the Kaha Station. Each treatment was replicated three times and the size of the fields varied from 4767 to 6200 sqm. In each field one replicate of the seven treatments was tested and the size of the plots was between 296 and 456 sqm. Each plot had three or four strips of cotton rows of five metres length. A double row of cotton of totally 10m length was the size of a subsample and three subsamples were collected per replicate, i.e. a total length of 30 m per treatment and replicate. Each treatment was replicated three times. The double rows were altered at each sampling day and attention was paid not to sample the same row twice. An example of a sampling pattern is given for one replicate in Figure 1. In order to increase the occurrence and even distribution of the key predator O. albidipennis, 28 onions were planted in each cotton row, which attract thrips as their main prey. Seven to nine days prior to the prespray sampling, the onions were pulled out and laid out for drying. This forced thrips and O. albidipennis to migrate to the cotton plants.
The application of the products was done with a handheld sprayboom with 6 nozzles, covering two cotton rows at each spray run. The amount of the remaining spray liquid in a plot was measured at the end of the application in order to calculate the exact actual rate and spray volume applied per plot. Details on the application and additional information are given in Table 2.
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Table 1. Treatment list in selectivity test of lufenuron, profenofos and its mixtures in cotton, Kaha, Egypt, 2000.
Treatment applied on date* Treatments Formu-
lation Rate
g a.i./ha 1 2 3 4 1 Untreated Control 2 Lufenuron EC 050 50 x 3 Lufenuron EC 050 100 x 4 Lufenuron EC 050 50 x 2 x x 5 Curyom (Lufenuron + Profenofos) EC 550 110 x
6 Curyom (Lufenuron + Profenofos) EC 550 330 x
7 Profenofos EC 720 1000 x *Treatment dates: 1 = June 9; 2 = June 10; 3 = June 11; 4 = June 17
A combined drop cloth / D Vac suction technique was used for measuring the impact of
pesticides on the natural enemy complex in cotton (Sechser et al. 1998). A plastic sheet (5 x 1 m), fixed on the two longitudinal edges to slightly longer wooden sticks was rolled out in the furrow between two cotton rows without, as much as possible, disturbing the plants. Cotton plants on both sides were then shaken vigorously by hand as short a time as necessary. Immediately after shaking the sheet was lifted up with the two wooden bars fixed on both lateral sides. Litter and larger leaf material was removed quickly and the plastic sheet kept slightly shaken to keep dislodged arthropods irritated and avoid their escape. The dropped arthropod material was then sucked up with a modified battery driven car vacuum cleaner with a paper bag inside. The paper bag was removed after each subsampling process (5 m double row of cotton), the arthropod content inside killed with a few droplets of ether and the paper bag transferred afterwards for several hours in a plastic bag. Storage in the ether atmoshere guaranteed complete kill of all arthropods.
At the end of the sampling operation all bags were brought to a laboratory and paper bags removed after a few hours from the plastic bags and filled into bigger paper bags for storage. Removal from the plastic bags is essential to avoid moulding which would make the whole material uncountable.
The counting was done under a binocular microscope with a 20x magnification. The content of one bag was processed through a set of sieves to separate the material from most of the remaining litter and to split it up in various classes of size. Species at defined developmental stages normally concentrate in one size class which makes the evaluation much easier. The arthropod material was identified down to the genus/species level for the most relevant predators and separated by adult and immature stages where appropiate. In addition, small and big immature stages of the most relevant predator species, the minute pirate bug O. albidipennis, were separately counted in order to evaluate any growth inhibiting effect. The results were reported as number of specimens per total sampled row length (i.e. 90 m) per treatment.
Table 2. Details of application in selectivity test of lufenuron, profenofos and its mixtures in cotton, Kaha, Egypt, 2000.
Treatment g a.i./ha
Repl. Plot size m2 Net amount spray
liquid/ha
Intended rate g or ml
a.i./ha
Net amount g or ml a.i./ha
applied A 423,4 B 457
1 Untreated
control C 403,2 A 423,4 139 50 47,5 B 403,2 187,5 50 47,1
2 Lufenuron
50 g C 403,2 173,6 50 41,6 A 423,4 170,1 100 85 B 403,2 173,6 100 99,2
3 Lufenuron
100 g C 403,2 185 100 93 A 423,4 189 - 187.8 2 X 50 47.5 - 47.2 B 295,7 224.9 - 233.4 2 X 50 56.5 - 58.7
4 Lufenuron
2x50 g C 403,2 208.3 - 189.7 2 X 50 52.3 - 47.5 A 423,4 189 110 104 B 403,2 176 110 96,8
5 Luf/Prof 10/100 g C 403,2 193,4 110 106,4
A 423,4 189 330 313,1 B 403,3 179,8 330 298,7
6 Luf/Prof 30/300 g C 403,2 198,4 330 313,1
A 423,4 185,4 1000 909,8 B 295,7 209,7 1000 1056,8
7 Profenofos
1000 g C 403,2 193,4 1000 974,9 A better distribution of predator populations was intended by standardizing parameters as much as possible (selecting fields in a close neighbourhood, same scheme of seeding, fertilizing and irrigation for all fields). Variations in population densities were further avoided by limiting the time of sampling to the cooler morning hours (09 to 11 h), since arthropods have the tendency to hide later on during the hot day time. Since the size of the trial did not allow all sampling on one day, the application of the treatments and all samplings were done in the same sequence over three days (Table 3).
The population monitoring started with a prespray sampling at three days before, and followed up at three, ten and twenty days after application. Since there was still some impact of lufenuron observed on nymphs of O. albidipennis, another evaluation was done at thirty days after application in all lufenuron treatments.
The effect of the treatments on the populations of the various predator groups was expressed as percentage reduction/increase according to Abbott. The data collected on number were subjected to transformations into square-root values and were compared for significant effects (5%) with Duncan's Multiple Range Test for variables. This test controls the Type I comparisonwise error rate, not the experimentwise error. Figures with the same letter are not significantly different.
Figu
re 1
. Ex
ampl
e of
tria
l lay
out i
n fie
ld 3
2 on
the
Kah
a St
atio
n w
ith 7
trea
tmen
ts a
nd e
xam
ple
of m
arki
ng o
f thr
eesu
bsam
ple
site
s in
one
repl
icat
e in
the
untre
ated
con
trol
- 3 D
AA
*3
DA
A10
DA
A20
DA
A30
DA
A
*DA
A =
day
s afte
r app
licat
ion
Luf
enur
on 2
x 5
0 g
Lu
fenu
ron
100
g
Lu
fenu
ron
50 g
Luf/P
rof
10/1
00 g
Luf/P
rof 3
0/30
0 g
Prof
enof
os 1
000
g
Unt
reat
ed c
ontro
l
S t r
i p
t r
e a
t e
d b
u t
n o
t s
a m
p l
e d
S
t r i
p
t r e
a t
e d
b u
t n
o t
s a
m p
l e
d
Leg
end:
Sam
plin
g da
tes
Luf/P
rof 3
0/30
0 g
Luf/P
rof 3
0/30
0 g
Lu
fenu
ron
50 g
Lu
fenu
ron
50 g
Lu
fenu
ron
50 g
Lu
fenu
ron
100
g
Lu
fenu
ron
100
g
Lu
fenu
ron
2 x
50 g
Lu
fenu
ron
2 x
50 g
Luf
/Pro
f 10/
100
g
Luf
/Pro
f 10/
100
g
Prof
enof
os 1
000
g
Prof
enof
os 1
000
g
Unt
reat
ed c
ontro
l
Unt
reat
ed c
ontro
l
125
Figu
re 1
. Exa
mpl
e of
tria
l lay
out i
n fie
ld 3
2 on
the
Kah
a St
atio
n w
ith 7
trea
tmen
ts a
nd e
xam
ple
of m
arki
ng o
f thr
ee su
bsam
ple
site
s in
one
repl
icat
e in
the
unte
rate
d co
ntro
l
Pres
pray
Appl
icat
ion
Post
spra
ysa
mpl
ing
inin
sam
plin
g in
trea
tmen
t no.
trea
tmen
t no.
trea
tmen
t no.
June
61s
t pre
spra
y sa
mpl
ing
1, 2
, 3(-
3 D
AA
)7
1st p
resp
ray
sam
plin
g4,
5, 6
(- 3
DA
A)
81s
t pre
spra
y sa
mpl
ing
7(-
3 D
AA
)9
1st a
pplic
atio
n2,
310
1st a
pplic
atio
n4,
5, 6
111s
t app
licat
ion
712
1st p
osts
pray
sam
plin
g1,
2, 3
(+3
DA
A)
131s
t pos
tspr
ay sa
mpl
ing
4, 5
, 6(+
3 D
AA
)14
1st p
osts
pray
sam
plin
g7
(+3
DA
A)
172n
d ap
plic
atio
n4
192n
d po
stsp
ray
sam
plin
g1,
2, 3
(+10
DA
A)
202n
d po
stsp
ray
sam
plin
g4*
, 5, 6
(+10
DA
A)
4*=
10 D
AA
1 (3
DA
A2)
212n
d po
stsp
ray
sam
plin
g7
(+10
DA
A)
293r
d po
stsp
ray
sam
plin
g1,
2, 3
(+20
DA
A)
303r
d po
stsp
ray
sam
plin
g4*
*, 5
, 6(+
20 D
AA
)4*
*= 2
0 D
AA
1 (1
3 D
AA
2)Ju
ly 1
3rd
post
spra
y sa
mpl
ing
7(+
20 D
AA
)3 9
4th
post
spra
y sa
mpl
ing
1, 2
, 3(+
30 D
AA
)10
4th
post
spra
y sa
mpl
ing
4***
, 5, 6
(+30
DA
A)
4***
= 30
DA
A1
(23
DA
A2)
DA
A =
day
s afte
r app
licat
ion
Trea
tmen
t no
1: U
ntre
ated
con
trol
Trea
tmen
t no
5: C
URI
OM
(Luf
enur
on+
Prof
enof
os) E
C 5
50 1
10 (1
0+10
0) g
ai/h
aTr
eatm
ent n
o 2:
Luf
enur
on E
C 0
50 5
0 g
ai/h
aTr
eatm
ent n
o 6:
CU
RIO
M (L
ufen
uron
+Pr
ofen
ofos
) EC
550
330
(30+
300)
g a
i/ha
Trea
tmen
t no
3: L
ufen
uron
EC
050
100
g a
i/ha
Trea
tmen
t no
7: P
rofe
nofo
s EC
720
100
0 g
ai/h
aTr
eatm
ent n
o 4:
Luf
enur
on E
C 0
50 2
x50
g ai
/ha
Tabl
e 3.
- Tim
e sc
hedu
le fo
r sel
ectiv
ity te
st o
f luf
enur
on, p
rofe
nofo
s and
thei
r mix
ture
s in
cotto
n, K
aha,
Egy
pt, 2
000
126
127
Results and discussion Occurrence (arthropod populations) The counts revealed a rather even distribution of all groups of predators in all fields (Tables 4-8). The figures demonstrated a rising population of all species. The nymphs of O. albidipennis and the larvae of C. carnea were the only species represented by immature stages, while all the others occurred as adults. O. albidipennis was the most numerous species during the whole trial period, occurring constantly at high numbers. The second predatory bug species, the mirid C. verbasci, occurred at rather low level and so did the lacewing C. carnea. The predatory beetles (ladybird and rove beetles) and spiders always yielded appreciable high numbers. Effects of pesticides Lufenuron As already known from previous data (Bourgeois 1994), lufenuron is safe to all adult stages of all predator species on Kaha Station (O. albidipennis, C. verbasci, Scymnus spp., C. undecimpunctata, Paederus alfierii and spiders) (Tables 4-8, Figure 2). Also, lufenuron did not effect egglaying of the minute pirate bug Orius. This was shown by the continuous presence of first instar nymphs, which must have hatched from freshly laid eggs (Figure 3).
In this study lufenuron interfered in the moulting process of Orius and Campylomma. There was a clear inhibition of nymphal development of both predatory bug species. The degree and duration of the suppression was dependant on the dosage. At the tested rates between 30 and 100 g ai/ha there was a reduction of nymphal populations after application, but less pronounced at 10 g ai/ha in a mixture with profenofos (Figure 3). In the rating at 3 days after application (DAA), all nymphal instar stages were still present in comparable numbers to the untreated control. The presence of first instar nymphs demonstrated that lufenuron has no transovarial or ovicidal effect. The growth inhibiting action becomes fully visible at 10 DAA by the lower numbers of older nymphal stages, showing that no further development beyond the first instar nymphs had taken place (Figure 4).
The least detrimental effect was observed in the 10 g lufenuron/100 g profenofos mixture, where a higher number of nymphal stages was counted as compared to the lufenuron rates of 30 to 100 g ai/ha.
The recovery becomes more obvious in the 20 DAA counts. At 20 DAA there is also some recovery at the 50 g ai/ha rate. In the plots with the 30 g lufenuron/300 g profenofos ai/ha plots the number of Orius nymphs is kept low by the negative impact of the higher profenofos rate of 300 g ai/ha. At 30 DAA recovery is achieved in the 10 to 30 g ai/ha rates, while it is still delayed at 50 g a.i./ha. First signs of recovery become also visible at 100 g. The lower figures of the older nymphs can be explained by the preceeding lower starting level in the 20 DAA counts.
The split application of 2 x 50 g ai/ha at 1 week interval does not offer any advantage over the single application of the full rate of 100 g ai/ha with regard to the impact on Orius nymphs. Whether this approach offers any advantage in the control strategy against lepidopterous cotton pests has to be clarified in separate trials.
At no time were nymphal stages wiped out completely by any of the tested rates of lufenuron (10-100 g ai/ha). The product did not cause any flare-up of pests during the 30 days observation period following the application, as could be proved by visual inspection of the plots.
The effect on Campylomma nymphs was the same as on Orius, but this predatory bug occurred in much lower numbers.
In the evaluations at 3 and 10 DAA, there was a strongly rising population of larvae of C. carnea. Young larvae are not yet affected by the potential impact of lufenuron on the moulting process, but this effect becomes visible on older instars in the counts 20 DAA. In the evaluations at 30 DAA the population densities of C. carnea was again the same in the untreated and treated fields.
Profenofos Profenofos at 1000 g ai/ha caused a temporary moderate reduction of Orius adults (3 DAA) and a stronger reduction of older nymphs (Table 5). Freshly hatched nymphs were present in the counts since the product has no ovicidal activity. Recovery was observed at 20 DAA. Profenofos is safe for larvae of Chrysoperla, adults of Paederus, and all stages of spiders (Tables 5-8). It is also safe to the ladybird beetle Scymnus, but causes some temporary suppression of Coccinella, the other species of this family. This is an extremely high tested rate of profenofos and most recommended rates in cotton are much lower.
Profenofos did not cause any flare up of pests during the 30 days observation period following the application (visual inspection of the plots). Methodology of testing The testing of growth inhibiting effects and the subsequent recovery is a difficult task under field conditions. The choice of the appropriate field size is already a problem. Either the size is too small, then immigrating stages can simulate a fading detrimental effect while in reality it is camouflaging a still existing negative impact. Too big field sizes carry the risk of pretending an ongoing growth inhibition, while in reality a recovered predator species had not yet succeeded in reaching the central parts of a plot. By choosing the parameter of evaluating the nymphs separated by their stages, a neutral and objective decision tool has become available to decide properly whether an inhibition effect has gone or not.
A prerequisite for this approach is the appropiate evaluation of the developmental stages. Predatory nymphs are very small and it is virtually impossible to count them with the conventional methods (visual observation, drop sheet counts in the furrow) in the field. By shifting the counting process to the laboratory, the exact evaluation of the sampled material is possible. Together with other measures in the execution of such a trial (comparable environment for all fields, planting of preferred host plants, quick execution of the sampling process) a high degree of accuracy can be also achieved under field conditions, as could be proved with this study. Acknowledgements The authors would like to thank M. Angst, M. Arslan-Bir and M. Gillham for revising the manuscript. Thanks are also due to D. R. Wille for his assistance in the statistical evaluation. References Bourgeois, F. 1994: MATCH (lufenuron), IPM Fitness and Selectivity. Ciba-Geigy Internal
Publication: 90 pp. Buholzer, F., Drabek, J., Bourgeois, F. & Guyer, W. 1992: CGA 184 699 a new acylurea
insecticide. Med. Fac. Landbouww. Univ. Gent 57/3a: 781-790. Sechser, B., Reber, B., Ruzette, M.A. & Ngo, N. 1998: A combined Drop Cloth/Vacuum
Sampling Technique for measuring the impact of several insecticides on the natural enemy complex in cotton in the USA and Egypt. IOBC/wprs Bulletin 21 (6): 101-108.
Tabl
e 4.
Eva
luat
ion
of c
otto
n pr
edat
or fa
una
3 da
ys b
efor
e ap
plic
atio
n, K
aha,
Egy
pt, 2
000
Unt
reat
edD
**Lu
fenu
ron
D**
Lufe
nuro
nD
**Lu
fenu
ron
D**
Lufe
nuro
n/D
**Lu
fenu
ron/
D**
Prof
enof
osD
**co
ntro
lPr
ofen
ofos
Prof
enof
os50
g a
i/ha
100
g ai
/ha
2x50
g a
i/ha
10/1
00 g
ai/h
a30
/300
g a
i/ha
1000
g a
i/ha
Ori
us
340
a26
7a
364
a36
9a
350
a44
5a
561
aad
ult
Ori
us im
mat
ure
36a
36a
30a
26a
29a
22a
39a
L1-L
2O
rius
imm
atur
e13
a27
a28
a33
a37
a18
ab15
bL3
-L5
Cam
pylo
mm
a14
a7
a12
a10
a17
a16
a13
aad
ult
Cam
pylo
mm
a2
a5
a4
a2
a3
a5
a2
aim
mat
ure
Chr
ysop
erla
15a
21a
15a
18a
22a
12b
13b
imm
atur
eSc
ymnu
s14
0ab
116
b17
5ab
143
ab22
0a
206
a18
3a
adul
tC
occi
nella
237
b31
4ab
380
ab30
2ab
466
a46
5a
311
bad
ult
Paed
erus
43ab
24b
57a
70a
47ab
74a
55a
adul
tA
rane
ae22
8a
190
a17
0a
245
a23
0a
248
a20
6a
imm
. + a
dult
D**
Dun
can'
s Mul
tiple
Ran
ge T
est:
Figu
res f
ollo
wed
by
the
sam
e le
tter a
re n
ot si
gnifi
cant
ly d
iffer
ent (
P>0.
05)
Num
ber o
f spe
cim
ens p
er 9
0 m
cot
ton
row
129
Tabl
e 5.
Eva
luat
ion
of c
otto
n pr
edat
or fa
una
3 da
ys a
fter
appl
icat
ion,
Kah
a, E
gypt
, 200
0
Unt
reat
edD
**Lu
fenu
ron
A*
D**
Lufe
nuro
nA
*D
**Lu
fenu
ron
A*
D**
Lufe
nuro
n/A
*D
**Lu
fenu
ron/
A*
D**
Prco
ntro
l%
%%
Prof
enof
os%
Prof
enof
os%
50 g
ai/h
a10
0 g
ai/h
a2x
50 g
ai/h
a10
/100
g a
i/ha
30/3
00 g
ai/h
a10
Ori
us
496
a46
46,
5a
460
7,3
a44
89,
7a
399
20a
257
48b
adul
tO
rius
imm
atur
e80
a12
7-5
9a
121
-51
a12
1-5
1a
6223
a37
54b
L1-L
2O
rius
imm
atur
e38
a27
29a
44-1
6a
365,
3a
2729
a7
82c
L3-L
5C
ampy
lom
ma
19a
1332
a9
53a
953
a15
21a
479
bad
ult
Cam
pylo
mm
a6
a4
33a
350
a2
67a
267
a0
100
aim
mat
ure
Chr
ysop
erla
45a
52-1
6a
54-2
0a
450
a66
-47
a57
-27
bcim
mat
ure
Scym
nus
174
ab13
423
b21
2-2
2a
194
-11
ab26
0-4
9a
281
-61
aad
ult
Coc
cine
lla30
3a
386
-27
a37
3-2
3a
331
-9a
339
-12
a27
68,
9b
adul
tPa
eder
us65
bc46
29c
123
-89
a85
-31
ab73
-12
bc98
-51
aad
ult
Ara
neae
320
a36
3-1
3a
321
0a
344
-8a
387
-21
a41
8-3
1a
imm
. + a
dult
A*
Abb
ott (
% re
duct
ion
or in
crea
se o
f pre
dato
r pop
ulat
ion)
D**
Dun
can's
Mul
tiple
Ran
ge T
est:
Figu
res f
ollo
wed
by
the
sam
e le
tter a
re n
ot si
gnifi
cant
ly d
iffer
ent (
P>0.
05)
Num
ber o
f spe
cim
ens p
er 9
0 m
cot
ton
row
130
Tabl
e 6.
Eva
luat
ion
of c
otto
n pr
edat
or fa
una
10 d
ays a
fter a
pplic
atio
n, K
aha,
Egy
pt, 2
000
Unt
reat
edD
**Lu
fenu
ron
A*
D**
Lufe
nuro
nA
*D
**Lu
fenu
ron
A*
D**
Lufe
nuro
n/A
*D
**Lu
fenu
ron/
A*
D**
Prof
enof
osA
*D
**co
ntro
l%
%%
Prof
enof
os%
Prof
enof
os%
%50
g a
i/ha
100
g ai
/ha
2x50
g a
i/ha
10/1
00 g
ai/h
a30
/300
g a
i/ha
1000
g a
i/ha
Ori
us
407
a33
019
a36
89,
6a
411
-1a
393
3,4
a26
335
ab22
744
bad
ult
Ori
us im
mat
ure
159
a99
38b
127
20ab
7056
b15
90
a99
38b
5267
cL1
-L2
Ori
us im
mat
ure
153
a1
99c
597
c1
99c
2683
b6
96d
1193
cdL3
-L5
Cam
pylo
mm
a8
a4
50a
13-6
3a
713
a15
-88
a10
-25
ab5
38b
adul
tC
ampy
lom
ma
35ab
1946
bc10
71c
974
c54
-54
a55
-57
a3
91c
imm
atur
eC
hrys
oper
la91
a74
19a
6529
a72
21a
8111
a66
27bc
5638
cim
mat
ure
Scym
nus
295
ab21
627
b33
1-1
2ab
277
6,1
ab40
0-3
6a
311
-5a
230
22ab
adul
tC
occi
nella
342
a35
0-2
a41
5-2
1a
384
-12
a44
0-2
9a
285
17ab
138
60c
adul
tPa
eder
us83
bc74
11c
175
-110
a14
2-7
1ab
115
-39
ab14
4-7
3a
116
-40
aad
ult
Ara
neae
646
a56
213
a55
015
a47
427
a56
013
a47
526
a42
035
aim
m. +
adu
lt
A*
Abb
ott (
% re
duct
ion
or in
crea
se o
f pre
dato
r pop
ulat
ion)
D**
Dun
can's
Mul
tiple
Ran
ge T
est:
Figu
res f
ollo
wed
by
the
sam
e le
tter a
re n
ot si
gnifi
cant
ly d
iffer
ent (
P>0.
05)
Num
ber o
f spe
cim
ens p
er 9
0 m
cot
ton
row
131
Tabl
e 7.
Eva
luat
ion
of c
otto
n pr
edat
or fa
una
20 d
ays a
fter a
pplic
atio
n, K
aha,
Egy
pt, 2
000
Unt
reat
edD
**Lu
fenu
ron
A*
D**
Lufe
nuro
nA
*D
**Lu
fenu
ron
A*
D**
Lufe
nuro
n/A
*D
**Lu
fenu
ron/
A*
D**
Prof
enof
osA
*D
**co
ntro
l%
%%
Prof
enof
os%
Prof
enof
os%
%50
g a
i/ha
100
g ai
/ha
2x50
g a
i/ha
10/1
00 g
ai/h
a30
/300
g a
i/ha
1000
g a
i/ha
Ori
us
291
a19
932
b22
024
ab22
822
ab23
719
ab21
526
b22
921
abad
ult
Ori
us i
mm
atur
e18
6a
2487
c18
90c
896
c66
65b
2885
c89
52ab
L1-L
2O
rius
imm
atur
e84
a33
61b
010
0c
693
c47
44b
594
c60
29b
L3-L
5C
ampy
lom
ma
57a
493
ab4
93ab
010
0b
1181
ab7
88a
689
aad
ult
Cam
pylo
mm
a37
b13
65bc
197
d6
84cd
99-1
67a
47-2
7b
61-6
5a
imm
atur
eC
hrys
oper
la49
a20
59b
2059
b26
47ab
2451
b26
47c
50-2
bim
mat
ure
Scym
nus
369
a24
633
c36
60,
8a
318
14ab
272
26bc
340
7,9
a28
922
abad
ult
Coc
cine
lla42
8a
337
21a
396
7,5
a31
726
a40
94,
4a
265
38a
145
66b
adul
tPa
eder
us11
9ab
8628
b17
3-4
5a
114
4,2
ab16
8-4
1a
175
-47
ab21
1-7
7a
adul
tA
rane
ae53
5a
402
25b
425
21b
415
22b
502
6,2
ab47
611
a54
0-1
aim
m. +
adu
lt
A*
Abb
ott (
% re
duct
ion
or in
crea
se o
f pre
dato
r pop
ulat
ion)
D**
Dun
can's
Mul
tiple
Ran
ge T
est:
Figu
res
follo
wed
by
the
sam
e le
tter a
re n
ot si
gnifi
cant
ly d
iffer
ent (
P>0.
05)
Num
ber o
f spe
cim
en p
er 9
0 m
cot
ton
row
132
Tabl
e 8.
Eva
luat
ion
of c
otto
n pr
edat
or fa
una
30 d
ays a
fte r
app
licat
ion,
Kah
a, E
gypt
, 200
0
Unt
reat
edD
**Lu
fenu
ron
A*
D**
Lufe
nuro
nA
*D
**Lu
fenu
ron
A*
D**
Lufe
nuro
n/A
*D
**Lu
fenu
ron/
A*
D**
cont
rol
%%
%Pr
ofen
ofos
%Pr
ofen
ofos
%50
g a
i/ha
100
g ai
/ha
2x50
g a
i/ha
10/1
00 g
ai/h
a30
/300
g a
i/ha
Ori
us
85a
89-5
a67
21ab
88-4
a44
41b
90-6
aad
ult
Ori
us im
mat
ure
45ab
48-7
ab31
31bc
1762
c76
-31
a55
-22
abL1
-L2
Ori
us im
mat
ure
131
a29
78b
2085
b27
79b
9635
a94
28a
L3-L
5C
ampy
lom
ma
11a
918
a3
73a
464
a11
0a
918
aad
ult
Cam
pylo
mm
a12
a5
58ab
375
b2
83b
48
ab6
50ab
imm
atur
eC
hrys
oper
la19
a9
53a
1332
a19
0a
190
a21
-11
aim
mat
ure
Scym
nus
116
b13
6-1
7b
146
-26
b24
8-1
13a
161
-45
ab21
8-8
8ab
adul
tC
occi
nella
152
a16
1-6
a20
5-3
5a
265
-74
a23
5-8
3a
187
-23
aad
ult
Paed
erus
72a
5819
a10
4-4
4a
95-3
2a
85-1
3a
125
-74
aad
ult
Ara
neae
325
a21
534
b19
540
b20
437
b18
936
b25
123
bim
m. +
adu
lt
A*
Abb
ott (
% re
duct
ion
or in
crea
se o
f pre
dato
r pop
ulat
ion)
D**
Dun
can's
Mul
tiple
Ran
ge T
est:
Figu
res f
ollo
wed
by
the
sam
e le
tter a
re n
ot si
gnifi
cant
ly d
iffer
ent (
P>0.
05)
Num
ber o
f spe
cim
en p
er 9
0 m
cot
ton
row
133
Figu
re 2
. Im
pact
of l
ufen
uron
and
its
mix
ture
s w
ith p
rofe
nofo
s on
pre
dato
rs in
cot
ton,
Kah
a, E
gypt
, 200
0
0
10
0
20
0
30
0
40
0
50
0
60
0
-33
10
20
30
Unt
reat
ed c
ontr
ol
Luf
enur
on 5
0 g
ai/h
a
Luf
enur
on 1
00 g
ai
Luf
enur
on 2
x 5
0 g
ai
Luf
enur
on/P
rofe
nofo
s 10
/100
gai L
ufen
uron
/Pro
feno
fos
30/3
00 g
ai Pro
feno
fos
1000
g a
i
Day
s af
ter
appl
icat
ion
Ori
us a
lbid
ipen
nis,
adu
lts(m
inut
e pi
rate
bug
)Number of specimen per 90 m cotton row
134
Figu
re 3
. Im
pact
of l
ufen
uron
and
its
mix
ture
s w
ith p
rofe
nofo
s on
pre
dato
rs in
cot
ton,
Kah
a, E
gypt
, 200
0
0
100
200
-33
10
203
0
Unt
reat
ed c
ontr
ol
Luf
enur
on 5
0 g
ai/h
a
Luf
enur
on 1
00 g
ai
Luf
enur
on 2
x 5
0 g
ai
Luf
enur
on/P
rofe
nofo
s 10
/100
gai L
ufen
uron
/Pro
feno
fos
30/3
00 g
ai Prof
enos
100
0 g
ai
Day
s af
ter
appl
icat
ion
Ori
us a
lbid
ipen
nis,
sm
all n
ymph
s (L
1-2)
(min
ute
pira
te b
ug)
(nym
phs
at 3
and
10
DA
A w
ere
pred
omin
antl
y L
1)Number of specimen per 90 m cotton row
135
Figu
re 4
. Im
pact
of l
ufen
uron
and
its
mix
ture
s w
ith p
rofe
nofo
s on
pre
dato
rs in
cot
ton,
Kah
a, E
gypt
, 200
0
0
100
200
-33
1020
30
Unt
reat
ed c
ontr
ol
Luf
enur
on 5
0 g
ai/h
a
Luf
enur
on 1
00 g
ai
Luf
enur
on 2
x 5
0 g
ai
Luf
enur
on/P
rofe
nofo
s 10
/100
gai L
ufen
uron
/Pro
feno
fos
30/3
00 g
ai Prof
enof
os 1
000
g ai
Day
s af
ter
appl
icat
ion
Ori
us a
lbid
ipen
nis,
big
nym
phs
(L3-
5)(m
inut
e pi
rate
bug
)Number of specimen per 90 m cotton row
136
