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)
i
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
ii
iii
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
iv
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 SintTruiden,
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]
v
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, R1058.8.42, 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 NiefernÖschelbronn,
e-mail: [email protected]
47. KOCH, Heribert, Dr., Landesanstalt für Pflanzenschutz und Pflanzenbau, Essenheimer Str.
144, D-55128 Mainz,
e-mail: [email protected]
vi
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 NiefernÖschelbronn,
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]
vii
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 NiefernÖschelbronn,
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]
viii
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 NiefernÖschelbronn,
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]
ix
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 pesticideeffects on predatory mites ....................................................................................................... 61
Van de Veire, M., Cornelis, W. & Tirry, L.
x
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 Trichogramma 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
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
1
2
-
-
-
-
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 (ToxicityExposure-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 offfield 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.
3
exposure of
NTAs possible?
yes
no
assess lethal effects (LR50) in lab tests on glass plates using
Aphidius rhopalosiphi and Typhlodromus pyri
TERin-field<1
no
or
TERoff-field<10?
yes
specify appropriate risk mitigation
for NTAs at risk
or
assess lethal and sublethal effects in extended labtests on
natural substrates using sensitive species
plus 1species if TERin-field is < 1 or
plus 2 species if TERoff-field is < 10
TERin-field<1
no
or
TERoff-field<5?
yes
specify appropriate risk mitigation
for NTAs at risk
or
assess effects in higher tier studies (elab, semi-field, field) with
appropriate species
yes
unacceptable
effects?
no
yes
no authorization granted
authorization possible
Figure 1: Decision-making scheme for non-target arthropods
4
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.,
6
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
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 rateresponse 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".
7
8
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
9
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
Glass
Apple ‘Priam’
Apple ‘Querina’
Grapevine ‘Regent’
Bean ‘Caruso’
Echinochloa
Sasa
Aspidistra
Perfekthion
X
X
–
X
X
X
–
–
Dithane Ultra
X
–
X
X
X
–
–
–
Basagran Super
X
–
–
–
–
X
X
X
Table 2. Test substances and test substrates to assess LR50-values for A. rhopalosiphi
Substrate
Glass
Apple ‘Priam’
Apple ‘Querina’
Grapevine ‘Regent’
Grapevine ‘Blauer Portugieser’
Bean ‘Caruso’
Perfekthion
X
X
X
X
–
X
Morestan
X
–
X
–
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.
10
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).
11
Typhlodromus pyri
LR50-values Perfekthion
40
35
LR 50 value
Perfekthion [ml/ha]
30
lower confidence limit
25
upper confidence limit
20
15
10
5
0
glass
glass
apple 'priam'
vine 'regent'
bean 'caruso'
bean 'caruso'
echinochloa
substrate
Figure 1: Determined LR50-values for Typhlodromus pyri and Perfekthion and confidence
limits on glass and leaves from different plants.
Typhlodromus pyri
LR50-values Dithane Ultra
300
Dithane Ultra [g/ha]
250
LR 50 value
lower confidence limit
200
upper confidence limit
150
100
50
0
glass
glass
bean 'caruso'
apple 'querina'
vine 'regent'
vine 'regent' glue
substrate
Figure 2: Determined LR50-values for Typhlodromus pyri and Dithane Ultra and confidence
limits on glass and leaves from different plants.
12
Typhlodromus pyri
LR50-values Basagran Super SG
14000
Basagran Super [g/ha]
12000
10000
8000
LR 50 value
lower confidence limit
upper confidence limit
6000
4000
2000
0
glass
sasa
aspidistra
echinochloa
substrate
Figure 3: Determined LR50-values for Typhlodromus pyri and Basagran Super and confidence
limits on glass and leaves from different plants.
Aphidius rhopalosiphi
LR50-values Perfekthion EC
0,45
0,4
Perfekthion [ml/ha]
0,35
0,3
0,25
0,2
0,15
LR 50 value
0,1
lower confidence limit
0,05
upper confidence limit
0
glass
apple 'querina'
apple 'priam'
vine 'regent'
bean 'caruso'
bean 'caruso'
substrate
Figure 4: Determined LR50-values for Aphidius rhopalosiphi and Perfekthion and confidence
limits on glass and leaves from different plants.
13
Aphidius rhopalosiphi
LR50-values Morestan
450
400
Morestan [g/ha]
350
300
250
200
150
LR 50 value
100
lower confidence limit
50
upper confidence limit
0
glass
apple 'querina'
vine 'regent'
bean 'caruso'
substrate
Figure 5: Determined LR50-values for Aphidius rhopalosiphi and Morestan and confidence
limits on glass and leaves from different plants.
2,1
Typhlodromus Basagran
1,4
2,5
Typhlodromus Dithane
1,8
6
Typhlodromus Perfekthion
2,2
3
Aphidius Perfekthion
1,7
max. ratio plant : glass
2,2
Aphidius Morestan
max. ratio plant : plant
1,1
0
1
2
3
4
5
6
7
Figure 6: Comparison of the maximum ratios of the LR50-values calculated for both species,
all tested plant protection products and plant surfaces.
14
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 undersurface, 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.
15
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 (DeStephaniPerez) (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 nontarget 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.
16
Pesticides and Beneficial Organisms
IOBC/wprs Bulletin Vol. 24 (4) 2001
pp. 17 - 24
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
17
18
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
Baythroid EC050
Cymbush DG
Decis EC 2,5
Karate
Mavrik 2 F
Pirimor G
Sumi Alpha
Sumi Alpha
Talstar Flo
formulation
active ingredient
EC
DG
EC
EC
SC
WG
EC
EC
SC
cyfluthrin
cypermethrin
deltamethrin
lambdacyhalothrin
fluvalinate
pirimicarb
esfenvalerate
esfenvalerate
bifenthrin
tested dose
(g a.i./ha)
10.0
15.0
5.0
5.0
36.0
125.0
5.0
5.0
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
19
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 %
20
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)
minimum maximum
mean
Set 1
exposure
field
Set 2
exposure
field
Rainfall
(mm)
day 0
day 1
day 3
day 0-1
day 1-3
12.3
10.0
11.0
10.0
6.3
20.0
20.6
16.1
20.6
20.6
16.2
15.3
13.6
15.3
13.5
(protected)
(protected)
(protected)
0.0 mm
6.4 mm
day 0
day 1
day 0-1
11.8
12.3
12.3
20.9
20.0
20.0
16.4
16.2
(protected)
(protected)
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.
21
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
name
observed
mortality
(%)
corrected
mortality
(%)
% wasp
on plants
# mummies/
unit
E
(%)
IOBC
class
set 1 – day 0
control
bifenthrin
cyfluthrin
cypermethrin
lambdacyhalothrin
10.0
46.7
13.3
13.3
20.0
40.8
3.7
3.7
11.1
24.8a
27.2a
15.3b
16.1b
23.5a
19.8a
21.5a
17.7a
21.2a
22.9a
35.7
13.9
-3.1
-2.8
2
1
1
1
set 1 – day 1
control
bifenthrin
10.0
40.0
33.3
29.9a
28.0a
22.3a
19.7a
41.1
2
set 1 – day 3
control
bifenthrin
6.7
10.0
3.6
31.0a
28.4a
23.7a
26.8a
-9.0
1
set 2 – day 0
control
deltamethrin
esfenvalerate
fluvalinate
pirimicarb
13.3
50.0
50.0
16.7
23.3
46.4
46.4
10.7
17.9
36.2ab
17.3d
26.9c
37.3a
29.8bc
42.6a
43.9a
23.8a
45.4a
50.8a
44.8
69.8
4.8
2.1
2
3
1
1
set 2 – day 1
control
deltamethrin
esfenvalerate
10.0
26.7
23.3
18.5
14.8
32.7a
28.0a
29.0a
33.1a
31.2a
30.5a
23.2
21.5
1
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
22
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.
23
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.
24
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): 10341041.
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
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
25
26
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).
27
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
Insecticides
Control
Spinosad
Tebufenozide
Azadirachtin
Topical treatment of pupae
% Adult
Eggs/female, % Eclosionc IOBC
Doses µ a.i./g pupae
a
(Concentrations mg a.i./l) emergence
7 days b
class
0
199.5 ± 54.9a 85.0 ± 1.5a
–
(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
–
–
–
(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
–
–
–
(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
–
–
–
0.005
0.005
0.005
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
Insecticides
Control
Spinosad
Tebufenozide
Azadirachtin
Doses µ a.i./g
pupae
(Concentrations
mg a.i./l)
0
Topical treatment of pupae
% Adult
% Attacked
Longevity,
emergence a
hosts c
days b
% Progeny
size d
IOBC
class
95.0 ± 5.0a
21.7 ± 2.9ab
86.0 ± 4.0a
98.0 ± 1.2a
–
(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
*
*
3
0.0026
2.6
(100)
90.0 ± 5.8a
3.3 ± 0.4f
83.3 ± 4.4a
5.2
(200)
75.0 ± 5.0a
1.8 ± 0.2g
85.0 ± 2.9a*
87.5 ± 2.5a*
3
*
*
3
91.7 ± 8.3a
12.8
(500)
80.0 ± 8.2a
1.6 ± 0.2g
80.0 ± 0.0a
25.5
(1000)
90.0 ± 5.8a
1.4 ± 0.1g
–
–
4
(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
(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
0.0026
0.0026
87.5 ± 2.5a
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
%
b
ADULT MORTALITY
Podisus maculiventris
100
24h
48h
72h
b
80
60
48 mg/l
40
180 mg/l
20
0
Control
Spinosad
Azadirachtin Tebufenozide
max field recommended rates
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
% Adult
Eggs/female % Eclosion b
mg a.i./l
mortality, 72 h 1 month a**
Control
0
0
359.6± 31.1a
70.3± 6.5a
Spinosad
120
100
Tebufenozide
180
0
213.0± 39.9b
68.8± 5.5a
Azadirachtin
48
0
283.9± 28.8ab 53.4± 5.7a
IOBC
class
–
4
2
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
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
Control
Spinosad
Tebufenozide
Azadirachtin
0
120
180
48
Insecticides
Concentrations
mg a.i./ l
Control
Spinosad
Tebufenozide
Azadirachtin
Insecticides
0
120
180
48
21.6±0.5a 88.8±6.3a
0.2±0.4b
–
22.2±1.1a 88.8±2.0a
21.2±1.8a 86.4±2.8a
INGESTION*
Doses µg a.i./ g adult
0
27.39 (120)
41.10 (180)
10.96 (48)
75.1±5.8a
–
71.3±2.9a
43.7±5.0b
Longevity, % Attacked % Progeny size f
days d
hosts e
31.4±0.7a 87.5±4.8a
0.5±0.1b
–
26.3±0.4a 91.3±3.1a
4.9±0.1b 64.1±8.2b
TOPICAL APPLICATION
(Concentrations mg a.i./
l)
Control
Spinosad
Tebufenozide
Azadirachtin
Longevity, % Attacked % Progeny size c
days a
hosts b
65.0±7.9a
–
70.0±5.4a
43.9±4.8b
Longevity, % Attacked % Progeny size i
hosts h
days g
28.1±0.5a
0b
27.8±1.0a
28.8±0.4a
92.5±2.8a
–
88.3±2.2a
96.5±1.7a
70.0±6.3a
–
73.9±3.4a
71.5±4.7a
IOBC
class
–
4
1
2
IOBC
class
–
4
1
3
IOBC
class
–
4
1
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
Control
Spinosad
Tebufenozide
Azadirachtin
Insecticides
Control
Spinosad
Tebufenozide
Azadirachtin
Insecticides
Control
Spinosad
Tebufenozide
Azadirachtin
Concentrations
mg a.i./ l
0
80
96
400
180
48
Concentrations
mg a.i./ l
0
80
96
400
180
48
% Mortality
at 48 h a
Eggs/female,
days b
%
Eclosion c
IOBC
class
42.0±1.5a
–
(*)
(* )
38.2±4.3a
39.6±3.1a
79.5±3.6a
–
(*)
(*)
80.5±3.8a
78.3±5.4a
–
–
1
3
1
1
0a
–
3.1±3.1a
87.5±5.1b
0a
0a
INGESTION*
% Mortality
at 48 hours d
% Mortality
at 6 days e
%
Eggs/female
Eclosion g
, days f
IOBC
class
0a
0
30.3±3.3a
0a
66.0±10.4b
(* )
–
–
–
24.9±10.7b
100±0.0c
(* )
0a
0a
36.2±3.0a
0a
0a
27.5±3.5a
TOPICAL APPLICATION
90.0±3.8a
(*)
–
(*)
88.2±2.7a
84.5±5.4a
–
2
–
4
1
1
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
0
5.40 (80)
27.02 (400)
12.16 (180)
3.24 (48)
0a
0a
4.1±4.1a
0a
0a
0a
4.1±4.1a
4.1±4.1a
0a
0a
24.4±3.5a
34.1±2.9a
37.1±2.2a
28.3±1.4a
30.4±3.6a
84.8±6.8a
83.0±4.6a
78.2±5.5a
90.2±9.8a
84.0±3.6a
–
1
1
1
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.
34
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 nonsteroidal 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 nontarget 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
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
35
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).
No. of hatched adult Aleochara
800
700
600
500
400
300
200
100
Hosts (Delia pupae) provided per week
250
0
0
500
750
1000
1250
1500
750
1500
2250
3000
3750
4500
Total number of hosts (Delia pupae) provided
Figure 1: Relation between the total number of hatched Aleochara bilineata beetles per test
vessel (mean ± st.dev.) and the host density.
37
40
35
% Parasitation
30
25
20
15
10
5
250
0
0
Hosts (Delia pupae) provided per week
500
1000
1500
1250
750
750
1500
2250
3000
3750
4500
Total number of hosts (Delia pupae) provided
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.
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 Ecotoxicology. 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 Pflanzenschutzmitteln 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
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
39
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
Quassin
Quassia
solution
Active agent
Extraction
Producer
Quassia extract
430 g/l
Alcohol
extract
Siegfried
Agro AG
CH-4800
Zofingen
According to
recipe by
Kreuter
(1995)
Quassia extract
of 250 g dry
Quassia wood
+ 2 l water
(steeped for 24 h and
cooked for 0,5 h)
Quassetum
Quassia extract
42 g/l
+ potash soap
100 g/l
+ Equisetum arvense
+ Artemisia vulgaris
Water
extract
Alcohol
extract
Grieder
Naturprodukte
CH-4497
Rünnenberg
Tested
Highest
recomapplication
rate
mended
application according to
Barrett et al.
rate for
orchards
(1994)
3.0 l/ha
1.2 l/ha
201.0 l/ha
80.4 l/ha
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.
42
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
Testspecies
Treatment
T. pyri
Control
Quassin
Quassia solution
Quassetum
Control
Quassin
Control
Quassia solution
Quassetum
Control
Quassin
Quassia solution
Quassetum
A. andersoni
E. finlandicus
Application
rate
in l/ha
1.2
80.4
120.0
–
1.2
–
80.4
120.0
–
1.2
80.4
120.0
Number of living (l), dead (d) and
Mortality
escaped (e) animals 7 days after treatment
rate (Abl
d
e
bott) in %
(mean ± Std) (mean ± Std) (mean ± Std) on day 7
–
16.4 ± 1.5
0.2 ± 0.5
3.4 ± 1.5
61
6.4 ± 1.5
0.4 ± 0.6
13.2 ± 1.9
79
3.4 ± 1.5
0.6 ± 0.9
16.0 ± 2.0
95
0.8 ± 1.2
16.8 ± 1.9
2.4 ± 1.1
–
18.2 ± 1.3
0.8 ± 0.8
1.0 ± 0.7
0
18.2 ± 0.5
0.4 ± 0.6
1.4 ± 0.6
–
19.2 ± 0.8
0.2 ± 0.5
0.6 ± 0.6
4
18.4 ± 1.1
0.4 ± 0.6
1.2 ± 0.8
94
1.2 ± 1.3
16.0 ± 1.9
2.8 ± 2.4
0
–
19.2 ± 1.1
0.8 ± 1.1
0
100
0.2 ± 0.5
19.8 ± 0.5
0
100
1.0 ± 1.2
19.0 ± 1.2
0
100
3.8 ± 2.2
16.2 ± 2.2
Table 3: Influence of Quassin, Quassia solution and Quassetum on reproduction of Typhlodromus
pyri and Amblyseius andersoni
Testspecies
Treatment
T. pyri
Control
Quassin
Quassia solution
Quassetum
Control
Quassin
Control
Quassia solution
Quassetum
A. andersoni
Application
rate
–
1.2
80.4
120.0
–
1.2
–
80.4
120.0
Number of eggs/female
from day 7 – 14
(mean ± Std)
11.84 ± 0.72
7.28 ± 1.19
7.17 ± 1.89
12*
13.58 ± 1.39
11.98 ± 1.80
15.72 ± 1.92
11.48 ± 1.16
**
* result from one female only
** only females survived, so that reproduction was not possible
Reduction of
number of
eggs/female in %
–
39
39
–
–
12
–
27
–
43
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
Testspecies
Treatment
T. pyri
Control
Quassin
Quassia solution
Quassetum
Control
Quassin
Quassia solution
Quassetum
Control
Quassin
Quassia solution
Quassetum
A. andersoni
E. finlandicus
Application
rate
in l/ha
–
1.2
80.4
120.0
–
1.2
80.4
120.0
–
1.2
80.4
120.0
Number of living (l), dead (d) and
Mortality
escaped (e) animals 7 days after treatment
rate (Abl
d
e
bott) in %
(mean ± Std) (mean ± Std) (mean ± Std) on day 7
–
16.6 ± 0.6
0.8 ± 0.8
2.6 ± 0.6
29
11.8 ± 2.1
1.0 ± 1.4
7.2 ± 2.2
19
13.4 ± 2.2
0.8 ± 1.8
5.8 ± 1.8
0
100
19.0 ± 0.7
1.0 ± 0.7
–
18.6 ± 0.9
0.2 ± 0.5
1.2 ± 1.1
6
17.4 ± 1.8
0.8 ± 1.3
1.8 ± 1.8
8
17.2 ± 1.3
0.4 ± 0.6
2.4 ± 0.9
0
100
19.2 ± 0.8
0.8 ± 0.8
–
17.8 ± 1.3
0.2 ± 0.5
2.0 ± 1.2
0
100
1.4 ± 1.7
18.6 ± 1.7
0
100
1.2 ± 1.6
18.8 ± 1.6
0
100
19.8 ± 0.5
0.2 ± 0.5
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
Testspecies
Treatment
Application
rate
in l/ha
T. pyri
Control
Quassetum
Quassetum
Quassetum
Control
Quassetum
Quassetum
Control
Quassetum
Quassetum
Quassetum
Quassetum
Quassetum
Control
Quassetum
Quassetum
Quassetum
Quassetum
Control
Quassetum
Quassetum
Quassetum
Quassetum
–
100
80
60
–
100
80
–
60
50
40
30
20
–
100
80
60
40
–
30
20
10
5
A. andersoni
E. finlandicus
Number of living (l), dead (d) and
Mortality
escaped (e) animals 7 days after treatment
rate (Abl
d
e
bott) in %
(mean ± Std) (mean ± Std) (mean ± Std) on day 7
–
16.2 ± 0.5
0.8 ± 0.5
3.0 ± 0.0
0
100
19.2 ± 0.8
0.8 ± 0.8
0
100
18.8 ± 0.8
1.2 ± 0.8
94
1.0 ± 2.2
16.4 ± 2.7
2.6 ± 2.0
–
18.4 ± 1.1
0.2 ± 0.5
1.4 ± 0.9
0
100
18.0 ± 1.2
2.0 ± 1.2
0
100
17.6 ± 0.9
2.4 ± 0.9
0
–
18.0 ± 1.0
2.0 ± 1.0
98
0.4 ± 0.6
17.0 ± 1.9
2.6 ± 1.8
82
3.2 ± 1.9
14.2 ± 2.3
2.6 ± 1.1
59
7.4 ± 2.3
9.2 ± 1.8
3.4 ± 2.0
46
9.8 ± 3.7
6.8 ± 3.2
3.4 ± 0.9
10
16.2 ± 2.2
2.0 ± 1.4
1.8 ± 1.5
–
17.2 ± 0.5
0.6 ± 0.6
2.2 ± 0.8
0
100
19.4 ± 0.9
0.6 ± 0.9
0
100
19.4 ± 0.9
0.6 ± 0.9
0
100
18.8 ± 0.5
1.2 ± 0.5
0
100
18.8 ± 0.8
1.2 ± 0.8
–
18.0 ± 1.6
0.2 ± 0.5
1.8 ± 1.3
99
0.2 ± 0.5
11.0 ± 1.4
8.8 ± 1.3
99
0.2 ± 0.5
7.2 ± 4.2
12.6 ± 3.8
80
3.6 ± 2.7
1.0 ± 1.0
15.4 ± 3.2
41
10.6 ± 3.6
0.8 ± 1.1
8.6 ± 3.9
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
Treatment
Control
Quassin
Quassin
Quassin
Control
Quassin
Quassin
Quassin
Quassin
Control
Quassia solution
Quassia solution
Quassia solution
Control
Quassia solution
Quassia solution
Application
rate
in l/ha
–
1.0
0.8
0.5
–
0.4
0.3
0.2
0.1
–
60.3
40.2
20.1
–
10.0
5.0
Number of living (l), dead (d) and
escaped (e) animals 7 days after treatment
l
d
e
(mean ± Std) (mean ± Std) (mean ± Std)
18.0 ± 1.4
0.4 ± 0.6
1.6 ± 1.1
0
1.4 ± 1.3
18.6 ± 1.3
0.2 ± 0.5
0.8 ± 0.8
19.0 ± 1.0
0.4 ± 0.6
1.0 ± 2.2
18.6 ± 2.6
17.0 ± 1.2
0.2 ± 0.5
2.8 ± 1.3
0.4 ± 0.6
1.6 ± 1.1
18.0 ±1.2
0.8 ± 0.8
1.6 ±1.1
17.6 ± 1.1
3.0 ± 2.5
1.4 ± 1.5
15.6 ± 2.2
8.8 ± 2.4
0.6 ± 0.6
10.6 ± 2.1
17.6 ± 1.1
0.2 ± 0.5
2.2 ± 0.8
0
0.4 ± 0.9
19.6 ± 0.9
0
1.6 ± 1.5
18.4 ± 1.5
0
0.8 ± 0.8
19.2 ± 0.8
18.0 ± 0.7
0.6 ± 0.6
1.4 ± 0.6
1.0 ± 0.7
1.0 ± 0.0
18.0 ± 0.7
7.4 ± 2.7
0.4 ± 0.6
12.2 ± 2.6
Mortality
rate (Abbott)
in %
on day 7
–
100
99
98
–
98
95
82
48
–
100
100
100
–
94
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. SETACEurope, 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
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 % .
47
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)
Quassin (l/ha)
Quassetum (l/ha)
26.8
0.4
40.0
53.6
0.8
80.0
80.4
1.2
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).
Mortality
%
Mortality (Abbott)
(Abbott) %
100
80
60
Quassia solution
Quassin
Quassetum
40
20
0
l/ha 26,8
53,6
80,4
0,8
1,2
40
80
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).
50
Table 2: Reproduction of C. carnea after exposure on Quassia residues during larval
development
Treatment
Control
Application
rate in l/ha
-
No. of
{{ }}
17 11
Fecundity a) Fertility % b)
33.3 ± 3.4
89.4 ± 5.1
Quassia
solution
26.8
53.6
80.4
18
15
16
11
13
9
35.6 ± 1.3
36.1 ± 3.9
32.5 ± 3.2
88.4 ± 4.3
89.7 ± 2.9
93.6 ±1.6
Quassin
0.8
1.2
15
12
11
14
36.8 ± 2.6
34.7 ± 3.4
89.9 ± 3.6
89.0 ± 1.7
40.0
80.0
13
15
12
8
39.0 ± 5.2
36.8 ± 1.0
86.1 ± 4.4
93.8 ± 2.7
Quassetum
a) mean no. of eggs eggs per female per day
b) mean hatching rate of the eggs
Mortality (Abbott) %
100
80
60
Quassin
Quassia solution
Quassetum
40
20
0
l/ha 26,8 53,6 80,4
0,4
0,8
1,2
40
80
120
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.
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
solution
26.8
53.6
80.4
9
12
10
9
14
13
37.1 ± 3.8
31.7 ± 2.9
32.7 ± 3.0
91.0 ± 1.1
92.1 ± 2.8
93.8 ± 2.0
Quassin
0.4
0.8
1.2
11
13
11
11
13
16
32.8 ± 4.6
32.8 ± 2.3
26.0 ± 2.2
89.3 ± 1.3
92.2 ± 1.4
91.2 ± 3.2
40.0
80.0
120.0
13
14
13
14
11
13
32.7 ± 2.7
34.4 ± 4.0
32.7 ± 1.7
94.6 ± 0.6
90.6 ± 0.8
91.9 ± 2.0
Quassetum
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
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.
53
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 2448 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)
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
% toxicity
(E)
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)
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
%
unhatched
eggs (R2)
0
3.59
3.01
1.75
3
0.1400
% toxicity
(E)
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
2.60 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
2.67 A
2.66 A
0.28 B
2.43 A
2.54 A
4
0.0008
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
S
R
Slope
(Standard
Error)
1.487
(0.484)
LC50
(Confidence
Interval)
296
(159 - 496)
Resistance
Factor
LC50 R/LC50 S
–
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
RxS
SxR
d.f.
Pr>f
% females
mortality
(M)
18.67 b
41.69 a
1
0.0224
Females
fertility
(R1)
0.72
0.73
1
0.9025
%
unhatched
eggs (R2)
5.60
9.96
1
0.0775
% toxicity
(E)
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: 7375.
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: Organophosphorous 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
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
61
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 compared. This revealed that there were no significant differences between these parameters (ttest; 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.
* : SDP = spray deposit
Active
ingredient
Acetamiprid
Amitraz
Avermectin
Avermectin
Chlorfenapyr
Clofentezin
Endosulfan
Fenpyroximate
Flufenoxuron
Imidacloprid
Novaluron
% effect
% mort.
Trade
Dose
%mort.
Rt/Rc % effect
% mort.
Rt/Rc
Rt/Rc
(E)
(SDP 30day)*
name (ppm AI) (SDP 5day)*
(E)
(SDP 15day)*
Mospilan
80
16.6
1
16.6
Mitac
400
81.5
0
100.0
67.7
0.3
91.0
64.7
1
Vertimec
5
58.9
1
58.9
20.0
1
20.0
2.5
1
Vertimec
10
61.1
1
61.1
55.0
1
55.0
5.0
1
Intrepid
240
23.9
1
23.9
Apollo
200
25.0
1
25.0
Thiodan
500
40.0
1
40.0
Naja
40
8.6
1
8.6
Cascade
11
49.0
1
49.0
35.0
1
35.0
Confidor
100
50.9
1
50.9
10.0
1
10.0
Rimon 10
50
26.0
1
26.0
EC
Pyrazophos
Afugan
220
32.6
1
32.6
Pyridaben
Sanmite
750
62.9
1
62.9
2.1
1
2.1
Spinosad
Tracer
250
23.9
1
23.9
Sulphur
Cumulus
5000
14.8
1
14.8
Tebufenpyrad Masaï
30
0.0
1
0.0
64.7
2.5
5.0
% effect
(E)
B
B
A
A
A
A
A
B
A
B
B
A
Toxicity
class
A
D
B
Table 1. Pesticides and concentrations tested on the predatory mite A. californicus. Nymphal mortality percentages of 15 pesticides after 7
days of exposure to pesticide spray deposits of 5, 15 and 30 days old. Total effect (mortality + oviposition effect) and toxicity class according
to the IOBC classification.
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
A semi-field test for evaluating the side-effects of plant protection
products on the aphid parasitoid Aphidius rhopalosiphi (DeStefaniPerez) (Hymenoptera, Braconidae) – First results
M. Moll1 & M. Schuld2
IBACON GmbH, Arheilger Weg 17, D-64380 Roßdorf, Germany;
2
GAB-Biotechnologie, Eutingerstr. 24, D-75223 Niefern-Öschelbronn, Germany
1
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 semifield 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
67
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 %.
20
18
Temperature [°C]
16
14
IBACON
12
10
GAB
8
6
4
2
0
17.07.2000
18.07.2000
19.07.2000
20.07.2000
21.07.2000
Date
Figure1. Average air temperature during exposure and parasitization phase of the semi-field
study
69
100
Relative Humidity [%]
90
80
70
60
IBACON
50
40
GAB
30
20
10
0
17.07.2000
18.07.2000
19.07.2000
20.07.2000
21.07.2000
Date
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).
80
Parasitation Rate [%]
70
60
50
IBACON
GAB
40
30
20
10
0
5 ml/ha
10 ml/ha
20 ml/ha
Perfekthion
Figure 3. Parasitation rate relative to the control
30 ml/ha
40 ml/ha
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
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;
71
72
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
73
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.
Inside surface
covered by black
netting
Aluminium frame
13 cm
13 cm
Upper and lower
surfaces covered
by foam
1,5 cm
Ventilation hole
Host/food introduction hole
Wasp introduction hole
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
74
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.
Emergence tube
parasitised egg circle
lines of honey/gelatine
20 mm
7 mm
120 mm
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).
75
2.
2.1
2.1.1
2.1.2
2.1.3
2.2
2.3
2.4
Part 2: Pupae initial toxicity test (less susceptible life stage, pupae within the
host eggs)
Experimental conditions
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.
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.
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).
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.
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.
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.
76
3.
3.1
3.1.1
3.1.2
3.1.3
3.2
3.3
Part 3: Persistence test (duration of harmful activity, exposure of adults to
pesticide residues applied on plants at intervals after treatment)
Experimental conditions
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).
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.
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.
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.
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
77
3.4
(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.
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
4.1.1
Experimental conditions
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.
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
4.1.2
78
4.1.3
4.2
4.3
4.4
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.
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.
Application of chemical/water
A stationary sprayer similar to that described under 1.6 may be used.
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).
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.
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.
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.
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.
5.1.1
5.1.2
5.1.3
5.2
Application of products
79
5.2.1
5.2.2
5.2.3
5.2.3.1
5.2.3.2
5.2.3.3
5.2.3.4
5.2.3.5
5.3
5.3.1
5.3.2
5.3.2.1
5.3.2.2
5.3.2.3
Test product(s)
The named usually formulated product under investigation.
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.
Mode of application
Applications should comply with good agricultural practice.
Type of application
According to the instructions of the (proposed) label. Normally a spray.
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.
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.
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.
Data on chemicals used against other pests
Interference with other chemicals should be avoided.
Mode of assessment, recording and measurements
Meterorological data
Records of temperature and humidity should be made during the whole trial
period, thermohygrograph may be used.
Type, time and frequency of assessment
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.
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.
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
80
5.3.3
5.3.4
5.4
6.0
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.
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.
Repeated treatments
Preparations that are treated more than once are tested by repeating the points 1, 2
and 3 for each treatment.
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.
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.
81
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.
82
Pesticides and Beneficial Organisms
IOBC/wprs Bulletin Vol. 24 (4) 2001
pp. 83 - 88
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
83
84
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/nonmarked 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.
85
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.
100
% pollina-ted flowers
Control
Nemacur
Confidor
80
60
40
20
0
1
2
3
4
5
6
7
Evaluation
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
86
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
M ± SE
M ± SE
M ± SE
M ± SE
M ± SE
M ± SE
M ± SE
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
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
Treatment
F2
F3
M
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
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.
87
70
Control
Nemacur
Confidor
60
Flight Activity
50
40
30
20
10
0
2
3
08/11/99
4
16/11/99
5
23/11/99
30/11/99
6
7
07/12/99
14/12/99
Evaluation Date
Fig. 2. Mean flight frequency (no. of worker bumblebees entering and leaving the hive/30
minutes). (Error bars = standard errors).
alive larvae
alive
k
70
60
No./ 50
hive 40
30
20
10
0
100
No./ 80
hive
60
40
20
0
1
Control
2
Nemacur
1
Control
3
Confidor
2
Nemacur
3
Confidor
alive pupae
50
40
No./
hive 30
20
10
0
Control
1
Nemacur
2
Confidor
3
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.
88
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.
Pesticides and Beneficial Organisms
IOBC/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
89
90
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.
91
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.
92
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
Sampling 1
Carabidae large
Carabidae small
Lycosidae
Araneae other
Staphylinidae
Collembola
Acari (mites)
Carabidae larvae
Field 2 centre
Three days after setting
13
3
124
44
14
73
7
0
Sampling 2
Carabidae large
Carabidae small
Lycosidae
Araneae other
Staphylinidae
Collembola
Acari (mites)
Carabidae larvae
Field 2 edge
2
0
127
36
5
75
0
0
12
3
66
20
35
14
1
0
6
1
93
12
7
18
5
0
Seven days after setting
32
10
81
51
6
349
2
2
6
2
78
48
4
1633
1
1
37
22
87
38
47
250
0
2
8
5
94
49
90
32
0
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).
93
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
Caelifera
Psocoptera
Thysanoptera
Sternorrhyncha
Heteroptera
Coleoptera
Hymenoptera
Lepidoptera
Diptera
Araneae
Acari
Field 2
Caelifera
Psocoptera
Thysanoptera
Stenorrhyncha
Heteroptera
Coleoptera
Hymenoptera
Lepidoptera
Diptera
Araneae
Acari
Tree 1
Tree 2
Tree 3
Sum
0
0
2
5
0
5
13
0
103
24
0
0
0
0
1651
0
8
102
0
23
0
4
0
6
0
38
2
5
8
0
24
29
0
0
6
2
1694
2
18
123
0
150
54
4
Tree 1
Tree 2
Tree 3
Sum
0
0
0
22
1
1
2
1
5
1
0
1
9
0
34
0
3
1
0
21
2
0
0
0
0
24
2
12
6
0
21
3
0
1
9
0
80
3
16
9
1
47
6
0
94
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
95
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 NonTarget 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., MeadBriggs, 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.
96
Pesticides and Beneficial Organisms
IOBC/wprs Bulletin Vol. 24 (4) 2001
pp. 97 - 101
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
97
98
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
Biolid E
Biolid E+
Folithion
Magister
Mineral oil
Mineral oil +
Fenitrothion
Fenazaquin
Oikos
Azadirachtin A
Formulation
%
80
80 +
48.5
18.32
Dosage (cc/hl)
3.2
150
1000
1000+150
35
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).
99
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
Biolid E
Biolid E +
Folithion
Magister
Oikos
T+1
% Cl
14,6 1
T+6
% Cl
0
1
T + 13
% Cl
0
1
T + 20
% Cl
0
1
T + 27
% Cl
0
1
76,5
4
59,5
3
20,3
1
0
1
0
1
36,0
0
2
1
7,3
0
1
1
41,5
0,7
2
1
40,7
0
2
1
30,0
20,0
2
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
Biolid E +
Folithion
Magister
Oikos
T+1
% Cl
T+6
% Cl
T + 13
% Cl
T + 20
% Cl
T + 27
% Cl
T + 34
% Cl
89,6
4
75,9
4
49,9
2
12,8
1
4,6
1
0
1
73,5
0
3
1
84,3
22,8
4
1
80,6
1,8
4
1
73,6
0
3
1
76,0
0
4
1
52,1
0
3
1
100
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
Oikos
T+1
%
18,6
Cl
1
T+6
%
23,5
Cl
1
T + 13
%
19,0
Cl
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
101
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.
102
Pesticides and Beneficial Organisms
IOBC/wprs Bulletin Vol. 24 (4) 2001
pp. 103 - 112
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, Planococcus 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
105
106
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
Euseius
stipulatus
Rodolia
cardinalis
Cryptolaemus
montrouzieri
Leptomastix
dactylopii
Lysiphlebus
testaceipes
Cales noacki
Costa-Comelles
et al., 1994
Garrido & del
Busto, 1986
Ripollés, 1986
Field,
Direct spray
Laboratory,
Direct spray
Laboratory,
Direct spray
Laboratory,
Dry residue
Laboratory,
Direct spray
Laboratory,
Direct spray
Ripollés, 1986
Garrido et al.,
1986
Garrido, 1992
Exposure
substrate
Stage
Variable
Citrus tree
Mixed
Initial toxicity
Citrus leaf
Pupae
Initial toxicity
Glass
Pupae
Initial toxicity
Glass
Adults
Initial toxicity
Citrus leaf
Citrus leaf
Parasitized
Initial toxicity
pupae
Parasitized
Initial toxicity
pupae
107
Table 2. Effects of different plant protection compounds on the selected natural enemies.
Lysiphlebus
testaceipes
1
3
1
4
3
1-2
3
1
1
2
3
1
1-2
1
1
2
2
4
4
2-3
3
2
3
1
1
1-2
4
1
1
1
3
3
1-2
2
1
2
1-2
2
3
1
3
3
4
4
1
3
3-4
3-4
3-4
3
1
2
1
3
1
2
4
3
1
3
3-4
1
1
2
2-3
1-2
1
2
3
Cales noacki
Leptomastix
dactylopii
ORGANOCHLORINE
Endosulfan (Thiodan)
2-3
3
Lindane (Agronexa-60)
2
ORGANOPHOSPATES
Acephate (Orthene)
4
4
Carbofenthion (Spider Spray)
Chlorfenvinfos (Birlane)
2-3
4
Chlorpyrifos (Dursban)
2
2
Diazinon (Diazinon)
2
Dimethoate (Perfektion)
2-3
4
Ethion (Ethion Emuls.)
2-3
1
Ethylazinfos (Azifene)
Etrimfos (Ekamet)
Fenitrothion (1)
3
Fenthion (Lebaycid)
3
2
Fentoate (Cidalina)
4
Fosalone (Zolone)
Fosfamidon (Dimecron)
3-4
Fosmet (Imidan)
2-3
4
Malathion (Volktion)
2
4
Mecarbam (Murfotox)
1-2
Metamidofos (Tamaron)
Methidathion (Ultracid)
2-3
3
Methylazinfos (Gusathion)
2-3
3-4
Methylchlorpyriphos (Reldan)
3
1
Methyloxydemeton(Metasystox)
2
3
Methylparathion (Folidol)
Methylpyrimifos (Actellic)
1-2
1-2
Mevinfos (Fosdrin)
Ometoate (Folimat)
3-4
Pyridafenthion (Ofunack)
Quinalfos(Ekalux)
2-3
4
Tetraclorvinfos (Gardona)
1
Tiometon (Ekatin)
2
Triazofos (Hostathion)
Triclorfon (Dipterex)
1
1
Rodolia
cardinalis
Cryptolaemus
montrouzieri
Euseius
stipulatus
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”.
1
3
1-2
3
2-3
2-3
1-2
2
1
3
3
3
2
3
3
1
4
3-4
2-3
3
1-2
1-2
1-2
4
3-4
2-3
4
3
3
3
3
2
Carbaryl (Sevin)
Carbosulfan (Marshal)
Etiofencarb (Croneton)
Mehtomyl (Lannate)
Pirimicarb (ZZ-Aphox)
CARBAMATES
3-4
1-2
2
2
4
4
1-2
2
PYRETHROIDS
3-4
2
1-2
2
4
1-2
2
1
Bifentrin (Talstar)
3-4
1
Cihalotrin (Karate)
Cipermethrin (Ambush C)
4
4
4
Deltamethrin (Decis)
4
3-4
4
4
Fenvalerate (Sumicidin)
3-4
4
Flucitrinate (Cybolt)
2
Fluvalinate (Klartan)
3-4
3
4
3
Permethrin(Ambush)
4
JUVENILE HORMONE ANALOGUES
Fenoxycarb (Insegar)
1-2
4
Pyriproxifen (Atominal)
1
4
4
INSECT GROWTH REGULATORS
Buprofezin (Applaud)
1-2
3
Diflubenzuron (Dimilin)
1
Flufenoxuron (Cascade)
2-3
Hexaflumuron (Consult)
1
Lufenuron (Match)
1
1
4
Teflubenzuron (Nomolt)
MOULTING ACCELERATING COMPOUNDS
Tebufenozide (Mimic)
1
NEONICOTINOIDS
Imidacloprid (Confidor)
2-3
4
ACARICIDES
Amitraz (Mitac)
4
1
Benzoximate (Artaban)
2
Bromopropilate (Neoron)
3-4
3
Clofentezine (Apollo)
1-2
Dicofol (Kelthane)
4
1
1
Fenazaquin (Magister)
4
3
Fenbutatin oxide (Torque)
2
1
1
Fenotiocarb (Panocon)
3
Cales noacki
Lysiphlebus
testaceipes
Leptomastix
dactylopii
Rodolia
cardinalis
Cryptolaemus
montrouzieri
Euseius
stipulatus
108
1
2-3
1
2
1
1
3-4
1
1
1
1
1
3
3-4
3
3
2
4
2
2-3
1
1-2
1
1
3
1
1
1
1
2-3
1-2
2-3
1
2
4
1
1-2
Hexithiazox (César)
Pyridaben (Sanmite)
Propargite (Omite)
Tetradifon (Tedion)
Captan (1)
Copper Oxychloride (1)
Zineb (1)
Avermectin (Vertimek)
Azadirachtin (1)
B. thuringiensis (Delfin)
Benfuracarb (Oncol)
Petroleum Spray Oil
Soap sprays
Butocarboxim (Darwin)
1
4
4
Cales noacki
Lysiphlebus
testaceipes
1
2-3
1-2
FUNGICIDES
1-2
1
1-2
1
OTHERS
2-3
1
1
1
1
1-2
1
4
Leptomastix
dactylopii
Rodolia
cardinalis
Cryptolaemus
montrouzieri
Euseius
stipulatus
109
2
1
1
1
1
1
2
1
1
1
2-3
3-4
2
1
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), organophosphates (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
110
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.
Active ingredient
Pest (toxicity)
Scales (4),
Cacoecimorpha
pronubana(4),
Flower moth (4)
Dimethoate
Aphids (2-4)
Malathion
Medfly (4)
Methidathion
Scales (3-4)
Methylpyrimifos
Scales (3-4)
Carbosulfan
Aphids (3-4)
Diflubenzuron
Leafminer (4)
Flufenoxuron
Spider mites (3-4),
leafminer (4)
Hexaflumuron
Leafminer (4)
Lufenuron
Woolly white fly (34), leafminer (4)
Imidacloprid
Leafminer (4)
Bromopropilate
Eryophyes sheldoni
(4)
Dicofol
Spider mites (4)
Hexithiazox
Spider mites (3-4)
Copper Oxichloride Phytophthora spp.
Avermectin
Leafminer (4)
B. thuringiensis
Cacoecimorpha
pronubana (4),
Flower moth (4)
Benfuracarb
Aphids (3-4),
leafminer (4)
Petroleum Spray Oil Scales (3-4), spider
mites (3-4)
Average toxicity against natural enemies
GTC (n)
SELCTV (n)
Chlorpyrifos
2.3 (6)
3.06 (34)
2.5 (6)
3.2 (6)
2.7 (6)
2.6 (4)
1.5 (1)
1.0 (1)
2.97 (209)
3.12 (256)
2.88 (42)
3.83 (12)
–
1.97 (77)
2.0 (2)
–
1.0 (1)
–
1.8 (4)
–
3.2 (3)
–
2.5 (4)
1.63 (8)
1.8 (5)
1.0 (2)
1.3 (3)
2.5 (1)
1.59 (68)
–
–
2.95 (18)
1.0 (5)
0.00 (2)
1.0 (1)
–
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
111
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 products 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.
112
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: 232248.
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. SamsøePetersen, 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
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
113
114
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
115
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.
300
150
250
number of parasitoids
200
150
50
100
50
0
number of predators
100
0
3
4
5
6
7
8
9
10
month
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.
116
average individuals/sample
A
***
4
**
*
3
***
2
**
1
0
3
4
5
6
7
8
9
10
month
***
***
***
***
***
6
7
8
9
10
month
average inviduals/sample
B
2
1,5
1
0,5
0
3
4
5
average individuals/sample
C
**
2,5
2
1,5
1
*
0,5
0
3
4
5
6
7
8
9
10
month
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).
117
A
average individuals/sample
***
5
***
4
3
***
2
**
*
1
0
3
4
5
6
7
8
9
10
month
average individuals/sample
B
2
1,5
***
**
1
0,5
*
0
3
4
5
6
7
8
9
10
month
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
118
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
individuals
Organic
%
larvae (adults)
Order Neuroptera (s.s.)
Family Chrysopidae
Family Coniopterygidae
Order Raphidioptera
Total no. of individuals
Diversity (Shannon Index)
C. carnea
Mallada sp.
Chrysopidae
indet.
individuals
%
larvae (adults)
139 (38)
3
21
80
1
9
64 (16)
51 (1)
11
38
25
5
(18)
(5)
224
0.7
8
2
100
(38)
(29)
210
1.4
18
14
100
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.
119
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: 3239.
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.): 225250.
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: 13111313.
120
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. SamsøePetersen, 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): 97106.
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
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
121
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.
123
Table 1. Treatment list in selectivity test of lufenuron, profenofos and its mixtures in cotton,
Kaha, Egypt, 2000.
Treatments
1 Untreated Control
2 Lufenuron
3 Lufenuron
4 Lufenuron
5 Curyom (Lufenuron
+ Profenofos)
6 Curyom (Lufenuron
+ Profenofos)
7 Profenofos
Treatment applied on
date*
1
2
3
4
Formulation
Rate
g a.i./ha
EC 050
EC 050
EC 050
50
100
50 x 2
EC 550
110
x
EC 550
330
x
EC 720
1000
x
x
x
x
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
1
Untreated
control
2
Lufenuron
50 g
3
Lufenuron
100 g
4
Lufenuron
2x50 g
5
Luf/Prof
10/100 g
6
Luf/Prof
30/300 g
7
Profenofos
1000 g
Repl. Plot size m2 Net amount Intended Net amount g
spray
rate g or ml or ml a.i./ha
liquid/ha
a.i./ha
applied
A
423,4
B
457
C
403,2
A
423,4
139
50
47,5
B
403,2
187,5
50
47,1
C
403,2
173,6
50
41,6
A
423,4
170,1
100
85
B
403,2
173,6
100
99,2
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
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
C
403,2
193,4
110
106,4
A
423,4
189
330
313,1
B
403,3
179,8
330
298,7
C
403,2
198,4
330
313,1
A
423,4
185,4
1000
909,8
B
295,7
209,7
1000
1056,8
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.
Profenofos 1000 g
Profenofos 1000 g
Luf/Prof 30/300 g
Luf/Prof 30/300 g
Lufenuron 100 g
Lufenuron 100 g
3 DAA
10 DAA
20 DAA
Lufenuron 2 x 50 g
Lufenuron 2 x 50 g
Lufenuron 2 x 50 g
30 DAA
Luf/Prof 10/ 100 g
Luf/Prof 10/100 g
Luf/Prof 10/100 g
Figure 1.ofExample
of trial
layout32
in on
field
on theStation
Kaha Station
7 treatments
and example
of marking
three
Figure 1. Example
trial layout
in field
the32Kaha
with 7with
treatments
and example
of marking
of of
three
subsample sites in one
subsample
sites
in
one
replicate
in
the
untreated
control
replicate in the unterated control
Legend:
Sampling dates
- 3 DAA*
Lufenuron 50 g
Untreated control
*DAA = days after application
Lufenuron 50 g
Untreated control
Lufenuron 100 g
Strip treated but not sampled
Lufenuron
Lufenuron50
50gg
Untreated control
Strip treated but not sampled
Profenofos 1000 g
Luf/Prof 30/300 g
125
1st prespray sampling
1st prespray sampling
1st prespray sampling
Treatment
Treatment
Treatment
Treatment
no 1:
no 2:
no 3:
no 4:
1, 2, 3
4, 5, 6
7
4
2, 3
4, 5, 6
7
Application
in
treatment no.
(+10 DAA)
(+10 DAA)
(+10 DAA)
(+20 DAA)
(+20 DAA)
(+20 DAA)
(+3 DAA)
(+3 DAA)
(+3 DAA)
1, 2, 3
(+30 DAA)
4***, 5, 6 (+30 DAA)
1, 2, 3
4*, 5, 6
7
1, 2, 3
4**, 5, 6
7
1, 2, 3
4, 5, 6
7
(- 3 DAA)
(- 3 DAA)
(- 3 DAA)
Postspray
sampling in
treatment no.
4***= 30 DAA1 (23 DAA2)
4**= 20 DAA1 (13 DAA2)
4*= 10 DAA1 (3 DAA2)
Treatment no 5: CURIOM (Lufenuron+ Profenofos) EC 550 110 (10+ 100) g ai/ha
Treatment no 6: CURIOM (Lufenuron+ Profenofos) EC 550 330 (30+ 300) g ai/ha
Treatment no 7: Profenofos EC 720 1000 g ai/ha
4th postspray sampling
4th postspray sampling
1st application
1st application
1st application
1st postspray sampling
1st postspray sampling
1st postspray sampling
2nd application
2nd postspray sampling
2nd postspray sampling
2nd postspray sampling
3rd postspray sampling
3rd postspray sampling
3rd postspray sampling
Untreated control
Lufenuron EC 050 50 g ai/ha
Lufenuron EC 050 100 g ai/ha
Lufenuron EC 050 2x50 g ai/ha
DAA = days after application
June 6
7
8
9
10
11
12
13
14
17
19
20
21
29
30
July 1
3
9
10
Prespray
sampling in
treatment no.
Table 3.- Time schedule for selectivity test of lufenuron, profenofos and their mixtures in cotton, Kaha, Egypt, 2000
126
127
Results and discussion
Occurrence (arthropod populations)
The counts revealed a rather even distribution of all groups of predators in all fields (Tables 48). 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.
a
a
a
a
a
a
ab
b
ab
a
340
36
13
14
2
15
140
237
43
228
190
24
314
116
21
5
7
27
36
267
50 g ai/ha
D** Lufenuron
a
b
ab
b
a
a
a
a
a
170
57
380
175
15
4
12
28
30
a
a
ab
ab
a
a
a
a
a
245
70
302
143
18
2
10
33
26
D** Lufenuron
a
a
ab
ab
a
a
a
a
a
230
47
466
220
22
3
17
37
29
a
ab
a
a
a
a
a
a
a
248
74
465
206
12
5
16
18
22
a
a
a
a
b
a
a
ab
a
206
55
311
183
13
2
13
15
39
D** Lufenuron/ D** Lufenuron/ D** Profenofos
Profenofos
Profenofos
100 g ai/ha
2x50 g ai/ha
10/100 g ai/ha
30/300 g ai/ha
1000 g ai/ha
Number of specimens per 90 m cotton row
a
364
a
369
a
350
a
445
a
561
D** Lufenuron
D** Duncan's Multiple Range Test: Figures followed by the same letter are not significantly different (P>0.05)
Orius
adult
Orius immature
L1-L2
Orius immature
L3-L5
Campylomma
adult
Campylomma
immature
Chrysoperla
immature
Scymnus
adult
Coccinella
adult
Paederus
adult
Araneae
imm. + adult
Untreated
control
Table 4. Evaluation of cotton predator fauna 3 days before application, Kaha, Egypt, 2000
a
a
b
a
b
a
a
b
a
a
D**
129
a
a
a
a
a
ab
a
bc
a
80
38
19
6
45
174
303
65
320
363
46
386
134
52
4
13
27
127
-13
29
-27
23
-16
33
32
29
-59
a
c
a
b
a
a
a
a
a
321
123
373
212
54
3
9
44
121
0
-89
-23
-22
-20
50
53
-16
-51
a
a
a
a
a
a
a
a
a
344
85
331
194
45
2
9
36
121
-8
-31
-9
-11
0
67
53
5,3
-51
a
ab
a
ab
a
a
a
a
a
387
73
339
260
66
2
15
27
62
A* Abbott (% reduction or increase of predator population)
D** Duncan's Multiple Range Test: Figures followed by the same letter are not significantly different (P>0.05)
Orius
adult
Orius immature
L1-L2
Orius immature
L3-L5
Campylomma
adult
Campylomma
immature
Chrysoperla
immature
Scymnus
adult
Coccinella
adult
Paederus
adult
Araneae
imm. + adult
-21
-12
-12
-49
-47
67
21
29
23
a
bc
a
a
a
a
a
a
a
418
98
276
281
57
0
4
7
37
-31
-51
8,9
-61
-27
100
79
82
54
a
a
b
a
bc
a
b
c
b
Untreated D** Lufenuron A* D** Lufenuron A* D** Lufenuron A* D** Lufenuron/ A* D** Lufenuron/ A* D** Pr
control
%
%
%
Profenofos %
Profenofos %
50 g ai/ha
100 g ai/ha
2x50 g ai/ha
10/100 g ai/ha
30/300 g ai/ha
10
Number of specimens per 90 m cotton row
496
a
464
6,5 a
460
7,3 a
448
9,7 a
399
20 a
257
48 b
Table 5. Evaluation of cotton predator fauna 3 days after application, Kaha, Egypt, 2000
130
a
a
a
ab
a
ab
a
bc
a
159
153
8
35
91
295
342
83
646
562
74
350
216
74
19
4
1
99
13
11
-2
27
19
46
50
99
38
a
c
a
b
a
bc
a
c
b
550
175
415
331
65
10
13
5
127
15
-110
-21
-12
29
71
-63
97
20
a
a
a
ab
a
c
a
c
ab
474
142
384
277
72
9
7
1
70
27
-71
-12
6,1
21
74
13
99
56
a
ab
a
ab
a
c
a
c
b
560
115
440
400
81
54
15
26
159
A* Abbott (% reduction or increase of predator population)
D** Duncan's Multiple Range Test: Figures followed by the same letter are not significantly different (P>0.05)
Orius
adult
Orius immature
L1-L2
Orius immature
L3-L5
Campylomma
adult
Campylomma
immature
Chrysoperla
immature
Scymnus
adult
Coccinella
adult
Paederus
adult
Araneae
imm. + adult
13
-39
-29
-36
11
-54
-88
83
0
a
ab
a
a
a
a
a
b
a
475
144
285
311
66
55
10
6
99
26
-73
17
-5
27
-57
-25
96
38
a
a
ab
a
bc
a
ab
d
b
420
116
138
230
56
3
5
11
52
35
-40
60
22
38
91
38
93
67
a
a
c
ab
c
c
b
cd
c
Untreated D** Lufenuron A* D** Lufenuron A* D** Lufenuron A* D** Lufenuron/ A* D** Lufenuron/ A* D** Profenofos A* D**
control
%
%
%
Profenofos %
Profenofos %
%
50 g ai/ha
100 g ai/ha
2x50 g ai/ha
10/100 g ai/ha
30/300 g ai/ha
1000 g ai/ha
Number of specimens per 90 m cotton row
407
a
330
19 a
368
9,6 a
411
-1
a
393
3,4 a
263
35 ab
227
44 b
Table 6. Evaluation of cotton predator fauna 10 days after application, Kaha, Egypt, 2000
131
a
a
a
b
a
a
a
ab
a
186
84
57
37
49
369
428
119
535
402
86
337
246
20
13
4
33
24
25
28
21
33
59
65
93
61
87
b
b
a
c
b
bc
ab
b
c
425
173
396
366
20
1
4
0
18
21
-45
7,5
0,8
59
97
93
100
90
b
a
a
a
b
d
ab
c
c
415
114
317
318
26
6
0
6
8
22
4,2
26
14
47
84
100
93
96
b
ab
a
ab
ab
cd
b
c
c
502
168
409
272
24
99
11
47
66
A* Abbott (% reduction or increase of predator population)
D** Duncan's Multiple Range Test: Figures followed by the same letter are not significantly different (P>0.05)
Orius
adult
Orius immature
L1-L2
Orius immature
L3-L5
Campylomma
adult
Campylomma
immature
Chrysoperla
immature
Scymnus
adult
Coccinella
adult
Paederus
adult
Araneae
imm. + adult
6,2
-41
4,4
26
51
-167
81
44
65
ab
a
a
bc
b
a
ab
b
b
476
175
265
340
26
47
7
5
28
a
a
c
b
a
c
c
11
a
-47 ab
38
7,9
47
-27
88
94
85
540
211
145
289
50
61
6
60
89
-1
-77
66
22
-2
-65
89
29
52
a
a
b
ab
b
a
a
b
ab
Untreated D**Lufenuron A* D** Lufenuron A* D** Lufenuron A* D** Lufenuron/ A* D** Lufenuron/ A* D**Profenofos A* D**
control
%
%
%
Profenofos %
Profenofos %
%
50 g ai/ha
100 g ai/ha
2x50 g ai/ha
10/100 g ai/ha
30/300 g ai/ha
1000 g ai/ha
Number of specimen per 90 m cotton row
291
a
199
32 b
220
24 ab
228
22 ab
237
19 ab
215
26 b
229
21 ab
Table 7. Evaluation of cotton predator fauna 20 days after application, Kaha, Egypt, 2000
132
ab
a
a
a
a
b
a
a
a
45
131
11
12
19
116
152
72
325
85
215
58
161
136
9
5
9
29
48
34
19
-6
-17
53
58
18
78
-7
b
a
a
b
a
ab
a
b
ab
195
104
205
146
13
3
3
20
31
40
-44
-35
-26
32
75
73
85
31
b
a
a
b
a
b
a
b
bc
204
95
265
248
19
2
4
27
17
37
-32
-74
-113
0
83
64
79
62
b
a
a
a
a
b
a
b
c
189
85
235
161
19
4
11
96
76
36
-13
-83
-45
0
8
0
35
-31
b
a
a
ab
a
ab
a
a
a
251
125
187
218
21
6
9
94
55
a
a
a
23
-74
-23
b
a
a
-88 ab
-11
50 ab
18
28
-22 ab
D** Lufenuron A* D** Lufenuron A* D** Lufenuron A* D** Lufenuron/ A* D** Lufenuron/ A* D**
%
%
%
Profenofos %
Profenofos %
50 g ai/ha
100 g ai/ha
2x50 g ai/ha
10/100 g ai/ha
30/300 g ai/ha
Number of specimen per 90 m cotton row
a
89
-5
a
67
21 ab
88
-4
a
44
41
b
90
-6 a
A* Abbott (% reduction or increase of predator population)
D** Duncan's Multiple Range Test: Figures followed by the same letter are not significantly different (P>0.05)
Orius
adult
Orius immature
L1-L2
Orius immature
L3-L5
Campylomma
adult
Campylomma
immature
Chrysoperla
immature
Scymnus
adult
Coccinella
adult
Paederus
adult
Araneae
imm. + adult
Untreated
control
Table 8. Evaluation of cotton predator fauna 30 days after application, Kaha, Egypt, 2000
133
Number of specimen per 90 m cotton row
-3
3
20
Days after application
10
30
Pro fe no fos 100 0 g ai
Lufe nuron/Pro fe no fos 30/300 g
ai
Lufe nuron/Pro fe no fos 10/100 g
ai
Lufe nuron 2 x 50 g ai
Lufe nuron 10 0 g ai
Lufe nuron 50 g ai/ha
Untre ate d control
Figure 2. Impact of lufenuron and its mixtures with profenofos on predators in cotton, Kaha, Egypt, 2000
0
100
200
300
400
500
600
Orius albidipennis, adults
(minute pirate bug)
134
Number of specimen per 90 m cotton row
-3
3
Days after application
10
20
30
Profe no s 1 00 0 g ai
Lufe nuron/Profe no fos 30 /30 0 g
ai
Lufe nuron/Profe no fos 10 /10 0 g
ai
Lufe nuron 2 x 50 g ai
Lufe nuron 10 0 g ai
Lufe nuron 50 g a i/ha
Untre ated contro l
Figure 3. Impact of lufenuron and its mixtures with profenofos on predators in cotton, Kaha, Egypt, 2000
0
1 00
2 00
Orius albidipennis, small nymphs (L1-2)
(minute pirate bug)
(nymphs at 3 and 10 DAA were predominantly L1)
135
Number of specimen per 90 m cotton row
-3
3
Days after application
10
20
30
Profe nofos 1000 g ai
Lufe nuron/Profe nofos 30/300 g
ai
Lufe nuron/Profe nofos 10/100 g
ai
Lufe nuron 2 x 50 g ai
Lufe nuron 100 g ai
Lufe nuron 50 g ai/ha
Untre ate d control
Figure 4. Impact of lufenuron and its mixtures with profenofos on predators in cotton, Kaha, Egypt, 2000
0
100
200
Orius albidipennis, big nymphs (L3-5)
(minute pirate bug)
136