working group „pesticides and beneficial organisms“ · bouqut, etienne; dow agrosciences,...

IOBC / WPRS

Working Group „Pesticides and Beneficial Organisms“

OILB / SROP

Groupe de Travail „Pesticides et Organismes Utiles“

Proceedings of the meeting

at

Ponte de Lima, Portugal

8th –10th October 2003

editor:

Heidrun Vogt

IOBC wprs Bulletin Bulletin OILB srop Vol. 27 (6) 2004

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 2004

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: [email protected]

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 92-9067-168-6.

Preface

The IOBC WG „Pesticides and Beneficial Organisms“ held its annual meeting at the Escola Superior Agrária, Ponte de Lima, Portugal, from 8th – 10th October 2003. The meeting was attended by 80 participants from 12 countries (Austria, Belgium, Canada, France, Germany, Italy, Poland, Portugal, Spain, Switzerland, The Netherlands, United Kingdom).

Oral and poster presentations covered a wide range of pesticide side-effects’ topics as well as the importance of beneficials for integrated control in different crops. Special group meetings took place to discuss and exchange results from ring tests for the validation, development and finalization of test guidelines. Due to the fact, that quite many of participants from Southern Europe took part at the meeting for the first time, there was a very intense exchange about the principles and research subjects of our WG, which aims at promoting the environmental and for beneficials safe use of pesticides by working out and providing as much knowledge a possible about side-effects of pesticides. From all contributions presented at the meeting 13 are published in this Bulletin and 4 abstracts are included. I thank all colleagues who supported me in reviewing the manuscripts.

The WG members enjoyed the very special atmosphere in the “Escola Superior Agrária” as the school has been established in a former monastery. It contributed to giving a relaxing atmosphere to the meeting and the participants liked to stay there together for discussing. Many thanks go to the Local Organizers, J. Raul Rodriques and José Ribeiro, and their team, caring that everything went smoothly during the meeting and giving us the opportunity during an excursion to learn a lot about the region with its fascinating landscape, towns and villages, its history, its agricultural production, its wines and traditional cuisine.

The meeting was financially supported by IOBC. Furthermore, the Organisation Committee and, on behalf of the WG “Pesticides and Beneficial Organisms”, the convenor wants to thank the Escola Superior Agrária de Ponte de Lima, Câmara Municipal de Melgaço, Câmara Municipal de Vila Nova de Çerveira, Estação de Vitivinicultura Amândio Galhano, Fundação para a Ciència e a Tecnologia, Sapec Agro and Syngenta CP Lda. for their support.

Heidrun Vogt (Convenor)

Dossenheim, 3rd November 2004

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List of Participants

1. ADELBERGER, INGE; Gab Biotechnologie GmbH, Eutinger Strasse 24, D-75223 Niefern-Öschelbronn, Germany. E-mail: [email protected]

2. AGUIAR, ANA, (PROF.); Secção Autónoma de Ciências Agrárias, Faculdade de Ciências da Universidade do Porto, CAMPUS AGRARIO, 4485-661 Vairão, Portugal. E-mail: [email protected]

3. ALBANO, SILVIA; Estação Agronómica Nacional, Quinta do Marquês, Av. Da República Nova, 2784-505 Oeiras, Portugal. E-mail: [email protected]

4. AMARO COSTA, CRISTINA; Estação Agronómica Nacional, Quinta do Marquês, Av. Da República Nova, 2784-505 Oeiras, Portugal. E-mail: [email protected]

5. AMARO, FERNANDA; EAN - Estação Agronómica Nacional, Quinta do Marquês, Av. Da República Nova, 2784-505 Oeiras, Portugal. E-mail: [email protected]

6. AMARO, PEDRO (Prof.). Secção Autónoma de Protecção Integrada, Instituto Superior de Agronomia, Tapada da Ajuda, 1349-017, Lisboa, Portugal.

7. BACELAR, M. SOFIA; Escola Superior Agrária de Ponte de Lima, 4990-706 Refóios do Lima, Ponte de Lima, Portugal. E-mail: [email protected]

8. BAIER, BARBARA (Dr.); Institute of Ecotoxicologie and Ecochemistry in Plant Protection, Federal Biological Research Centre for Agriculture and Forestry (BBA), Königin-Luise-Straße 19, D-14195 Berlin, Germany. E-mail: [email protected]

9. BAKKER, FRANK (Dr.), Universiteit van Amsterdam, Dept. of Pure & Applied Ecology, Sect. Population Biology/MITOX, Kruislaan 320, NL-1098 SM Amsterdam, The Netherlands. E-mail: [email protected]

10. BARTELS, ANJA; AGES (Austrian Agency for Health and Food Safety), Institute for Plant Protection Products, Dept. Environmental Risk Assessment, Spargelfeldstraße 191, A-1226 Wien, Austria. E-mail: [email protected]

11. BARTH, MARKUS; BioChem agrar, Kupferstr. 6, D-04827 Gerichshain, Germany. E-mail: [email protected]

12. BLAKE, ROBIN; Ecological Sciences; Jealott’s Hill Int. Research Station Bracknell, Berkshire, SL6 6BG, United Kingdom. E-mail: [email protected]

13. BOAVIDA, CONCEIÇÃO; IMPACTEST, Avenida Almirante Reis, 204-7º Dto, 1000-056 Lisboa, Portugal. E-mail: [email protected]

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14. BOUQUT, ETIENNE; Dow AgroSciences, Buropolis BP 229, 1240, routes des Dolines, 06904 Sophia Antipolis cedex, France. E-mail: [email protected]

15. BOSTANIAN, NOUBAR; Integrated Pest Management and Biodiversity / Lutte intégrée et Biodiversité Agriculture et Agroalimentaire Canada / Agriculture and Agri-Food Canada, St-Jean-sur-Richelieu, Quebec, J3B 3E6, Canada. E-mail: [email protected]

16. BROWN, KEVIN (Dr); Ecotox Ltd., PO Box 1, Tavistock, Devon, United Kingdom. E-mail: [email protected]

17. BRUEHL, CARSTEN; Ecological Sciences; Jealott’s Hill Int. Research Station Bracknell, Berkshire, SL6 6BG, United Kingdom.E-mail: [email protected]

18. CAVACO, MIRIAM; DGPC (Direcção Geral de Protecção das Culturas), Tapada da Ajuda, Edifício 1, 1349-018 Lisboa, Portugal. E-mail: [email protected]

19. COSTA-LOURENÇO, INÊS; Rua Pedro Soares, Apartado 158, 7801-902 Beja, Portugal. E-mail: [email protected]

20. COSTA, JORGE; DRAEDM (Direcção Regional de Agricultura de Entre-Douro e Minho – Div. Protecção das Culturas), Rua da Restauração, 336, 4050-501 Porto, Portugal. E-mail: [email protected]

21. COULSON, MIKE; Ecological Sciences; Jealott’s Hill Int. Research Station Bracknell, Berkshire, SL6 6BG, United Kingdom. E-mail: [email protected]

22. DAVID, PHILIPS (DR.); Environmental Risk Assessment Team, Central Science Laboratory, Sand Hutton, York, YO41 1LZ, United Kingdom. E-mail: [email protected]

23. DELGADO, RUI; Syngenta CP Lda, Avenida de Berna, 52, 2º, 1050-043 Lisboa, Portugal. E-mail: [email protected]

24. DINTER, AXEL (Dr); Dupont de Nemours (Germany); Dupont Str. 1, D-61352 Bad Homburg, Germany. E-mail: [email protected]

25. DREXLER, ANDERA; BASF AG., Agricultural Products Division, P.O. Box 120, D-67114 Limburgerhof, Germany. E-mail: [email protected]

26. DRIJVER, CORA; Plant Protection Service, PO Box 9102, 6700 HC Wageningen, The Netherlands. E-mail: [email protected]

27. FERREIA-DA-COSTA, PAULO; Portugal Dow AgroSceinces, Alamead dos Oceanos n° 4.5405 A (Parque das Nações), 1990-389 Lisboa, Portugal. E-mail: [email protected]

28. FIGUEIREDO, ELISABETE; SAPI/DPPF, Instituto Superior de Agronomia, Tapada da Ajuda, 1349-017 Lisboa, Portugal. E-mail: [email protected]

29. FREITAS, JOSÉ; DRATM, Centro de Estudos Vitivinícolas do Douro, 5050-071 Peso da Régua, Portugal.

30. FUSSELL, SUZANNAH; MAMBO-TOX LTD., Biomedical Sciences Building, Bassett Crescent East, Southampton SO16 7PX, United Kingdom. E-mail: [email protected]

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31. GARRIDO, JOÃO; Estação Vitivinícola "Amândio Galhano" Apartado 23, 4970 Arcos de Valdevez, Portugal. E-mail: [email protected]

32. GIMENO, CARMEN (BIOLOGIST); TRIALCAMP, Pedro de Luna 9, 46017 Valencia, Spain. E-mail: [email protected]

33. GODINHO, MARIA; Estação Agronómica Nacional, Quinta do Marquês, Av. Da República Nova, 2784-505 Oeiras, Portugal. E-mail: [email protected]

34. GONÇALVES, CATARINA; Estação Agronómica Nacional, Quinta do Marquês, Av. Da República Nova, 2784-505 Oeiras, Portugal.

35. GUERNER-MOREIRA, JOAQUIM; DRAEDM, Direcçao Regional de Agricultura de Entre-Douro e Minho, Div. Protecçao das Culturas, Rua da Restauraçao, 336, 4050-501 Porto, Portugal. E-mail: [email protected]

36. HALSALL, NIGEL (Dr.); MAMBO-TOX LTD., Biomedical Sciences Building, Bassett Crescent East, Southampton SO16 7PX, United Kingdom. E-mail: [email protected]

37. HEIMBACH, UDO (Dr.); Federal Biological Research Centre for Agriculture and Forestry (BBA), Institute for Plant Protection in Arable Crops and Grassland, Messeweg 11/12, D-38104 Braunschweig, Germany. E-mail: [email protected]

38. HUGGETT, DAVID (DR.); Syngenta, Ecological Sciences, Jealott’s Hill Int. Research Station Bracknell, Berkshire, SL6 6BG, United Kingdom. E-mail: [email protected]

39. JACAS MIRET, JOSEP-ANTON (Dr); Universitat Jaume I ; Departament de Ciències Experimentals, Campus del Riu Sec, E-12071 Castelló de la Plana, Spain. E-mail: [email protected]

40. JANSEN, JEAN-PIERRE; Department of Biological control and Plant genetic ressources, Agricultural Research Centre, Gembloux, 2, Chemin de Liroux, 2, 5030 Gembloux, Belgium. E-mail: [email protected]

41. KLEIN, SABINE; IBACON GmbH, Arheilger Weg 17, D-64380 Roßdorf, Germany. E-mail: [email protected]

42. LAWRENCE, ALAN; Terrestrial Ecotoxicology, Huntingdon Life Sciences, Woolley Road, Alconbury, Huntingdon, Cambridgeshire, PE28 4HS, United Kingdom. E-mail: [email protected]

43. MALLETT, MICHAEL; CEMAS (Cem Analytical Services Ltd.), Glendale Park, Fernbank Road, North Ascot, Berkshire, SL5 8JB, United Kingdom. E-mail: [email protected]

44. MANUCCI, FREDERICA; Via Ca’Del Vento, 21 Bagnacavallo-Ravenna, Italy. E-mail: [email protected]

45. MAUS, CHRISTIAN (DR.); BayerCropScience AG, Ecotoxicology, Bldg. 6620, Alfred-Nobel-Str. 50, D-40789 Monheim, Germany. E-mail: [email protected]

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46. MCEWEN, PETER (Dr); Insect Investigations Ltd., Units 10-12, CBTC 2, Off Parkway, Capital Business Park, Wentloog, Cardiff, CF3 2PX, United Kingdom. E-mail: [email protected]

47. MEDINA, MARIA DEL PILAR (Dr.); Bravo Murillo, 257, 5º drcha, 29020 Madrid, Spain. E-mail: [email protected]

48. MÉGEVAND, BENOIT (Dr).; IMPACTEST, Avenida Almirante Reis, 204-7º Dto, 1000-056 Lisboa, Portugal. E-mail: [email protected]

49. MELANDRI, MASSIMILIANO; Via Ca’ Del Vento, 21 Bagnacavallo-Ravenna, Italy. E-mail: [email protected]

50. MENDES, FELISBELA; DGPC (Direcção Geral de Protecção das Culturas), Tapada da Ajuda, Edifício 1, 1349-018 Lisboa, Portugal. E-mail: [email protected]

51. MEXIA, ANTÓNIO M.M. (Prof.); Secção Autónoma de Protecção Integrada, Instituto Superior de Agronomia, Tapada da Ajuda 1349-017, Lisboa, Portugal; Estação Agronómica Nacional, Quinta do Marquês, Av. Da República Nova, 2784-505 Oeiras, Portugal. E-mail: [email protected] and [email protected]

52. MILES, MARK; DowAgroSciences, European Development Centre, 3 Milton Park, Abingdon, Oxon, United Kingdom. E-mail: [email protected]

53. MIRANDA, NELSON R.; Escola Superior Agrária de Ponte de Lima, 4990-706 Refóios do Lima, Ponte de Lima, Portugal. E-mail: [email protected]

54. MOLL, MONIKA (Dr.); IBACON GmbH, Arheilger Weg 17, D-64380 Roßdorf, Germany. E-mail: [email protected]

55. MOURA, M. LUÍSA; Escola Superior Agrária de Ponte de Lima, 4990-706 Refóios do Lima, Ponte de Lima, Portugal. E-mail: [email protected]

56. MÜTHER, JUTTA (Dr.); GAB Biotechnologie GmbH, Eutinger Strasse 24, D-75223 Niefern-Öschelbronn, Germany. E-mail: [email protected]

57. NAVE, ANABELA; AAPIM, Associação de Agricultores para Produção Integrada de Frutos de Montanha, Av. Monsenhor Mendes do Carmo, 23 - R/chão Esq., 6300-586 Guarda, Portugal. E-mail: [email protected]

58. NIENSTEDT, KARIN (Dr.); Springborn Smithers Laboratories (Europe) AG, Seestrasse 21, 9326 Horn, Switzerland. E-mail: [email protected]

59. OLSZAK, REMIGIUSZ (Dr.); Institute of Pomology and Floriculture, Pomologiczna 18, 96-100 Skierniewice, Poland. E-mail: [email protected]

60. PASQUALINI, EDISON (Dr.); Universitá degli Studi di Bologna, DISTA (Dipartimento di Scienze e Tecnologie Agroambientali), Via Filippo Re, 6, I-40126 Bologna, Italy. E-mail: [email protected]

61. PETERS, ARNE (Dr.); e-nema GmbH, Klausdorfer Str. 28-36, 24223 Raisdorf, Germany. E-mail: [email protected]

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62. PHILLIPS, DAVID; Environmental Risk Assessment Team, Central Science Laboratory, Sand Hutton, York. YO41 1LZ, United Kingdom. E-mail: [email protected]

63. PHILLIPS, SARAH (Dr.); Insect Investigations Ltd., Units 10-12, CBTC 2, Off Parkway, Capital Business Park, Wentloog, Cardiff, CF3 2PX, United Kingdom. E-mail: [email protected]

64. PIJNAKKER, JULIETTE; Praktijkonderzoek Plant & Omgeving, Kruisbroekwg, 5, 2670 AA Naaldwijk, The Netherlands. E-mail: [email protected]

65. POULLOT, DELPHINE; Enigma, Hameau de St. Véran, F-84190 Beaumes de Venise, France. E-mail: [email protected]

66. RIBEIRO, JOSÉ J.A.; Escola Superior Agrária de Ponte de Lima, 4990-706 Refóios do Lima, Ponte de Lima, Portugal. E-mail: [email protected]

67. RODRIGUES, J. RAUL; Escola Superior Agrária de Ponte de Lima, 4990-706 Refóios do Lima, Ponte de Lima, Portugal. E-mail: [email protected]

68. RODRIGUES, SOFIA; Estação Agronómica Nacional, Quinta do Marquês, Av. Da República Nova, 2784-505 Oeiras, Portugal.

69. RODRIGUES, VASCO; Escola Superior Agrária de Ponte de Lima, 4990-706 Refóios do Lima, Ponte de Lima, Portugal. E-mail: [email protected]

70. RÖHLIG, UTA; BioChem agrar, Kupferstr. 6, D-04827 Gerichshain, Germany. E-mail: [email protected]

71. SCHMITZER, STEPHAN; IBACON GmbH, Arheilger Weg 17, D-64380 Roßdorf, Germany. E-mail: [email protected]

72. SERRANO, CARLOS (Biologist); TRIALCAMP, Pedro de Luna 9, 46017 Valencia, Spain. E-mail: [email protected]

73. TORNIER, INGO (Dr.); GAB Biotechnologie GmbH, Eutinger Strasse 24, D-75223 Niefern-Öschelbronn, Germany. E-mail: [email protected]

74. TORRES, LAURA MONTEIRO (Prof); Departamento de Protecção de Plantas, Universidade de Trás-os-Montes e Alto Douro, Apartado 202, 5001-911 Vila Real Codex, Portugal. E-mail: [email protected]

75. URBANEJA, ALBERTO (Dr.); Dep. Protección Vegetal y Biotecnología, Instituto Valenciano de Investigaciones Agrarias (IVIA), Apartado Oficial, Carretera de Moncada-Náquera Km. 4.5, 46113 Moncada, Valencia, Spain. E-mail: [email protected]

76. VAN DE VEIRE, MARC (Dr.); Faculty of Applied Biological and Agricultural Sciences, University of Gent, Laboratory of Agrozoology, Coupure Links 653, B-9000 Gent, Belgium. E-mail: [email protected]

77. VINALL, STEPHEN; Mambo-Tox Ltd., Biomedical Sciences Building, Bassett Crescent East, Southampton SO16 7PX, United Kingdom. E-mail: [email protected]

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78. VIÑUELA, ELISA (Prof.); Protección de Cultivos, E.T.S.I. Agrónomos, E-28040 Madrid, Spain. E-mail: [email protected]

79. VOGT, HEIDRUN (Dr.); Federal Biological Research Centre for Agriculture and Forestry (BBA), Institute for Plant Protection in Fruit Crops, Schwabenheimer Straße 101, D-69221 Dossenheim, Germany. E-mail: [email protected]

80. WAINWRIGHT, MELANIE; Terrestrial Ecotoxicology, Huntingdon Life Sciences, Woolley Road, Alconbury, Huntingdon, Cambridgeshire, United Kingdom. PE28 4HS. E-mail: [email protected]

81. WALKER, HAZEL; Ecotox Ltd., PO Box 1, Tavistock, Devon, United Kingdom. E-mail: [email protected]

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Contents Preface......................................................................................................................................... i List of participants..................................................................................................................... iii Natural control against pests on vegetables in Portugal: important species

and their role. Mexia, A., Figueiredo, E. & do Céu Godinho, M .............................................................. 1

Predators of Phyllocnistis citrella Stainton (Lepidoptera: Gracillariidae) on citrus

in Spain: role of lacewings and ants. Jacas, J. A. & Urbaneja, A. ............................................................................................... 9

Comparative effects of several insect growth regulators and spinosad on the different

developmental stages of the endoparasitoid Hyposoter didymator (Thunberg). Schneider, M. Smagghe, G. & Viñuela, E. ....................................................................... 13

Duration of the toxicity of abamectin and spinosad on the parasitic wasp Encarsia

formosa Gahan in Northern and Southern Europe. Van de Veire, M., Viñuela, E., Bernardo, U., Tirry, L., Adan, A. & Viggiani, G. ........... 21

Toxic effects of indoxacarb to a predacious mirid and two species of predacious mites.

Bostanian, N.J., Vincent, Ch., Hardman, J.M. & Larocque, N. ...................................... 31 Toxicity of five insecticides on predatory mites (Acari: Phytoseiidae) in vineyards in

two Portuguese regions Rodrigues, R., Gonçalves, R., Silva, C. & Torres, L. ...................................................... 37

Effects of the fungicide zoxamide, alone and in combination with mancozeb, to

beneficial arthropods under laboratory and field conditions Miles, M. & Green, E. ..................................................................................................... 45

Development of an extended-laboratory method to test bait insecticides

Medina, P., Pérez, I., Budia, F., Adán, A. & Viñuela, E. ................................................ 59 Side effects of surfactants and pesticides on entomopathogenic nematodes assessed

using advanced IOBC guidelines Peters, A. & Poullot, D. ................................................................................................... 67

Influence of insecticide coated seeds on larvae of Poecilus cupreus (L.) (Coleoptera;

Carabidae) using different container sizes and quantities of substrate Heise, J., Heimbach, U. & Schrader, S. .......................................................................... 73

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POSTER PRESENTATIONS

Laboratory tests of the impact of insect growth regulators on Anthocoris nemoralis F. Luigi Caroli & Edison Pasqualini. .................................................................................. 81

Laboratory evaluation of four fungicides, two insecticides and an insecticide/acaricide

to Agistemus fleschneri Summers (Acari: Stigmaeidae) Bostanian, N. J., Racette, G. & Larocque, N. ................................................................. 87

Variation in response of earthworms and soil microflora to reference test substances

Mallett, M. J., Hayward, J C & Davies, N. A. ................................................................ 93 ABSTRACTS Influence of some pesticides on fecundity and longevity of Coccinella septempunctata and

Adalia bipunctata (Col., Coccinellidae) under laboratory conditions Olszak, R.W, Ceryngier, P. & Warabieda, W. ............................................................... 105

Non-target arthropod field studies: asking the right questions for their purpose

Brown, K. ...................................................................................................................... 107 Sampling methods in orchard trials: Assessment of treatment effects through beating and

inventory sampling Müther, J. & Vogt, H. .................................................................................................... 109

Comparison between two different collecting methods of Anthocoris nemoralis F. in

investigations about side-effects of pesticides in field tests Vergnani S., Melandri M., Manucci F., Civolani S. & Pasqualini E. ........................... 111

Pesticides and Beneficial Organisms IOBC/wprs Bulletin Vol. 27 (6) 2004

pp. 1 - 8

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Natural control against pests on vegetables in Portugal: important species and their role António Mexia1,2, Elisabete Figueiredo2 & Maria do Céu Godinho1,3 1 Estação Agronómica Nacional, Quinta do Marquês Av. da República, 2784-505 Oeiras 2 Instituto Superior de Agronomia, Tapada da Ajuda, Lisboa 3 Escola Superior Agrária de Viseu, Estrada de Nelas, 3500 Viseu Abstract: The authors present the available knowledge about the major beneficial arthropod species found in vegetable fields and greenhouses in Portugal based on fieldwork performed to assess their efficacy as natural control agents since the middle of the eighties. The experimental results have been used to develop decision rules and pest control schemes adjusted to the Portuguese’s agroecosystems in order to develop and spread Integrated Pest Management (IPM). However, information in literature for local existing species with a key role as biological control agents is almost inexistent in many crops and the new developed pesticides do not include relevant technical information about side effects related to such species in many cases. In such circumstances an additional effort to adapt existing methodologies like those made available by IOBC Working Group on side effects must be followed up and included in the scientific efforts of this working group. Key-words: natural control, protected crops. Introduction Sustainable protected vegetable agroecosystems in Mediterranean Climate are based on natural control towards the richness in autochthonous fauna. Those have an important role as control pest factor. Greenhouse structures and certain production techniques that are usual practices by the growers can potency that role avoiding some unnecessary pesticide sprays. In the present work the authors present a briefly characterisation of the greenhouse system in Portugal especially about those aspects related with the enhancement of the natural enemies role. In a second point the authors point out some available information related with grower’s characterisation in function of the adopted crop protection strategy and technician profiles for those growers. The authors discuss some key points considered determinant to the success of biological control based on autochthonous natural enemies. Information about natural enemies (predators and parasitoids) is listed for greenhouses and open field crops (processing tomato and cabbages).

In the last part some aspects are suggested based on the lack of information about side effects on some important species present in Mediterranean region. Greenhouses agroecosystem in Mediterranean climate – the Portuguese Oeste region case The classic greenhouse structures in Oeste region of Portugal are based on wood covered by polyethylene without environment conditioning. Growers open the lateral windows in the beginning of the day and close them by the evening. All of those tasks are done manually.

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The landscape of the region (Torres Vedras, Lourinhã e Peniche) located at 50 km north of Lisbon is mostly covered with vegetables growing in greenhouses alternating with open-field crops as green beans, potato, lettuce, cabbages and leek.

This structure allows the insects’ movements through the openings during a long period of the day. In this context all protection strategies should take into account the immigrating population of the different pests especially whiteflies and caterpillars (Figueiredo & Mexia, 2000). Most of the pest groups, because of their polyphagous character, attack other vegetable crops outside and these crops assume stock role. This situation is the same for beneficial organisms, especially mirid bugs that are very abundant in the region. This group is important because of the efficient predatory activity which is, in many times, crucial to control whitefly in the spring-summer season in the beginning of the crop cycle (e.g. Carvalho & Mexia, 2000).

In these circumstances, natural control against the most important pests as whiteflies, caterpillars, leafminers, aphids and mites is very important to reduce population abundances.

Experimental work has revealed several times that key-pests in vegetable crops in this region have distinct patterns of importance. This question is more evident in leafminers’ case. In fact, economic importance of this pest is extremely related with parasitoids and some generalist predators’ abundance (Godinho & Mexia, 2000). Protection strategies adopted by the growers at the farm functioning level A study about grower’s characterisation in function of the adopted crop protection strategy and technician profiles definition for some of those growers was developed in order to test hypotheses concerning the restrictions in the adoption of the related technologies in protected vegetable crops in the Oeste region. The ISA/SAPI team carried out this study in the end of nineties (1997-2000). This characterisation was made through growers’ surveys in two phases. The last sample was defined according to:

(i) pest control strategies: traditional chemical control, ‘conditioned’ chemical control, IPM and crop protection in organic agriculture;

(ii) crop: tomato, lettuce and greenbeans. According to the results there was a huge difference in the growers’ attitude and on the

practices related with crop protection. These can be one of the factors influencing natural enemies abundance and complex biodiversity and consequently their capacity to control pest populations (Rodrigo et al., 2000). Natural control against key – pests in horticultural systems Vegetable protected crops in the Oeste region

Greenhouses in the Oeste region of Portugal have been considered as a very rich agro ecosystem in natural control agents (Godinho, 1997; Marques et al., 1999) as it happens in general in Mediterranean climate. Studies conducted since 1990 by SAPI/DPPF team of Instituto Superior de Agronomia pointed out some parasitoid complexes and generalistic predators important for the control of the key-pests (Table 1). The majority of them were much more abundant in IPM greenhouses than in those under traditional chemical control. In the last greenhouses the abundance of natural control agents began to increase at the end of the crop cycle after chemical sprays stopped (Mexia et al., 1999). For example, in these IPM greenhouses natural control is as much necessary for maintaining leafminer’s population

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below economic threshold (Godinho & Mexia, 2000; Lourenço et al., 2002; Marques et al., 1999). Table 1. Parasitoid complexes and predators found in vegetable protected crops in the Oeste region of Portugal.

Key-pest Natural control agent Reference

Whiteflies Trialeurodes vaporariorum West.

Encarsia formosa Gahan Encarsia pergandiella (Howard) Encarsia tricolor Foerster Amitus fuscipennis Macgown

Marques & Mexia, 1998; Marques et al., 1999

Caterpillars Helicoverpa armigera (Hbn.) Chrysodeixis chalcites (Esper.) Thysanoplusia orichalcea (Fab.) Autographa gamma (L.) Peridroma saucia (Hbn.)

Telenomus laeviceps Foerster Trichogramma sp. Hyposoter didymator Thunberg Cotesia kazak (Telenga) Cotesia plutellae (Kurdyumov) Euplectrus flavipes Fonscolombe Microplitis mediator (Haliday) Meteorus pulchricornis (Wesmael)Macrocentrus sp. near collaris (Spinola) Voria ruralis (Fallén) Aleiodes sp. (2 sp.) Ctenochares bicolorus (L.)

Marques et al., 1999; Figueiredo & Mexia, 2000

Leafminers Liriomyza huidobrensis Blanchard

Diglyphus isaea (Walker) Diglyphus crassinervis Erdös Diglyphus poppoea Walker Diglyphus minoeus (walker) Dacnusa sibirica Telenga Halticoptera sp. Chrysocharis sp. Epiclerus sp. Hemiptarsenus fulvicollis Trichopria sp. Pteromalid (not Halticoptera sp.)

Marques et al., 1999; Godinho & Mexia, 2000; Godinho et al., 1994; Godinho, 1997

Aphids Aphis gossypii Glover Nasonovia ribisnigri (Mosley) Myzus persicae (Sulzer) Aphis craccivora Koch Macrosiphum euphorbiae (Thomas) Aulacorthum solani (Kaltenbach)

Aphidius colemani Viereck Aphidius ervi Haliday Aphidius matricariae Haliday Ephedrus sp. Lysiphlebus fabarum (Marshall) Lysiphlebus testaceipes (Cresson) Trioxys angelicae (Haliday) and hyperparasitoids

Marques et al., 1999; Valério, 1999; Valério et al., 2003

Predators Dicyphus cerastii Wagner Aphidoletes aphidimyza Rondani Chrysoperla carnea Stephens Orius laevigatus Fieber Orius albidipennis (Reuter) Coenosia attenuata Stein

Marques et al., 1999 Prieto et al., 2003

4

Vegetable protected crops in other Portuguese regions

The natural control agents’ complexes were also studied in other regions of Portugal producing vegetable protected crops, mainly in Algarve (in the south of the mainland territory), Madeira Island and Azores islands (Table 2). The majority of these species are common to the four vegetable protected crop regions (Oeste and these three) but the importance of each one in each complex is not the same, at least in some cases (example: whitefly parasitoid and predator complexes).

Table 2. Natural control agents in crops in other important protected vegetable production regions in Portugal (Algarve, Madeira and Azores). Region Natural control agent Reference

Algarve (Tv, Bt)

Eretmocerus mundus Encarsia pergandiella, E. lutea, E. sophia E. tricolor Amitus fuscipennis

Queirós et al., 2003

Madeira (Tv, Bt, Ba)

Clitostethus arcuatus (Tv) Amitus fuscipennis (dominant) (Tv) Encarsia formosa (Tv), E. tricolor (Tv), E. pergandiella

(Tv, Bt), E. hispida (Tv, Bt), E. lutea (Bt) Eretmocerus mundus (Tv, Bt, Ba) Euderomphale cortinae (Ba)

Aguiar,1999; Aguiar et al., 1995; Félix, 1999 W

hite

flies

Azores Clistothecus arcuatus (but mainly in brassicas) Encarsia formosa

Odelta Oliveira, pers. com.

Madeira Trichogramma cordubensis, T. evanescens Hyposoter didymator Pimpla hypochondriaca Ophion sp.

Garcia et al., 1998; Aguiar, 1999; Félix, 1999

Cat

erpi

llars

Azores Trichogramma cordubensis Telenomus sp.

Garcia et al., 1995 and 1997

Algarve Diglyphus isaea Diglyphus poppoea Synacra sp. Dacnusa sibirica Hemiptarsenus sp. Pnigalio sp.

Muzavor et al., 2003; Gonçalves & Anunciada, 1998; Neves et al., 1997; Godinho et al., 1994

Leaf

min

ers

Madeira Diglyphus isaea Chrysocharis entedonides Aprostocetus flavifrons Dacnusa sibirica (introduced) Dacnusa pubescens Chorebus (3 sp. including C. morae, endemic)

Aguiar, 1999; Félix, 1999

Madeira Lysiphlebus fabarum (dominant till introduction of L. testaceipes)

Lysiphlebus testaceipes (recently introduced) Praon sp., Praon volucre Aphidoletes aphidimyza Syrphus sp.

Aguiar, 1999, Félix, 1999

Aph

ids

Azores Hym.: Braconidae: Aphidiinae (not identified) Aphidoletes aphidimyza Scymnus levaillanti Episyrphus balteatus Metasyrphus corollae


Tv = Trialeurodes vaporariorum; Bt = Bemisia tabaci; Ba = Bemisia near affer

5

Table 2 (cont.). Natural control agents in crops in other important protected vegetable production regions in Portugal (Algarve, Madeira and Azores). Region Natural control agent Reference

Algarve Orius laevigatus Duarte & Almeida, 1999

Madeira Macrolophus sp. Cyrtopeltis tenuis Reuter Coenosia attenuata Stein coccinellids, chrysopids, anthocorids

Aguiar, 1999; Félix, 1999; Félix et al., 1997; Prieto et al., 2003

gene

ralis

tic p

reda

tors

Azores Coenosia attenuata Stein Macrolophus sp. Cyrtopeltis tenuis Reuter Hemerobius sp. Tachyponus chrysomelinus Tachyponus nitidulus Atheta fungi Oligota parva Lepthyphantes tenuis Oedothorax fuscus Araneae Brachypelma vagans

Prieto et al., 2003; Odelta Oliveira, pers. com.

Tv = Trialeurodes vaporariorum; Bt = Bemisia tabaci; Ba = Bemisia near affer Open field crops in the Oeste and Ribatejo Portuguese regions

Processing tomato Processing tomato crop is being studied in Ribatejo region by SAPI/DPPF team only since 2000, and so, information about beneficials is somehow preliminary. Important key-pests in processing tomato are: noctuid caterpillars, mites and in some years, when the summers are cooler, the aphids can also cause problems aborting flowers. Beneficials found in Portugal in this ecosystem by this team and previous references from others studying in particular some pests are summarized on Table 3.

Cabbages Information about natural control in cabbages crops is scarce but some key-pests (as Pieris spp. caterpillars) have been studied. SAPI/DPPF team began to study this agroecosystem in 2002. Therefore, information obtained by this team is very limited and is not published yet. Available results are summarized on Table 4. Suggestions to the working group Iberian Peninsula is not a homogenous unit. The complexes of natural control agents are not always coincident or the most important ones in each ecosystem are not the same. For instance, the mirids are all over the Peninsula an important predator for whiteflies but the dominant species are not the same in Catalonia, Almeria-Murcia and Oeste (Carvalho & Mexia, 2000; Castañé et al., 2000; Sánchez et al., 2003). These differences accentuate going far away, along Mediterranean climate region to South France, Italy, Greece, Turkey, Israel and North Africa. So, the species selected to study pesticide side-effects should be chosen between the most important ones in each crop and region, which is not simple!

6

Table 3. Natural control agents present in processing tomato in Ribatejo region.

Key-pest Natural control agent Reference

noctuid caterpillars (Helicoverpa armigera and Plusiinae)

Trichogramma evanescens, T. cordubensis, T. turkestanica T. pintoi, T. bourarachae, eggs T. rhenana Telenomus sp., T. laeviceps Cotesia kazak Hyposoter didymator Copidosoma floridanum larvae/pupae Brachymeria tibialis Euplectrus flavipes

Araújo, 1990; Polaszesk, 1996, pers. com.; Silva, 1999; Figueiredo et al., 2003; Figueiredo et al., 2003

mites Homeopronematus anconai Pronematus ubiquitus Phytoseiulus persimilis Amblyseius californicus Amblyseius barkeri Amblyseius imbricatus Amblyseius messor Amblyseius stipulatus Typholodromus phialatus Typholodromus rhenanoides Typholodromus transvaalensis

Ferreira & Carmona, 1995

Table 4. Natural control in cabbages crops in different Portuguese regions.

Region Key-pest Natural control agent Reference

Pieris spp.

Cotesia glomeratus Pteromalus puparum Anilatus ebenina

Mainland

Delia radicum Staphylinidae (probably Aleochara sp.)

Figueiredo & Araújo, 1985; Ferreira, pers. com., 2003

Plutella xylostella Diadegma semiclausum Diadromus collaris

Pieris spp.

Trichogramma evanescens Cotesia glomeratus Pteromalus puparum

Madeira

Brevicoryne brassicae Diaeretiella rapae – Alloxysta victrix

Garcia et al., 1998; Aguiar, 1999

aphids Diptera: Cecidomyiidae Hymenoptera: Braconidae Hymenoptera: Cynipoidea: Figitidae

Azores

whiteflies Clitostethus arcuatus


Acknowledgements The authors are thankful to Eng. Odelta Oliveira for providing information about natural control in Azores vegetable crops. References Aguiar, AM.F. 1999. Artrópodes auxiliares na Ilha da Madeira. – In: Carvalho, J.P. (ed.)

Contribuição para a protecção integrada na Região Autónoma da Madeira, DRA/POSEIMA Madeira, Funchal: 309-331.

7

Aguiar, A.M.F., Polaszek, A., Brazão, C.I. & Félix, A.P. 1995. Algumas notas sobre parasitóides (Hymenoptera: Aleyrodidae) na Ilha da Madeira. III – Enc. Nac. Protecção Integrada, Lisboa, 18-19 Dez. 1995, Resumos: 47.

Araújo, A.M.C.M. 1990. Luta biológica contra Heliothis armigera no ecossistema agrícola “tomate para indústria”. – Ph D. thesis, Univ. Évora, 380 pp.

Carvalho, P.P. & Mexia, A. 2000. First approach on the potential role of Dicyphus cerastii Wagner (Hemiptera: Miridae), as control agent in Portuguese greenhouses. – Bull OILB/SROP, 23(1): 261-264.

Castañé, C., Alomar, O, Goula, M. & Gabarra, R. 2000. Natural populations of Macrolophus caliginosus and Dicyphus tamaninii in the control of the greenhouse whitefly in tomato crops. – Bull. OILB/SROP, 23(1): 221-224.

Duarte, G. & Almeida, L. 1999. Interrelações entre planta hospedeira (Chrysanthemum maximum) / praga (Frankliniella occidentalis) / predador (Orius laevigatus). – Actas V Enc. Nac. Protecção Integrada, Bragança, 27-29 Out. 1999: 313-317.

Félix, A.P. 1999. Subprograma de luta contra Trialeurodes vaporariorum. – In: Carvalho, J.P. (ed.) Contribuição para a protecção integrada na Região Autónoma da Madeira, DRA/POSEIMA Madeira, Funchal: 45-83.

Félix, A.P., Duarte, P.J. & Mexia, A. 1997. Estufas de demonstração de protecção integrada em horticultura na Região Autónoma da Madeira. – Actas IV Enc. Nac. Protecção Integrada, Angra do Heroísmo, 3-4 Out. 1997: 505-510.

Ferreira, M.A. & Carmona, M.M. 1995. Acarofauna do tomateiro em Portugal. – In: Advances Entomol. Ibérica: 385-392.

Figueiredo, D. & Araújo, J. 1985. Factores de mortalidade de Sesamia nonagrioides Lef. (Lepidoptera: Noctuidae) em Portugal. I - Parasitóides. – Bol. San. Veg. Plagas, 22: 251-260.

Figueiredo, E., Albano, S., Salvado, E., Godinho, M.C, Gonçalves, C., Queirós, M.R., Amaro, F. & Mexia, A. 2003. Complexo de parasitóides de noctuídeos (Lepidoptera: Noctuidae) em tomate de indústria. – Resumos III Congr. Nac. Entomol. Aplic., Ávila, 20-24 Out. 2003: 330.

Figueiredo, E. & Mexia, A. 2000. Parasitoid complex associated with lepidoptera on horticultural protected crops in the Oeste region of Portugal. – Bull. OILB/SROP, 23(1): 205-208.

Garcia, P., Oliveira, L. & Tavares, J. 1995. Comparative biology of three Trichogramma sp. (Hym.: Trichogrammatidae) populations captured in Azores. – Bol. Mus. Municipal Funchal, supl. 4: 311-318.

Garcia, P., Oliveira, L. & Tavares, J. 1997. Estudo do parasitismo dos ovos de Autographa gamma (Lep.: Noctuidae) em culturas de tomate e beterraba. – Actas IV Enc. Nac. Protecção Integrada, Angra do Heroísmo, 3-4 Out. 1997: 215-220.

Garcia, P., Oliveira, L., Vieira, V. & Tavares, J. 1998. Parasitóides entomófagos da ilha da Madeira: distribuição e hospedeiros. – Bol. Soc. Port. Entomol., supl. 6: 433-440.

Godinho, M.C. 1997. Protecção integrada em culturas de estufa: contribuição para o estudo das larvas mineiras. – MSc. Thesis, ISA/UTL, Lisboa, 166 pp.

Godinho, M., Gonçalves, A., Lima-Leite, E., Anunciada, L. & Mexia, A. 1994. Leafminers (Liriomyza spp.) problems in greenhouses in Portugal: evolution and present situation. – Bull. OILB/SROP, 17(5): 199-205.

Godinho, M. & Mexia, A. 2000. Leafminers (Liriomyza spp.) importance in greenhouses in the Oeste region of Portugal and its natural parasitoids as control agents in IPM programs. – Bull. OILB/SROP, 23(1): 157-161.

8

Gonçalves, M.A. & Anunciada, M.L. 1998. A mineira das folhas Liriomyza spp. (Diptera: Agromyzidae) e os seus parasitóides no feijão-verde em cultura protegida. – Bol. Soc. Port. Entomol., supl. 6: 193-204.

Lourenço, I., Rodrigues, S., Figueiredo, E., Godinho, M.C., Marques, C., Amaro, F. & Mexia, A. 2002. The effect of crop protection strategy in pest and beneficials incidence in protected crops. – Med. Fac. Landbouww. Univ. Gent, 67(3): 569-573.

Marques, C. & Mexia, A. 1998. Mosquinhas brancas e seus parasitóides em estufas da região Oeste – estudo preliminar. – Bol. Soc. Port. Entomol., supl. 6: 265-269.

Marques, C., Nunes, A.P., Almeida, M.L., Godinho, M.C., Figueiredo, E., Amaro, F., Carvalho, P. & Mexia, A. 1999. Manual de protecção integrada em culturas protegidas. Principais pragas e auxiliares na região Oeste. – ISA Press, Lisboa, 61 pp.

Mexia, M., Marques, C., Figueiredo, E., Amaro, F., Godinho, M.C., Centeno, M., Almeida, M.L. & Nunes, A.P. 1999. Melhoria da produção hortícola em estufa no Oeste. Relatório final do projecto PAMAF 2034, ISA/UTL-MADRP/DRARO. – Lisboa, 55 pp.+ anexos.

Muzavor, L., Almeida, L. & Gonçalves, M.A. 2003. Importância das plantas infestantes como hospedeiros alternativos das mineiras das folhas, Liriomyza spp. e seus parasitóides. – 6º Enc. nac. Protecção Integrada, 14-16 Maio 2003, ESA Castelo Branco, Castelo Branco (in press).

Neves, M.S., Gonçalves, M.A. & Anunciada, M.L. 1997. Liriomyza spp. (Dip.: Agromyzidae) e seus parasitóides Diglyphus spp. (Hym.: Eulophidae) no feijão-verde em estufa. – Actas IV Enc. Nac. Protecção Integrada, Angra do Heroísmo, 3-4 Out. 1997: 251-258.

Prieto, R., Figueiredo, R., Miranda, C. & Mexia, A. 2003. Coenosia attenuata Stein (Diptera: Muscidae) prospecção e actividade em Portugal. – Resumos III Cong. Nac. Entomol. Aplic., Ávila, 20-24 Out. 2003: 331.

Queirós, M. R., Figueiredo, E. & Mexia, A. 2003. Complexo de parasitóides de mosquinhas brancas em culturas protegidas no Algarve e no Oeste. – 6º Enc. nac. Protecção Integrada, 14-16 Maio 2003, ESA Castelo Branco, Castelo Branco (in press).

Rodrigo, I., Amaro, F., Godinho, M.C. & Mexia, A. 2000. Crop protection techniques in horticultural greenhouses farming systems: a sociological approach of farmers’ adoption. – Bull. OILB/SROP, 23(1): 39-42.

Sánchez, J.A., Martínez, J.I. & Lacasa, A. 2003. Distribución geográfica, abundancia y plantas hospedantes de míridos depredadores (Heteroptera: Miridae) en la región de Murcia, de intéres para el control biológico de plagas en cultivos hortícolas. – Resumos III Cong. Nac. Entomol. Aplic., Ávila, 20-24 Out. 2003: 359.

Silva, I.M.MS. 1999. Identification and evaluation of Trichogramma parasitoids for biological pest control. – Ph.D. thesis, Univ. Wageningen, Wageningen, 151 pp.

Valério, E. 1999. Os parasitóides e o seu potencial na limitação natural de afídeos (Homoptera: Aphidoidea) em culturas protegidas na região Oeste. – Rel. Final PRAXIS/XXI/BIC/16990/98, 48 pp.

Valério, E., Cecílio, A. & Mexia, A. 2003. Biodiversidade de parasitismo espontâneo de afídeos em horticultura protegida em diferentes sistemas de protecção das plantas. – 6º Enc. nac. Protecção Integrada, 14-16 Maio 2003, ESA Castelo Branco, Castelo Branco (in press).


pp. 9 - 12

9

Predators of Phyllocnistis citrella Stainton (Lepidoptera: Gracillariidae) on citrus in Spain: role of lacewings and ants Josep A. Jacas1 & Alberto Urbaneja2 1 Universitat Jaume I; Campus del Riu Sec; E-12071-Castelló de la Plana (Spain).

E-mail: [email protected] 2 Institut Valencià d’Investigacions Agràries; Ctra. Montcada-Nàquera km 5;

E-12113-Montcada (Spain). E-mail: [email protected] Extended Abstract* Spain is the largest producer of citrus for the fresh market worldwide (FAO, 2003). Many potential pests occurring in this crop are kept under excellent biological control by their natural enemies (Ripollés et al., 1995). Most of these natural enemies are specialists [e.g. Rodolia cardinalis Mulsant (Coleoptera: Coccinellidae)] and their role is widely recognized among citrus entomologists. Because of this status, investigations on side effects of pesticides on these species are routinely undertaken (Jacas & García Marí, 2001). Nevertheless, the invasion of Spanish citrus orchards by the citrus leafminer [Phyllocnistis citrella Stainton (Lepidoptera: Gracillariidae)] during the last decade of the twentieth century gave us the chance to quantify the contribution of opportunistic parasitoids and predators to mortality inflicted by natural enemies (both indigenous and exotic) on this pest. Recent studies where mortality caused by natural enemies took into account the effects of predation, parasitism and feeding punctures pointed at predation as the most important mortality factor overall (Pomerincke, 1999; Urbaneja et al., 2000a). In Spain, predation represented up to 60 % of total mortality caused by natural enemies (Urbaneja et al., 2000a). Nevertheless, it was not possible to relate predation rates to P. citrella numbers along the season. It appeared that generalist predators were not responding to variation in the density of P. citrella but to flushing. Predation rates were significantly related to densities of young receptive flushes, and this might be related to continuous availability of preferential hosts, such as aphids. Therefore, it was concluded from that study that generalist predators feeding on aphids, such as lacewings, and aphid-mutualists, such as ants, which also feed on citrus leafminer, were probably responsible for observed predation rates. For this reason, a study aimed at quantifying the role of Chrysoperla carnea Stephens (Neuroptera: Chrysopidae) and ants (Hymenoptera: Formicidae) as predators of P. citrella was undertaken. Key-words: Citrus, Phyllocnistis citrella, Chrysoperla carnea, ants, predation, conservation biological control. Chrysoperla carnea

Many field studies conducted in Spain have pointed at C. carnea as a predator of P. citrella (Garijo et al., 1995; González, 1996; Lucas, 1995; Ripollés, 1997; Urbaneja et al. 2000a, 2000b, 2001). In a laboratory assay, it was possible to rear this species when fed P. citrella * A full version of this study available at the Spanish Journal of Agricultural Research (2004, in press): Which

role do lacewings and ants play as predators of the citrus leafminer in Spain?

10

third instars only (Urbaneja et al., 2001). Nevertheless, mortality was high. Only one individual out of 40 tested could reach adulthood, and it took 42 days to complete its development. Immatures consumed on average 77.3 ± 22.7 larvae (n = 10) (Urbaneja et al., 2001) until pupation.

In a field study, where C. carnea populations, as well as both P. citrella and aphids, were monitored during one growing season, it was possible to correlate lacewings and aphids when a time-lag was considered. Nevertheless, no relationship could be established between this predator and P. citrella (Figure 1) (Urbaneja et al., 2001). Therefore, we concluded that C. carnea could accidentally feed on P. citrella, but could not be considered a key predator of this pest.

Figure 1. Flushing patterns (number of flushes/ m2) and incidence of P. citrella (mines per leaf) and aphids (individuals/leaf) in a Navelina-orange orchard in Elx in 1999. Ants The role of ants as predators of P. citrella was studied in an exclusion field study (Urbaneja et al., 2000b). No statistically significant differences could be found between control trees, where ant movement between canopy and ground was allowed, and ant-free trees, where ant movement was prevented by use of a pesticide treatment applied to the trunk. Once again, we could see ants feeding on P. citrella immatures, but their role was purely accidental. Other predators In addition to C. carnea and ants, other species have been cited in the Mediterranean as possible predators of P. citrella. These include spiders, anthocorids, thrips, and coccinellids (Garijo et al., 1995; González, 1996; Lucas, 1995; Ripollés, 1997; Urbaneja et al. 2000a, 2000b, 2001). Spiders are indeed very abundant in citrus orchards (Amalin et al., 1996, 2001a, 2001b; Pomerinke, 1999; Urbaneja et al. 2000a, 2000b, 2001), and the relevance of their role remains unclear.

050

100150200250300350400

17/2 3/3 24

/313

/427

/413

/526

/5 8/6 23/6 7/7 28

/717

/8 1/9 23/9

15/10 5/1

1

Date

Flus

hes/

m2

0

10

20

30

40

# in

divu

dual

s

Total flush Aphid-infested flush P. citrella-infested flush C. carnea

11

Conclusions Although we have not been able to prove the efficiency of either C. carnea or ants as predators of P. citrella, the establishment of this pest in our orchards has given us the chance to become aware of the existence of an important guild of undetermined generalist predators, and opportunistic parasitoids. Conservation of indigenous natural enemies should be seriously taken into account and not considered just a mere complement of other biological control strategies in citrus. Acknowledgements This work was partially funded by the Instituto Nacional de Investigación Agraria y Alimentaria (INIA), the Comisión Interministerial de Ciencia y Tecnología (CICYT), and the Conselleries d’Agricultura, Pesca i Alimentació and Cultura i Educació of the Valencian Government. References Amalin, D.M., Peña, J.E. & Mcsorley, R. 1996. Abundance of spiders in lime groves and their

potential role in suppressing the citrus leafminer population. – In: M.A. Hoy (ed.). Proceedings, International Meeting: managing the citrus leafminer, 22-25 April 1996, Orlando, Florida, University of Florida, Gainesville, Florida: p. 72.

Amalin, D., Peña, J.E., Mcsorley R.E. & Reiskind. J. 2001a. Predation by Hunting Spiders on Citrus Leafminer, Phyllocnistis citrella (Lepidopera: Gracillariidae). – Journal of Entomological Science, 36: 199-207.

Amalin, D., Peña, J.E., Reiskind J. & Mcsorley, R.E. 2001b. Predatory behavior of three species of Hunting Spiders attacking Citrus Leafminer. – Journal of Arachnology, 29: 72-81.

FAO (Food and Agriculture Organization) 2003. FAOSTAT Database. – http://faostat.fao.org/faostat/collections?language=ES

Garijo, C., García, E. & Wong, E. 1995. Experiencias sobre el comportamiento y el control de Phyllocnistis citrella en Andalucía. – Phytoma, 72: 94-102.

González, L. 1996. Estudio de diferentes parámetros y correlaciones de interés para el seguimiento de las poblaciones y el daño del minador de los brotes de los cítricos Phyllocnistis citrella Stainton, (Lepidoptera. Gracillariidae, Phyllocnistinae). – Levante Agrícola, 336: 232-246.

Jacas, J.A. & García-Marí, F. 2001. Side-effects of pesticides on selected natural enemies occurring in citrus in Spain. – IOBC/wprs Bulletin 24 (4): 103-112.

Lucas, A. 1995. El minador de las hojas de los cítricos (Phyllocnistis citrella Stainton). Distribución y control en la Región de Murcia. – Phytoma, 72: 103-114.

Pomerinke, M.A. 1999. Biological control of citrus leafminer, Phyllocnistis citrella (Lepidoptera: Gracillariidae) in southwest Florida. – PhD Thesis. University of Florida. Immokalee, FL, USA.

Ripollés, J.L. 1997. Estrategia de lucha contra el minador de los cítricos bajo el punto de vista del control integrado de plagas (I). – Levante Agrícola, 340: 258-276.

Ripollés, J.L., Marsà, M. & Martínez, M. 1995. Desarrollo de un programa de control integrado de las plagas de los cítricos en las comarcas del Baix Ebre-Montsià. – Levante Agrícola 332: 232-248.

12

Urbaneja, A., Llácer, E., Tomás, Ó., Garrido, A. & Jacas, J. 2000a. Indigenous natural enemies associated with Phyllocnistis citrella (Lepidoptera: Gracillariidae) in Eastern Spain. – Biological Control 18, 199-207.

Urbaneja A., Muñoz, A., Jacas, J. & Garrido, A. 2000b. Incidencia de las hormigas como depredadores sobre el minador de las hojas de los cítricos, Phyllocnistis citrella. – Levante Agrícola, 352: 338-346.

Urbaneja A., Muñoz, A., Garrido, A. & Jacas, J. 2001. Incidencia de Chrysoperla carnea (Stephens) (Neuroptera: Chrysopidae) en la depredación de Phyllocnistis citrella Stainton (Lepidoptera: Gracillariidae). – Boletín de Sanidad Vegetal Plagas, 27: 65-73.

Urbaneja, A., Muñoz, A., Garrido, A. and Jacas, J. (in preparation). Predation of Phyllocnistis citrella Stainton (Lepidoptera: Gracillariidae) on orange and lemon in Spain: the importance of generalist predators and their conservation.


pp. 13 - 19

13

Comparative effects of several insect growth regulators and spinosad on the different developmental stages of the endoparasitoid Hyposoter didymator (Thunberg) Marcela Schneider1,3, Guy Smagghe2 & Elisa Viñuela1 1 Unidad de Protección de Cultivos, E.T.S.I.Agrónomos. Ciudad Universitaria, s/n.

28040-Madrid. Spain. 2 Laboratory of Agrozoology, Ghent University. Coupure Links 653. B-9000-Ghent. Belgium. 3 Present address: Centro de Estudios Parasitológicos y de Vectores CEPAVE,Calle 2 N 584,

1900 La Plata. Argentine. Abstract: Mature third-instar larvae of the parasitoid Hyposoter didymator (Thunberg) should be considered as a susceptible stage because as demonstrated in this study, when they exit from the dead host to spin the silken cocoon, they are very exposed to pesticides due to their low mobility and absence of concealment inside the host. The IOBC classification for standard tests (residual exposure) was also used in the other exposure routes, to classify insecticides according to the total effect caused in the enemy. Topically applied at the maximum field recommended concentrations in Spain, the insect growth regulators (IGRs) tebufenozide (TEB) and methoxyfenozide (MET), were not toxic to this developmental stage (IOBC class 1). The IGRs diflubenzuron (DFB), pyriproxyfen (PYR) and azadirachtin (AZA) and the naturalyte spinosad (SPIN) were slightly, moderately or very harmful, and they were classified as IOBC classes 2 (AZA), 3 (PYR) and 4 (DFB and SPIN). In contrast with results reported for other natural enemies, pupae were rather susceptible when treated with 1µl of AZA and DFB (IOBC class 2) or PYR and SPIN (IOBC class 4). Only TEB and MET were harmless (IOBC class 1). Adults of the parasitoid were more tolerant to the studied IGRs which were classified as IOBC class 1 in residual or topical tests, and IOBC classes 1 and 2 in ingestion assays. Fresh residues of SPIN however, were very toxic to the wasp adults (IOBC class 4) but they had little residual toxicity at 10 days post application (IOBC class 1). In topical or ingestion assays, the toxicity of SPIN was very high to adults (IOBC classes 4). The immature stages (concealed inside the host) were not affected by AZA, DFB, MET, TEB and SPIN topically applied to parasitized L3 larvae of the host and all of them were classified as IOBC class 1. Key-words: Hyposoter didymator, adults, third-instar larvae, pupae, immature stages, spinosad, tebufenozide, methoxyfenozide, diflubenzuron, azadirachtin, pyriproxyfen, laboratory Introduction The impact of pesticides on the efficiency of natural enemies present in crops, has to be determined when biological control is going to be part of integrated pest management programs (IPM), because unless this step is accomplished, we do not know which products are compatible.

Hyposoter didymator (Thunberg) (Hymenoptera: Ichneumonidae) is a commonly observed noctuid endoparasitoid in Spain (Bahena et al., 1999; Schneider & Viñuela, 1999; Torres-Vila et al., 2000). Typically, Hyposoter females only lay one egg into the dorso-lateral region of the host larval body (noctuid larvae belonging to genus Spodoptera, Autographa, Helicoverpa, Chrysodeixis, etc), the three larval instars develop internally and the mature L3 larva kills the host and emerges to spin a silken protective pupal cocoon and pupates (Bahena et al, 1999). As a result of that, in this enemy, not only adults which are normally considered

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the most sensitive stage to pesticides (Hassan, 1994), but also mature last-instar larvae, could be more affected by pesticides than other developmental stages.

Amid the rational pesticides which have been embraced by IPM practitioners due to their efficacy as pesticides together with selective toxicity characteristics and favourable environmental profiles, are the insect growth regulators (IGRs) tebufenozide (TEB), methoxyfenozide (MET), pyriproxyfen (PYR), diflubenzuron (DFB) and azadirachtin (AZA), as well as the naturally derived spinosad (SPIN). All these insecticides are rather compatible with natural enemies [see for example Van de Veire et al. (1996), Darvas & Polgar (1998), Willians et al. (2003)], even though some of them can be toxic to immature stages of certain beneficials [see for example Hassan et al., 1994; Darvas & Polgar, 1998]. The ecdysone agonists TEB and MET, with no structural resemblance to the molting hormone 20E, trigger in lepidopterans the same response by binding to the ecdysone receptor complex (Palli & Retnakaran, 2001; Retnakaran et al., 2003). The limonoid AZA, present in the Meliaceae tree Azadirachta indica A. Juss, interferes with the normal metamorphosis process in insects (Schmutterer, 1995). The benzoylphenylurea (BPU) DFB prevents chitin production, resulting in several changes in the elasticity and firmness of the endocuticle (Ishaaya & Horowitz, 1999). The juvenile hormone (JH) analogue PYR gives strong suppressions of embryogenesis, metamorphosis and adult formation (Ishaaya & Horowitz, 1995). The naturalyte SPIN, obtained from a soil actinomycete, is a neurotoxic insecticide and exposure results in cessation of feeding followed later by paralysis and death (Salgado, 1998).

Recommendations of IOBC working group “Pesticides and Beneficial Organisms” aim to evaluate conditions under which the natural enemy is likely to encounter the worst posssible exposure to toxicant (Sterk et al., 1999). In this study we report on the impact of pesticides which are being marketed as safer to beneficials than classical pesticides, on immature stages of H. didymator when they were applied to parasitized larvae of the host as well as on mature last-instar larvae exited from the host and we compare the effects with those observed in pupae and adults. Results from these experiments should help to integrate chemical and biological control in agroecosystems where this enemy is present, because many attempts to suppress pest populations by biological measures have failed because of deleterious effects of chemicals on beneficials. Materials and methods Insect colony The different developmental stages of H. didymator from a colony maintained in a controlled environment chamber (25 ± 2ºC, 75 ± 5% relative humidity, 16L:8D photoperiod) were used in assays. The parasitoid was routinely reared on Spodoptera littoralis (Boisduval) third instars using a procedure developed in our laboratory (Schneider, unpublished data), and adults were provided with pure honey and water ad libitum. Host larvae were fed a Poitout based artificial diet, adults a 15% solution of honey in water and both were kept under the same environmental conditions described above. Insecticides The commercials Align® (3.2% AZA, non-oil EC, Sipcam Inagra, Valencia, Spain), Dimilin® (25% DFB, WP, AgrEvo, Valencia, Spain), Juvinal® (10% PYR, EC, Kenogard, Barcelona, Spain), Tracer® (48% SPIN, DowAgrosciences, Madrid, Spain) and Mimic® (24% TEB, SC, Rohm and Haas, Barcelona, Spain), and the experimental RH-2485 (24% MET, SC, Rohm and Haas, Spring House, PA), were used in assays. Depending on the application technique, aqueous or acetone fresh solutions (in acetone 99.5% Panreac® for topical assays) were prepared prior to the experiments. Insecticides were only applied at their maximum field

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recommended concentrations (MFRC) as registered in Spain for TEB (144 mg/l a.i.), AZA (48 mg/l a.i.), DFB (100 mg/l a.i.), PYR (75 mg/l a.i.) and SPIN (120 mg/l a.i.), or at the concentration recommended by the producer for the non registered MET (144 mg/l a.i.). Treatment of adults Young adults of H. didymator were exposed to residues of different ages of aqueous solutions of every insecticide on glass surfaces, topically treated on the pronotum with 1 µl of the acetone insecticide solutions once anaesthetized with cold temperatures or fed aqueous solutions of the different insecticides continuously in the drinking water during the life span (Schneider et al., 2003a). Four to 6 replicates of 5-10 females and 10-20 males per insecticide and control were used, because male longevity is shorter than that of females (Schneider et al., 2000). Treatment of pupae Pupae <24-h-old were topically treated with 1 µl of the acetone insecticide solutions using a Burkard microappplicator (Schneider et al., 2003b). Five replicates of 5 pupae per insecticide were used. Treatment of immature stages Effects on immature stages of the parasitoid were evaluated by treating topically, three days after parasitization, parasitized L3 larvae of the host as described above. Following treatment, host larvae were provided with untreated food and water until the emergence of the mature L3 larvae of the parasitoid. Six replicates of 5 host larvae per insecticide were used. Treatment of mature third-instar larvae Mature last-instar larvae, recently exited from the dead host, were collected from stock culture and topically treated as described above. Experiment consisted of 4 replicates of 5 larvae per insecticide. Statistics Evaluation of results was based on lethal and sublethal effects of the insecticides. In parasitized host larvae, we recorded the percentage of emergence of mature L3 larvae, their mortality and the longevity of adults; in mature third-instar larvae we evaluated larval mortality, percentage of adults, female longevity and beneficial capacity of the wasps. Data were analyzed by one way analysis of variance (ANOVA) using Statgraphics (STSC, 1987). The means were separated by the LSD multiple range test (P<0.05) When the premises of ANOVA were violated, a Kruskal-Wallis test was applied (Milliken & Johnson, 1984).

For the rest of assays, assessment and analysis of data was done as described by Viñuela et al. (2001), Medina et al. (2003) and Schneider et al. (2003b) for pupae and Schneider et al. (2003a) for adults.

For the toxicity rating of pesticides (based in the total effect caused in the enemy), the IOBC classification for laboratory standard tests (residual exposure) (Hassan, 1994): 1 (harmless, <30%), 2 (slightly harmful, 30-79%), 3 (moderately harmful, 80-99%), 4 (harmful, >99%), was also used for the other exposure routes used in our study (ingestion, topical application, treatment of parasitized host larvae), in order to have a common scale for comparison. Results and discussion The IOBC toxicity ratings of the different assays, are shown in Table 1. Treatment of adults The results reported by Schneider et al. (2003a) show that AZA, DFB, MET, PYR and TEB were compatible with adults of the parasitoid when they were exposed to residues or topically

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treated. SPIN however, is a neurotoxic insecticide (Salgado, 1998) and this has contributed undoubtedly to the harmfulness and fast action exhibited in adults of H. didymator, as it could be expected based on the many effects found in different parasitoids (Williams et al., 2003). However, old residues of SPIN were compatible with adults, because photolysis is the main route of degradation in nature (DowElanco, 1994). In contrast, when adults of H. didymator were fed continuously the insecticide, only the two MAC compounds, TEB and MET were compatible. Table 1: Laboratory IOBC toxicity ratings for the studied pesticides, to the different developmental stages of Hyposoter didymator

Adults5

Residual

Insecti-cides

MFRC1

mg a.i./l Immatu

re stages2

Mature L3 larvae3

Pupae4

Fresh 5-d-old 10-d-old Topic Ingest-

ion IOBC toxicity classes

AZA 48 1 2 2 1 1 1 1 2 DFB 100 1 4 2 1 1 1 1 2 MET 144 1 1 1 1 1 1 1 1 PYR 75 - 3 4 1 1 1 1 2 SPIN 120 1 4 4 4 2 1 4 4 TEB 144 1 1 1 1 1 1 1 1

1 MFRC= maximum field recommended concentrations. 2 L3 parasitized S. littoralis host larvae topically treated. 3 Larvae topically treated. 4 Based on the following publications: Medina et al. (2003) and Schneider et al. (2003b). Pupae <24-h-

old, topically treated with 1µl of the different insecticide solutions. 5 Based on the following publication: Schneider et al. (2003a). 6 Natural enemy response classified using the IOBC laboratory scale: 1= harmless (<30% reduction), 2=

slightly harmful (30-79% reduction), 3= moderately harmful (80-99% reduction), 4= harmful (>99% reduction)

So, residual effects, irrespective of the age of the residue, and topical toxicity of all the

IGR studied were negligible against adults of H. didymator and these could be classified as IOBC class 1. In contrast, fresh residues of SPIN were very toxic to the wasp (IOBC class 4) but the detrimental effect decreased drastically with the elapse of time and 10-d-old residues were totally harmless to the wasp (IOBC class 1). However, the insecticide exhibited a very high topical toxicity on the wasp and it was classified as IOBC class 4. Due to their high lepidopteran specificity (Retnakaran et al., 2003), only MET and TEB showed no oral toxicity to adults of the parasitoid. The rest of the studied IGRs (DFB, AZA and PYR), had a minimal oral toxicity to the natural enemy, and only slightly lowered the beneficial capacity of H. didymator females being classified as IOBC class 2. Only SPIN caused a high mortality towards both sexes and it was IOBC class 4. Treatment of pupae Even though the protected developmental stages of parasitoids are most often less sensitive to pesticides than the others (Jacas & Viñuela, 1994) AZA, DFB, PYR and SPIN were in general more toxic to H. didymator young pupae than to adults (IOBC classes 2-2-4 and 4 respectively) (Schneider et al., 2003b) and only MET and TEB could be classified as IOBC class 1. These

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findings are in agreement with many researchers who have reported that IGR pesticides are much more harmful to immature developmental stages of the enemies than to adults, that AZA is less toxic to parasitoids than to pests (Sipcam Inagra, 1996; Viñuela et al., 2000) and that SPIN can cause deleterious effects in parasitoids (Williams et al., 2003). Treatment of immature stages Immatures stages of the parasitoid were not affected by the insecticides when they were topically applied to parasitized host larvae at the maximum rate for field applications. Only emergence of mature L3 larvae was significantly decreased in every case, but the reduction compared to controls was small, so every insecticide was classified as IOBC class 1 (Table 2). However, when parasitized host larvae were fed sublethal concentrations (1 mg a.i./l) of AZA, DFB, MET, TEB and SPIN, the parasitoid was indirectly affected because of the early death of the host. The IOBC classes were: AZA (3) and the others (2) (Schneider & Viñuela, 1999). Table 2: Effects of different IGRs and spinosad on immature stages of H. didymator when L3 host larvae were topically treated with the insecticides

Longevity, days Insecticides MFRC1

mg a.i./l % L3

emergence % L3

mortality Female Male Control 0 98.3±1.6a 3.3±3.3a 28.5±0.8a 15.4±1.3a AZA 48 76.0±2.0b 6.6±4.2a 28.4±0.7a 15.3±1.2a DFB 100 77.0±2.1b 10.0±4.5a 28.3±0.5a 15.8±1.2a MET 144 76.8±2.4b 6.6±4.2a 27.5±0.9a 16.2±1.0a SPIN 120 80.0±2.2b 6.6±4.2a 28.5±0.9a 15.1±1.3a TEB 144 76.6±2.5b 6.6±4.2a 28.1±0.6a 15.4±1.2a

1 MFRC= maximum field recommended concentrations. Within the same column, data followed by the same letter are not statistically different (P=0.05, ANOVA, LSD)

Table 3: Effects of different IGRs and spinosad topically applied to mature third-instar larvae of H. didymator

Insecticides MFRC1

mg a.i./l% Larval mortalitya

% Adult emergenceb

Female longevity,

daysc

% Attacked hostsd

% Progeny sizee

Control 0 4.0±4.0a 92.0±4.9a 24.5±1.3a 91.0±1.6a 97.8±0.3a AZA 48 28.0±10bc 57.0±4.9cd 5.6±1.3c 42.5±2.4b 95.1±0.6de DFB 100 16.0±4.0ab 0.0±0.0g – – – MET 144 8.0±4.9a 90.0±6.12a 24.2±1.1a 90.6±1.0a 97.8±0.3ab PYR 75 56.0±7.5d 10.0±10.0ef 6.3±1.8c 46.6±3.0b 85.8±1.9f SPIN 120 64.0±7.5d 0.0±0.0g – – – TEB 144 8.0±4.9a 89.2±7.0ab 21.6±1.5ab 89.5±1.6a 98±0.2ab

1MFRC= maximum field recommended concentrations. Within the same column data followed by the same letter are not statistically different (P=0.05, a,d

ANOVA, LSD; b,c,e Kruskall Wallis)

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Treatment of mature third-instar larvae The results of our study showed that AZA, DFB, PYR and SPIN topically applied to third-instar larvae of H. didymator caused several lethal and sublethal effects to the parasitoid (Table 3).

Larval mortality in treated units was significantly higher than that of controls for AZA, DFB, PYR and SPIN, and adult emergence was significantly decreased by every insecticide except MET and TEB. The effect on adult emergence was very drastic for DFB, PYR and SPIN, being 0% for DFB and SPIN and 10% for PYR. Female longevity of survivors was especially lowered by AZA and PYR. The beneficial capacity of the wasps was also affected in that the number of attacked hosts was significantly lowered by AZA and PYR and it could not be studied for DFB and SPIN because number of survivors was zero. In contrast, the two MAC compounds, TEB and MET, were harmless to the parasitoid and did not affect any life parameter.

Insecticides were then classified as IOBC class: MET and TEB (1), AZA (2), PYR (3), DFB and SPIN (4).

Acknowledgements This work was financially supported by the Spanish Ministry of Education and Culture (projects AGF99-1135 and AGL2001-1652-C02-02 to E. Viñuela) and a doctoral fellowship to M. I. Schneider from CONICET (Argentine Ministry of Education and Culture). G. Smagghe acknowledges a postdoctoral grant from the Fund for Scientific Research (FWO, Brussels). References Bahena, F., Budia, F., Adán, A., Del Estal, P. & Viñuela, E. 1999: Scanning electron

microscopy of Hyposoter didymator in host Mythimna umbrigera larvae. – Annals of the Entomological Society of America 92: 144-152.

Darvas, B. & Polgar, L.A. 1998: Novel type insecticides: specificity and effects on non-target organisms. – In: Insecticides with novel modes of action. Ishaaya, I. & Degheele, D. (eds), Springer. Berlin: 188-259.

DowElanco. 1994: Spinosad technical guide. 24 pp. Hassan, S.A. 1994: Activities of the IOBC/WPRS Working Group “Pesticides and Beneficial

Organisms”. – IOBC/wprs Bulletin 17 (10): 1-5. Ishaaya, I. & Horowitz, A.R. 1995: Pyriproxyfen, a novel insect growth regulator for

controlling whiteflies: mechanism and resistance management. – Pesticide Science 43: 227-232.

Ishaaya, I. & Horowitz, A. R. 1999: Insecticides with novel modes of action: an overview. – In: Insecticides with novel modes of action. Ishaaya, I. & Degheele, D. (eds.), Springer. Berlin: 1-24.

Hassan S.A.; Bigler F.; Bogenschütz H. et al. 1994: Results of the sixth pesticide testing programme by the IOBC/WPRS Working Group “Pesticides and Beneficials Organisms”. – Entomophaga 39: 107-119.

Jacas, J. & Viñuela, E. 1994: Side-effects of pesticides on Opius concolor, a parasitoid of the olive fruit fly. – IOBC/wprs Bulletin 17(10):143-146.

Medina, P., Schneider, M.I., Smagghe, G., Budia, F., Gobbi, A., Tirry, L. & Viñuela, E. 2003: Toxicity of several modern pesticides to the parasitoid Hyposoter didymator and the predator Chrysoperla carnea: significance of penetration and excretion. – Proceedings 1st International Symposium on Biological Control of Arthropods: 489-496. Honolulu. January 2002.

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Milliken, G.A. & Johnson, D.E. 1984: Analysis of messy data. Vol. I: designed experiments. – Van Nostrand Reinhold, New York.

Palli, S. R. & Retnakaran A. 2001: Ecdysteroid and juvenile hormone recepterors: properties and importance in developing novel insecticides. – In: Biochemical sites of insecticide action and resistance. Ishaaya, I. (ed), Springer-Verlag, Heidelberg: 107-132

Retnakaran A., Krell P., Feng Q. & Arif B. 2003: Ecdysone agonists: mechanism and importance in controlling insect pests of agricultural and forestry. – Archives of Insect Biochemistry and Physiology 54: 187-199.

Salgado, V. L. 1998: Studies on the mode of action of spinosad: insect symptoms and physiological correlates. – Pesticide Biochemistry and Physiology 60: 91-102.

Schmutterer, H. (ed.) 1995: The Neem Tree. Source of unique natural products for integrated pest management, medicine, industry and other purposes. – VCH, Weinheim, Germany: 696 pp.

Schneider, M.I., Budia, F., Gobbi, A., de Remes Lenicov, A.M. & Viñuela, E. 2000: Topic toxicity of tebufenozide, spinosad and azadirachtin on pupae of the parasitoid Hyposoter didymator. – Boletín de Sanidad Vegetal Plagas 26:1-9 (In Spanish).

Schneider, M.I., Smagghe, G., Gobbi, A. & Viñuela, E. 2003b: Toxicity and pharmaco-kinetics of insect growth regulators and other novel insecticides on pupae of Hyposoter didymator (Thunberg 1822) (Hym., Ichneumonidae), a parasitoid of early larval instars of Noctuid pests. – Journal of Economic Entomology 96: 1054-1065.

Schneider, M.I., Smagghe G. & Viñuela, E. 2003a: Susceptibility of Hyposoter didymator (Hym., Ichneumonidae) adults to several insect growth regulators and spinosad by different exposure methods. – IOBC/wprs Bulletin 26 (5): 111-122.

Schneider, M.I. & Viñuela, E. 1999: Evaluation of tebufenozide on immature stages of Hypo-soter didymator, a parasitoid of noctuid larvae. – Mededelingen Faculteit Landbouw-kundige en Toegepaste Biologische Wetenschappen, Universiteit Gent 64/3a: 287-295.

Sipcam Inagra, 1996. Align®: Technical information. Sterk, G.; Hassan, S.A.; Baillod, M. et al. 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.

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Torres-Vila, L.M., Rodríguez-Molina, M.C., Palo, E., Del Estal, P. & Lacasa, A. 2000: Parasitoid complex of Helicoverpa armigera Hb. on tomato, in Guadiana plain (Extrema-dura. Spain). – Boletín de Sanidad Vegetal Plagas 26(3): 323-333. (In Spanish).

Van De Veire, M., Smagghe, G. & Degheele, D. 1996: Laboratory test method to evaluate the effect of 31 pesticides on the predatory bug, Orius laevigatus (Het: Anthocoridae). – Entomophaga 41: 235-243.

Viñuela, E., Adán, A., Smagghe, G., González, M., Medina M.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 and Technology 10: 165-177.

Viñuela, E., Medina M.P., Schneider, M.I., González, M., Budia, F., Adán, A. & Del Estal, P. 2001: 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. – IOBC/wprs Bulletin 24 (4): 25-34.

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Duration of the toxicity of abamectin and spinosad on the parasitic wasp Encarsia formosa Gahan in Northern and Southern Europe Marc Van de Veire1, Elisa Viñuela2, Umberto Bernardo3, Luc Tirry1, Ángeles Adan2 & Gennaro Viggiani3 1 Faculty of Agricultural and Applied Biological Sciences, University of Ghent, Coupure

Links 653, B - 9000 Ghent (Belgium) 2 Unidad de Proteccion de Cultivos, E.T.S.I. Agronomos, Universidad Politécnica Madrid,

E - 28040 Madrid (Spain) 3 IPP Istituto CNR per la Protezione delle Piante – Sezione di Portici, Via Università, 133,

I - 80055 Portici (NA) (Italy) Abstract: The duration of the toxicity of two biopesticides, abamectin and spinosad, on the wasp Encarsia formosa Gahan, was studied at three geographical locations: Belgium (Ghent, 50°N), Spain (Madrid, 40°N), Southern Italy (Naples, 40°N), in the spring (March/April) and in the summer (June/July) of 2003, with a “worst case” laboratory test for studying the residual toxicity on adult wasps. In the spring test, for both products, statistically significant differences were found between Belgium and Southern Italy. In Belgium, abamectin was still toxic (persistence class C) after 30 days, whereas in Italy the compound was not toxic anymore after this period. Spinosad was still highly toxic in Spain (persistence class D) and Belgium (persistence class C), 30 days after treatment, while it was no longer toxic in Italy. Light intensity measurements with a StowAway logger (broad light spectrum: UV, visible light, infrared) demonstrated much higher maximum daily light intensities (Lumen/ m²) in Southern Italy than in Spain and in Belgium. In the summer test, abamectin turned out to be of very short persistence in Italy (persistence class A). In Belgium and Spain, abamectin still had to be categorised in persistence class C, five days after treatment; 14 days after treatment, no toxicity was found anymore at both locations. Five days after treatment, spinosad was toxic at all locations (persistence class D). In Southern Italy, the product was no more toxic, 15 days after treatment, but in Spain it was still highly toxic (persistence class D). Maximum daily light intensities during the summer experimental period were much higher in Naples than in Madrid (where three sides of the greenhouse had been covered by black gauze because of the high temperatures) and Ghent. It appears that the shorter persistence of abamectin and spinosad in Southern Italy maybe attributed to the higher light intensities in that region, both in the spring and in the summer period. The results indicate that the evaluation of the toxicity of compounds, which are sensitive to photodegradation, should be done with great care. Key-words: persistence of pesticides, geographical locations, light intensity, abamectin, spinosad, mortality, reproduction, Encarsia formosa, classes for persistence Introduction The biopesticides abamectin (Jansson and Dybas, 1998) and spinosad (Thompson et al., 1997) are bacterial fermentation products with potential for use in IPM programs in glasshouse vegetables. Abamectin provides effective corrective control of the two-spotted spider mite Tetranychus urticae Koch, the western flower thrips Frankliniella occidentalis Perg. and leafminers (Liriomyza spp.). Spinosad can be used for control of leafminers, several caterpillar species and the western flower thrips. Avermectins and emamectin are prone to rapid photolytic breakdown (Feely et al., 1992; Lasota and Dybas, 1991). Spinosad is also known to last for a short interval in the field (Thompson et al., 1997).

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Little scientific information is currently available on the photodegradation of abamectin and spinosad. General technical data on the application or side-effects of the compounds do not discriminate between growing seasons or geographical locations. Therefore we have done simultaneous persistence studies for these compounds at three geographical locations in Europe (Belgium, Spain, Italy). As test organism, the parasitic wasp Encarsia formosa Gahan was chosen. An adapted persistence test (Van de Veire and Tirry, 2003) for the wasp E. formosa was carried out at the 3 locations. Equally sensitive wasps were tested with the same laboratory test equipment, the same compounds and concentrations and the same evaluation method for determination of the persistence. Materials and methods Locations Experiments were carried out in three different countries: Belgium (Ghent), Spain (Madrid) and Italy (Portici, Naples); the latitudes, altitudes of the locations and distances from the sea were respectively:50°N/4m/50km, 40°N/646m/350km and 40°N/60m/2 km. Plants Tomatoes, grown in small experimental greenhouses were used for the tests. The tomato cultivars differed between countries: Ghent: cv. Groove; Madrid: cv. Bodar, Portici: cv. Cuore di bue. Greenhouses In Ghent and Portici, separate greenhouses of respectively 15 m² and 82 m² were used. In Madrid, the experimental greenhouse (15 m²) was surrounded by adjacent units in three sides; due to the 2003 high summer temperatures, these adjacent greenhouses were covered with black gauze, thus decreasing the light in our experimental greenhouse, which only received sunlight through the roof in this period. Insects The E. formosa strains used in the experiments in Ghent and Madrid were both purchased from Biobest NV, while in Portici a lab strain was used. However, determination of the LC50 at both locations revealed that the Portici wasps were equally sensitive to abamectin as the Biobest strain. Insecticides The products abamectin (Vertimec, 1.8% EC, Novartis Agro) and spinosad (Tracer, 48%SC in Italy and Belgium; Spintor 48% SC, DowAgroSciences in Spain) were used for the tests. Test method At all locations, StowAway LI loggers (Onset Computer Corporation) were used for registration of the light intensity, while temperature and relative humidity (RH) were measured with Hobo loggers (Onset Computer Corporation); the loggers were hung in the middle of the plants during the whole experimental period. Tomatoes were sprayed with a hand sprayer on the underside of the leaves, with 10 mg a.i./l of abamectin and 125 mg a.i./l of spinosad (maximum recommended field concen-trations) till the point of run-off. Tomato leaves were collected from the plants at 5, 15 and 30 DAT (days after treatment). These leaves were taken in the vicinity of the light logger in order to ensure a good estimation of the light intensity. The spraying and sampling dates in spring and summer experimental period are given in tables 1 and 2. For testing the toxicity of both compounds, tomato leaves with aged residues (5, 15 or 30 DAT) were put in “Cornelis” cells (Van de Veire and Tirry, 2003). Then, E. formosa wasps were transferred to the cell (10 adults/cell). The cells were put in climatic chambers, with

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following abiotic conditions: 22-24°C, 60-80% RH, 16/8 (L/D). Forced ventilation was used at all locations. Tests were done in 5 replicates. The mortality of the wasps was checked at different times after exposure, up to 7 days. The mean % mortality, corrected for control mortality, was then calculated. Data were analysed by one-way ANOVA. Means were separated by a least significant difference (LSD) multiple range test (p = 0.05). When the premises of ANOVA were violated, a Kruskal Wallis test was applied. Table 1. Spring experiments: pesticide spraying and leaf sampling dates at the 3 locations.

Spraying date Leaf sampling date 5 DAT 15 DAT 30 DAT Ghent (Belgium) 13.03.03 18.03.03 28.03.03 12.04.03 Madrid (Spain) 10.04.03 15.04.03 25.04.03 09.05.03 Portici (Italy) 28.03.03 02.04.03 12.04.03 27.04.03

DAT = days after treatment Table 2. Summer experiments: pesticide spraying and leaf sampling dates at the 3 locations.

Spraying date Leaf sampling date 5 DAT 15 DAT 30 DAT Ghent (Belgium) 10.07.03 15.07.03 30.07.03 10.08.03 Madrid (Spain) 09.06.03 14.06.03 24.06.03 09.07.03 Portici (Italy) 23.06.03 28.06.03 08.07.03 –

DAT = days after treatment

In Portici, in cases where survival exceeded 50 %, the reproduction of the treated wasps was studied. Ten females were put individually in a small Petri dish (diameter: 5.5 cm) containing a layer of agar medium on which a bean leaf disc was placed, containing approximately 50 whitefly larvae (3rd, 4th stage). After 24 hours, the E. formosa females were removed; the number of parasitized larvae was counted 14 days later.

For the evaluation of the persistence, the 4 evaluation categories of the IOBC WG “Pesticides and Beneficial Organisms” (Sterk et al., 1999) were used: A = short lived (< 5 days); B = slightly persistent (5-15 days); C = moderately persistent (16-30 days); D = persistent (> 30 days).

Results and discussion Maximum temperatures and relative humidities at the 3 locations (Ghent, Portici and Madrid), during the two experimental periods (spring and summer) are given in Figures 1-4. Maximum light intensities per day during spring and summer at the studied locations, are given in Figures 5 and 6 respectively. Spring In spring conditions, abamectin can be classified as slightly persistent (persistence class B) in Portici whereas in Ghent and Madrid, the product has to be classified as moderately persistent (class C) (Fig. 7). These data correlate well with the light intensity at the different locations Light intensity was significantly higher in Portici (Kruskall Wallis; p = 0.05) than in Ghent and Madrid (Fig. 5). When abamectin is used in Southern Italy in greenhouses in the spring, the waiting period for introducing Encarsia wasps of 2 weeks can be recommended, whereas

24

in Belgium and Spain, under the given conditions, a period of 3 to 4 weeks should be taken into account before introducing the wasps. In Madrid however, the circumstances in the experimental greenhouse may differ quite seriously from those in the currently used com-mercial greenhouses, i.e. much more light can enter the commercial greenhouses. It is most likely that under practical conditions of commercial tomato growing in Madrid, a similar photolysis pattern of the product like in Portici can be expected.

Spinosad is more persistent than abamectin. Even in Portici, approx. 60% mortality of the wasps was found after 2 weeks. In Madrid, the persistence of spinosad turned out to be high (persistence class D = persistent) (Fig. 8). Even in Belgium, spinosad was still moderately toxic 30 DAT, but in Portici, the product caused less than 30% mortality 30 DAT.

For spinosad, waiting periods of 3 to 4 weeks can be recommended before (re)intro-ducing E. formosa.

It should also be noted that the aforementioned tests are laboratory tests, in which a maximum exposure of the wasps to the spray residues is realised. In higher tier tests, e.g. a semi-field or a field test, the exposure time of wasps to the spray deposits might be less, because they can avoid contact with the leaf surface bearing the toxic residues. It is thus possible that the period after pesticide applications for introducing the parasitic wasps may be shorter than those recommended on the basis of the laboratory tests. Summer In the summer, the persistence of abamectin differs strongly from that in spring. Both in Ghent and Naples, the product caused less than 30% mortality, so that the product can be classified in the persistence class A (short lived) (Fig. 9). In Madrid, most probably due to the lower light intensities during the test (which were significantly different from Portici and Ghent; Kruskall-Wallis, p = 0.05; Fig.6), the product residue still causes 100% mortality of the wasps at 5 DAT. However, at 15 DAT week, no (Ghent, Madrid) or little (Portici) toxicity was found. In summer, E. formosa wasps can thus be introduced in the greenhouse shortly after an abamectin treatment. Under the conditions of the tests, only in Spain the introduction should be done 2 weeks after the abamectin treatment. It is obvious that under practical conditions of commercial tomato growing in this area, much more light enters the greenhouse and the waiting period before (re)introducing E. formosa is most likely much shorter.

Spinosad turned out to be persistent (class D) in Madrid (Fig. 10). The product still killed 75% of the parasitoid wasps 30 days after treatment. As the light intensities in Madrid in spring and summer were similar, it is not surprising that the persistence was similar for both periods. The product was a little less persistent in Naples than in Belgium (persistence class B in Italy; class C in Belgium). These results point out that care has to be taken with spinosad when (re)introducing E. formosa. Spinosad was more persistent than abamectin, at all locations. A waiting period of 2 (Portici) to 4 weeks (Ghent, Madrid) should be considered, before introducing E. formosa wasps.

The reproduction of surviving wasps was only tested in Naples. In the spring experiment, wasps which survived abamectin spray deposits of 15 days and spinosad spray deposits of 30 days, reproduced like the untreated control wasps (ANOVA, P= 0.05). In the summer experiment, wasps surviving aged residues of abamectin of 5 days, and spinosad residues of 15 days also reproduced like the untreated control wasps (ANOVA, P= 0.05). Conclusions The comparative study for persistence of 2 biopesticides strongly revealed the effect of light on the activity lifespan of the compounds. As temperatures did not significantly differ between locations in the summer period (ANOVA, p=0.05), it is hardly realistic that a

25

different absorption pattern of the products by the leaves would have played a major role. Absorption may have taken place, but probably at a comparable rate at every location. Absorption of a compound in a leaf can be the reason why a spray deposit on a leaf can be less toxic to an organism e.g. E. formosa, than a spray deposit on an inert substrate e.g. a glass plate.

The results strongly indicate that the difference in persistence between the locations in spring and summer must be attributed to the different light intensities at these locations.

Although the StowAway LI loggers only measured a part of the UV light spectrum, they were indicative for measuring a given amount of light, part of UV, visible and infrared. As the locations also differed in altitude and distance from the sea, it could be possible that relatively more UV light was present in Naples, resulting in a quicker photolysis. Unfortunately, based on the results and the equipment used in the present study, it is not possible to make a firm statement on this. Acknowledgements. Thanks are due to Hendrik Van Caenegem for critically reading the manuscript. Research support by IWT (Institute for the Promotion of Innovation by Science and Technology in Flanders, Ministery of the Flemish Community, Agricultural Research). Project no. S-6205. E. Viñuela aknowledges the gift of tomato plants from IRTA (Cabrils, Spain). U. Bernardo wants to thank Dr. Iodice Luigi for his help at rearing E. formosa parasitoids. References Feely, W.F., Crouch, L.S., Arison, B.H., VandenHeuvel, W.J.A., Colwell, L.F. & Wislocki,

G. 1992: Photodegradation of 4”-(epimethylamino)-4”-deoxyavermectin B 1a thin films on glass. – J. Agric. Food Chem. 40: 691-696.

Jansson, R.K. & Dybas, R.A. 1998: Avermectins: Biochemical Mode of Action, Biological Activity and Agricultural Importance. Ch. 9. – In: Ishaaya, I. And Degheele, D. (Eds.). Insecticides with Novel Modes of Action. Springer-Verlag, Berlin, Germany.

Lasota, J.A. & Dybas, R.A. 1991: Avermectins, a novel class of compounds: Implications for use in arthropod pest control. – Annu. Rev. Entomol. 36: 91-117.

Sterk, G. Hassan, S.A., Baillod, Bakker, F., Bigler, F., Blümel, S, Bogenschütz, H, Boller, E. et al. 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.

Thompson, G.D., Michel, K.H., Yao, R.C., Mynderse, J.S., Mosburg, C.T. & Worden, T.V. 1997: The discovery of Saccaropolyspora spinosa and a new class of insect control products. – Down Eearth 52: (1): 1-5.

Van de Veire, M. & Tirry, L. 2003: Side-effects of pesticides on four species of beneficials used in IPM in glasshouse vegetable crops: “worst case” laboratory tests. – IOBC/wprs Bulletin 26 (5): 41-50.

26

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Fig. 6. Maximum light intensity (Lumen/m²) per day during the experimental period in the summer at the 3 locations.

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Fig.7. Mortality (%) of E. formosa wasps 5, 15 and 30 days after application of abamectin in the spring, at the 3 locations.

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pp. 31 -35

31

Toxic effects of indoxacarb to a predacious mirid and two species of predacious mites Noubar J. Bostanian1, Charles Vincent1, John M. Hardman2, Nancy Larocque1 1 Horticulture Research and Development Centre, Agriculture & Agri-Food Canada,

430 Gouin Blvd, Saint Jean-sur-Richelieu, Qc., Canada, J3B 3E6 2 Atlantic Food and Horticulture Research Centre, Agriculture & Agri-Food Canada,

32 Main street, Kentville, N.S. Canada, B4N 1J5 Abstract: Indoxacarb is a novel oxidiazine pro-insecticide that is toxic to Hyaliodes vitripennis (Say), a predacious mirid, found in Quebec apple orchards. The LC50 to H. vitripennis is about one half the recommended field dose of 54 ppm AI liter-1. Following an application, the intoxicated mirids remained motionless as their prolegs and posterior appeared to have paralyzed. Twenty four hours later, they appeared smaller, shrunken and severely dessicated. Indoxacarb had no adverse effects on Amblyseius fallacis (Garman) and Agistemus fleschneri Summers adults, number of eggs laid by treated adults of both species and percent hatch of treated eggs of these two species. Key-words: Toxicity, Amblyseius fallacis, Agistemus fleschneri, Hyaliodes vitripennis, indoxacarb, apple orchards. Introduction The voltage-gated sodium channel is an important site of action for a number of synthetic neurotoxic insecticides such as DDT, the pyrethroids, isobutylamides and dihydropyrazoles (Wing et al. 2000). Mass spectrometric studies have shown that indoxacarb is readily metabolized in lepidopteran larvae to the decarbomethoxylated form in the midgut (Wing et al. 2000). The decarbomethoxylated form is the actual sodium channel blocker and the S-enantiomer is twice as active as the racemate. Therefore, indoxacarb is a pro-insecticide and it is converted to the sodium channel blocker by bioactivation in the target insect. Based on structural similarities and neurophysiological action, it is postulated to act like the dihydropyrazole insecticides (Saldago 1990). The mode of entry into the target pest is through ingestion, followed by direct spray contact, with exposure to a dried residue being the least effective. Insects exposed to this compound stop feeding within a few hours, become less mobile and may show slight tremors and convulsions. Its toxicity seems to be more pronounced in Lepidoptera, hence, the possibility of its use in agro-ecosystems to manage lepidopteran pests with minimal impact to non-target organisms (Wing et al. 1998). We report in this study the toxicity of this compound to three non-target arthropods: Hyaliodes vitripennis (Say) (Heteroptera: Miridae) a predacious mirid and two predacious mites: Amblyseius fallacis (Garman) (Acari: Phytoseiidae) and Agistemus fleschneri Summers (Acari: Stigmaeidae).

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Materials and methods Hyaliodes vitripennis Nymphs and adults were collected individually in 30 ml plastic cups from a commercial orchard (St. Paul-d’Abbotsford, Quebec, Canada). The nymphs were collected in mid July, while adults were collected from the end of July to early August. In the laboratory, nymphs and unsexed adults were transferred in pairs to cages. The cages were 500 ml plastic containers with a large opening of 3.5 cm (diameter) on the cover and a smaller 2.5 cm (diameter) opening on the side wall. The openings were covered with a 40 micron mesh PeCap® (Tetko Inc. NC, USA) polyester screen. Two pieces of ‘J cloth towels’ (ca. 8 cm2) were placed at the bottom of the container and covered with a Whatman #2 filter paper (9 cm diameter). An apple leaf with its pedicel inserted in wet cotton was placed on top of the filter paper. Twenty to 25 adult two-spotted spider mites, Tetranychus urticae Koch (food for the predacious mirid), were transferred onto each leaf along with a pair of H. vitripennis at the same stage of development. The entire set up was treated with indoxacarb 30% DP (Avaunt®, Steward®, Dupont Canada, Toronto On). Indoxacarb was applied with a thin layer chromatography sprayer set at 10.34 kPa. At that pressure the amount of residue on the leaf was 2.00 mg cm-2.The concentrations decreased in geometric progression from 108 ppm active ingredients (AI) to 0.84 ppm AI. In the control, tap water was used and applied in the same manner as the insecticide. A total of 18 insects per concentration were used. After treatment, the cages were transferred to a growth chamber set at 25°C, 70% relative humidity and 16:8 h light:dark cycle. Mortality counts were made 24 h post treatment. Insects unable to move their appendages following probing with a needle were considered dead. Amblyseius fallacis Residual toxicity on field treated leaves. Indoxacarb was applied in an apple orchard at St. Paul-d’Abbotsford, Quebec, Canada. Applications were made with a diaphragm-pump, hand-gun system that delivered 54 ppm AI at 1380 kPa (72.9 g AI 1350 liters-1 ha-1). Six treated leaves from the outer canopy were collected from each replicate at 0 h and 24 h after treatment and brought to the laboratory. The leaves were placed lower surface up on wet polyfoam in seedling trays (73 x 15.7 x 2.5 cm). The wet polyfoam maintained the leaves fresh. The petioles were covered with wet J-cloth® (Chicopee Inc., Benson, NC, USA). Tangle-Trap® (The Tanglefoot Co. Michigan, USA) was applied to serve as a mite-proof barrier around a 3 cm diameter circular arena in the centre of the leaf. Six adult female A. fallacis were introduced in each arena with 30 two-spotted spider mites as food. The trays were placed in a growth chamber set at 24°C, 80% relative humidity and 16:8 h light:dark cycle. Mortality counts were made according to Abbott (1925) at 24 h and 48 h. Death was defined by the inability of mites to move their appendages when touched with a camel hair brush. Predators captured in the Tangle-Trap® on treated leaves were considered to have been repelled by the indoxacarb when compared with predators caught in the Tangle-Trap® on leaves treated with water. Topical toxicity to females: survival and fecundity. Ninety 24 h old A. fallacis females were placed one per arena and treated with indoxacarb (54 ppm AI).The leaves with the mites were treated with a thin layer chromatography sprayer set at 10.34 kPa. At that pressure the amount of residue on the leaf was 2.00 g cm-2. The trays containing the treated leaves with the arena were placed in a growth chamber set at 24°C, 80% relative humidity and 16:8 h light:dark cycle. The number of female mites alive for 48 h at 24 h intervals was noted, as well as, the number of eggs laid at 24 h intervals for 96 h.

33

Topical toxicity to eggs. Thirty A. fallacis females with two-spotted spider mite eggs (food ad libitum) were placed three per arena for 24 h and the number of eggs laid was recorded. The females were removed, and the eggs were treated as explained above with indoxacarb. Three days later egg hatch was recorded. To avoid cannibalism, newly hatched A. fallacis nymphs were removed from the arena at 24 h intervals for the duration of the study. Agistemus fleschneri Adult females were collected from a commercial orchard in August at St. Alexandre, Quebec. They were transported in paper bags placed in a cooler to the laboratory within 30 minutes. Toxicity to females. Thirty A. fleschneri females were transferred with a fine camel hair brush to double-sided tape (Cantech®, Montreal, Canada) on a microscope slide. Each slide was immersed for 5 seconds in indoxacarb (54 ppm AI) and allowed to dry for 30 minutes. The slides were then placed in a growth chamber at 21°C, 80% relative humidity and 16:8 h light:dark cycle. After 24 h of exposure, female mortality was determined by touching its abdomen. If the legs moved, than the female was considered alive. Female fecundity and egg hatch. The median portion of an apple leaf was cut in a rectangular form (4 cm x 3 cm) and 20 female two-spotted spider mites were transferred on each leaf to lay eggs (food for the predator) for 24 h. After 24 h, the adult two-spotted spider mites were removed and three A. fleschneri were introduced onto the leaves. All were treated with a thin layer chromatography sprayer set at 10.34 kPa and replicated 10 times. The experimental setting was then placed in a growth chamber at 21°C, 80% relative humidity and 16:8 h light:dark cycle. After 48 h of exposure, all female A. fleschneri were killed and their eggs were counted under a binocular stereoscope. Percent egg hatch was estimated by recording the number of hatched eggs in a growth chamber for six days. To minimize cannibalism newly hatched nymphs were removed as soon as they were counted. Throughout this study the control was treated with tap water. Probit analysis was done on mortality data with Polo PC (LeOra Software 1994). Percentages were compared with the Fisher exact test (Zar 1984). Results and discussion Indoxacarb is toxic to H. vitripennis and the LC50 is at about one half of the recommended dose of 54 ppm AI for orchards (Fig 1). There is no significant difference between the LC50 values for nymphs (25.9 ppm AI, range 19.0-46.9 ppm AI, χ2 = 4.32) to adults (29.9 ppm AI, range 19.0-46.9 ppm AI, χ2 = 4.12). We also noted that following an application, dying insects were difficult to diagnose as they remained motionless because their prolegs and posterior were paralyzed. Within 24 h, intoxicated insects appeared smaller, shrunken, and severely desiccated. In contrast indoxacarb has no residual toxicity to A. fallacis at 54 ppm AI (Table 1). However, when compared with the control, a significant number were caught in the Tangle-Trap® indicating the repellency of indoxacarb when the leaves were treated and aged in the orchard for 24 h. Zero mortality of A. fallacis and A. fleschneri females were recorded 24 h after a topical treatment at the field rate (54 ppm AI). In both species, there was no difference in the number of eggs laid and the percent of those eggs that hatched. The results of this study suggest that in apple orchards, indoxacarb will not have adverse effects to A. fallacis and A. fleschneri. However, there is a high probability, that it will be toxic to the predacious mirid H. vitripennis.

34

Nymph: R2 = 0.91

Adult: R2 = 0.87

12345678

0.5 1 1.5 2 2.5 3

Log (1+dose) ppm

Mor

talit

y

Probit

99.997.784.1

50.015.9

2.3

0.1

Figure 1. Indoxacarb effects on the nymphs (•) and adults (o) of Hyaliodes vitripennis. Table 1. Percent mortality and repellency of indoxacarb residues to A. fallacis females

Treatment n % Mortalityc % Repellencyc Time of leaf collection after treatment 24 h 48 hd 24 h 48 hd

0 Indoxacarba

30% DP 105 2a 5a 12a 24a

Controlb 108 0a 2a 10a 16a 24 Indoxacarba

30% DP 109 2a 3a 24a 30a

Controlb 108 0a 2a 10b 16b a Dose applied in an orchard: 54 ppm AI (72.9 g AI ha-1). b Water. c Percentages followed by the same letter in a column for the same interval of time are not

significantly different (P=0.05), based on Fisher exact test. d Cumulative mortality (24 + 48 h). Acknowledgments The authors thank Dupont Canada for graciously providing the indoxacarb and financially supporting the study with Agriculture & Agri-Food Canada (MII contract / grant number: 99-5922). This is Contribution # 335/2003.07.02R of the Horticultural Research & Development Centre, Agriculture and Agri-Food Canada, St. Jean-sur-Richelieu, Quebec, Canada. References Abbott, W. 1925: A method of computing the effectiveness of an insecticide. – J. Econ.

Entomol. 18: 26-27. LeOra Software 1994: POLO-PC: Probit and Logit Analysis. – Berkley, CA. Robertson, J.L. & Preisler, H.K. 1992: Pesticide bioassays with arthropods. – CRC Press,

Boca Raton, Florida, 127 pp.

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Saldago, V.L. 1990: Mode of action of insecticidal dihydropyrazoles: selective block impulse generation in sensory nerves. – Pestic. Sci. 23: 389-411.

Wing K.D., Sacher, M., Kagaya, Y., Tsurubuchi, Y., Mulderig, L., Cannair, M. & Schnee, M. 2000: Bioactivation and mode of action of the oxadiazine indoxacarb in insects. – Crop Prot. 19: 537-545.

Wing K.D., Schnee, M.E., Sacher, M. & Connair M. 1998: A novel oxadiazine insecticide is bioactivated in lepidopteran larvae. – Arch. Insect Biochem. Physiol. 37: 91-103.

Zar, J.H. 1984: Biostatistical Analysis. 2nd edition. – Prentice Hall, Englewood Cliffs, NJ, 718 pp.


pp. 37 - 44

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Toxicity of five insecticides on predatory mites (Acari: Phytoseiidae) in vineyards in two Portuguese regions Raul Rodrigues1, Rui Gonçalves1, Carlos Silva2 & Laura Torres3 1 Escola Superior Agrária de Ponte de Lima, Refóios do Lima, 4990-706 Ponte de Lima,

Portugal, E-mail: [email protected] 2 Escola Superior Agrária de Castelo Branco, Quinta da Senhora de Mércules, 1006-909

Castelo Branco, Portugal 3 Universidade de Trás-os-Montes e Alto Douro, Dep. Protecção de Plantas, Quinta de

Prados, 5000-911 Vila Real, Portugal. Abstract: Amongst the spider mites that cause important damages in Portuguese vineyards, the two-spotted mite Tetranychus urticae Koch is the most important species in the southern regions. However, in recent years it was found that the population of red spider mite Panonychus ulmi (Koch) is increasing and becoming a problem in vineyards mainly of the northwest region. To evaluate the toxicity of five insecticides (Bacillus thuringiensis, tebufenozide, flufenoxuron, phosalon and delta-methrin) on predatory mites (Acari: Phytoseiidae), two field tests were carried out during summer 2002, with a fully randomized design and five replicates per treatment, using commercial formulations at recommended field rates. The control plot was treated with water. Assessments of the phytoseiids were performed four days before the treatments, and the remaining mobile stages were counted 4, 7, 14, 21 and 35 days after the treatments. Phytoseiids mortality was calculated with the Henderson-Tilton formula and the toxicity of the insecticides was determined based on the standard for field methods of the IOBC/wprs Working group “Pesticides and Beneficial Organisms”. The results were similar in both trials: phosalon and deltamethrin had a poor selectivity (harmful) on the phytoseiid mites, Bacillus thuringiensis, tebufenozide and flufenoxuron showed a good selectivity to these predators. The most abundant Phytoseiid species identified were Phytoseius plumifer Canest. & Fanzag (91.8%) in Minho region and Typhlodromus phialatus Athias-Henriot (96.7%) in Castelo Branco region. Key-words: vineyards; integrated pest management; predatory mites; insecticides, side-effects Introduction The red spider mite Panonychus ulmi (Koch) and the two-spotted mite Tetranychus urticae Koch are the principal phytophagous mites that cause damages of economic importance in apple orchards. In Portuguese vineyards the two-spotted-mite Tetranychus urticae Koch is the most important species in the Southern regions. However, in recent years it was found that the population of red spider mite Panonychus ulmi (Koch) is increasing and becoming a problem in vineyards mainly of the northwest region. The chemical control normally used against this pest is not selective, so that not only the predator population decreases, but also resistance may occur (Costa-Comelles, 1986).

Members of the family Phytoseiidae show a remarkable ability to reduce red spider mite infestations. There are many behavioural aspects that need to be considered in the phytophagous and predacious mites. Recognizing these behaviours and the side effects of pesticides on predatory mites can increase the success of biological control. Therefore, successful utilization of biological control could depend on the compatibility of the natural predators with pesticides.

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Studies on the side effects of pesticides on phytoseiid mites in Portugal have began in 1995 at the University of Trás-os-Montes and Alto Douro, as part of the Project, Integrated Protection in apple crop against red spider mite in the Interior North of Portugal (Rodrigues, 2000; Torres, 1999). Further research to evaluate side effects of pesticides on all sensitive stages of the phytoseiid mites has started recently in the Higher School of Agriculture at Ponte de Lima in vineyards and apple orchards and will be carried out during three years (2002 to 2004) in Minho and Beira Interior regions. The main objective of this research is to evaluate the side effects of pesticides on all sensitive stages of the phytoseiids according to the standard method of the IOBC Working Group “Pesticides and Beneficial Organisms” as validated and published by the Joint Initiative (Blümel et al., 2000).

Material and method Five insecticides used against grape berry moth, Lobesia botrana (Lepidoptera: Tortricidae), were tested in single application in two portuguese regions, according to Blümel et al., 2000. Vineyards localization/trial design Experiments were conducted during the Summer period at Ponte de Lima – Minho region (trial 1) and Castelo Branco – Beira Interior region (trial 2). The characterization of the vineyards is summarised in Table 1. The plots were arranged in a fully randomized block design and the control plot was treated with water. Table 1: Characterization and location of trials

Ponte de Lima (Trial 1)

Castelo Branco (Trial 2)

Geographical coordinates Lat. 41.47º N Long. 8.32º W

Lat. 39.8º N Long.7.5ºW

Cultivars Loureiro Rufete Plantation density 1111 plants/ha (3 x 3 m) 3333 plants/ha (2 x 1.,5) Age 9 years 10 years Soil surface Rang: herbicides

Between rows: spontaneous herbaceous species. Frequent cuttings.

Rang: herbicides Between rows: spontaneous herbaceous species. Frequent cuttings.

Training system Single curtain Double curtain Trial area: 0.35 ha 0.135 ha

Vines per replicate 3 8 Test products/spraying equipment The test compounds were applied at recommended rates by the manufacturer. The commercial formulations of compounds and the applied doses are given in Table 2. The vines were sprayed with the insecticides by a knapsack using a hand-lance until run-off, using a volume of 1000 l/ha.

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Table 2. Insecticides used in both trials

Active ingredient Trade name Commercial product/ha

B. thuringiensis (3,2%) Dipel® 1,00 kg

phosalon (30% p/p) Zolone® 2,00 kg

deltamethrin (25 g/l) Decis® 0,30 l

flufenoxuron (100 g/l) Cascade® 0,50 l

tebufenozide (240 g/l) Mimic® 0,30 l

Sampling and data analysis The assessment of the mobile stages of Phytoseiid mites per leaf, was performed in laboratory with a stereoscopic microscope. The leaves were detached in each replicate at five times: four days before the treatements (T0), and 4, 7, 14 and 28 days after treatments (T4, T7, T14 and T 28). In each assessment 25 leaves per replicate (125 leaves per treatement) were evaluated.

Effects on the predatory mites were calculated with the Henderson-Tilton formula (Henderson & Tilton, 1955) and divided into four categories corresponding to the standard for field methods of the IOBC Working group “Pesticides and Beneficial Organisms”. According to this formula the percentage of mortality % = [ 1- (K1xP2)/(K2xP1)] x 100, where K1 = total number of target species before treatment in the control plot, K2 = total number of target species after treatment in the control plot, P1 = total number of target species before treatment in the test plot and P2 = total number of target species after treatment in the test plot.

According to the principles of IOBC, four evaluation categories (% mortality or reduction in beneficial capacity) were used: 1 = harmless (< 25%), 2 = slightly harmful (25-50%), 3 = moderately harmful (51-75%) and 4 = harmful (>75%) (Hassan, 1994). Experimental plots were maintained without pesticide sprays during the observations period.

After the treatment, mean values of mobile stages of predatory mites/leaf were counted at each time of assessment in all 5 replicates and analysed statistically. Data were checked for normal distribution and homogeneity of the variances and analysed by an univariate variance analysis (ANOVA, HSD Tukey-test performed at 5% level; SPSS 11.0 for Windows, SPSS Inc.; Chicago, IL). The identification of Phytoseiidae species was made using adequate keys (Chant & Yoshida-Shaul, 1987; Kreiter & Bourdonnaye, 1993; García-Mari et al., 1994).

Results and discussion

Phytoseiid species identified The total number of identified phytoseiid specimens was 3051, with 2097 from trial 1 and 954 from trial 2.

The relative abundance of phytoseiid species present in both trials is summarized in Table 3. The most abundant species were Phytoseius plumifer (91,8%) in trial 1 and Typhlodromus phialatus (96,7%) in trial 2.

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P. plumifer, T. phialatus, Kampimodromus aberrans and Typhlodromus pyri are considered as generalist predators and can survive in the absence of phytophagous mites; however, Euseius stipulatus is a generalist predator of phytophagous mites, but a specialist feeder on pollen (McMurtry & Croft, 1997).

Behaviour of generalist phytoseiids may be strongly influenced by leaf anatomy and climatic conditions. Phytoseius species usually occur on plants with highly pubescent leaves (Sabelis & Bakker, 1992). Local conditions in Minho region i.e. high relative humidity and very hairy-leaved grapevine varieties (as Loureiro), seem to be suitable for P. plumifer.

In contrast, T. phialatus is considered the most abundant and the most frequent species in meridional vineyards of Portugal (Ferreira, 1995; Teixeira, 1995) and Spain (Garcia-Marí et al., 1987). This species was thought to be strictly endemic to Mediterranean regions, but it is much more widespread since it was detected in Aquitanie, Burgundy and Poitot-Charentes, although always in low numbers (Kreiter & Bourdonaye, 1993) Table 3. Phytoseiid species presents in terms of their abundance

Trial 1 Trial 2 Phytoseiid species N % N %

Phytoseius plumifer Canest. & Fanzago 1925 91.8 - -

Typhlodromus pyri Scheuten 80 3.8 7 0.7

Kampimodromus aberrans (Oudemans) 69 3.3 25 2.6

Euseius stipulatus (Athias-Henriot) 23 1.1 - -

Typhlodromus phialatus Athias-Henriot - - 922 96.7

Mean density of predatory mites The mean densities of phytoseiid mites per leaf during the experiments are summarized in Figure 1 (trial 1) and Figure 2 (trial 2). Trial 1 In the preliminary sampling (T0) not any significant differences were detected in predacious mite densities (F= 0.004, p= 1.000). Four days after the treatments (T4) the mean density of phytoseiid mites per leaf was significantly different among plots (F=14.160, p= 0.000). At this time, predacious mite densities were significantly less in phosalon and deltamethrin treatments compared to the control plots and the remaining treatments. In the subsequent assessments, performed 7, 14, 21 and 35 days after the treatments, the mean density of phytoseiid mites per leaf was always significantly less in the phosalon and deltamethrin treatments compared to the control, tebufenozid, B. thuringiensis, and flufenoxuron. Trial 2 As in trial 1, predatory mites were homogeneously distributed within the plots before the treatments (F= 1.190, p= 0.343). In the first assessment, performed four days after treatment (T4), all insecticides caused a decline in predacious mite densities, however, predator densities also declined in control plots (F = 9537, p= 0,000). The mean number of phytoseiids

41

per leaf were significantly lower in the deltamethrin and phosalon treatments than in untreated and B. thuringiensis plots, however phosalone, tebufenozide and flufenoxuron did not differ significantly. In the assessments performed seven and 14 days after the reatments, the mean

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F= 0,004p= 1,000

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F= 48,220p= 0,000

F= 32,599p= 0,000

F= 77,383p= 0,000

F= 41,194p= 0,000

F= 90,995p= 0,000

Fig. 1. Mean density of Phytoseiidae per leaf in the different plots during the trial (T0 to T35), Means in same columns (for each assessment) followed by different letters are statistically significantly different (p<0,05, HSD-Tukey multiple comparison). Ponte de Lima, 2003.

0,00

0,50

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T0 T4 T7 T14 T21 T35 Tmean(T4 to T35)

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ile s

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F=1,190p= 0,343

F=9,537p= 0,000

F=4,860p= 0,000

F=5,774p= 0,001

F=17,761p= 0,000

F=14,277p= 0,000

F=17,893p= 0,000

Fig. 2. Mean density of Phytoseiidae per leaf in the different plots during the trial (T0 to T35), Means in same columns (for each assessment) followed by different letters are statistically significantly different (p<0,05, HSD-Tukey multiple comparison). Castelo Branco, 2003.

42

number of phytoseiid mites per leaf was again significantly lower in the phosalone and deltamethrin treatment compared to the control. In all plots, except deltamethrin and phosalone, an increase of phytoseiid mites densities was observed in the assessments performed 21 and 35 days after the treatments. At these dates, mean densities of phytoseiidmites were still significantly lower in deltamethrin and phosalone treatments than in the control, tebufenozide, B. thuringiensis and flufenoxuron plots, whereas these latter did not differ significantly. Toxicity The mortalities according to Henderson-Tilton for each trial are reported in Tables 4 and 5. Table 4. Toxicity classification of the insecticides, tested in Ponte de Lima (Trial 1) according to the IOBC Working Group “Pesticides and Beneficial Organisms” (% = reduction in comparison to the control plot, Henderson-Tilton; Cl = Class of toxicity).

T4 T7 T14 T21 T35 Tmean Active ingredient % CL % CL % CL % CL % CL % CL

Bacillus thur. -47,5 1 -0,2 1 5,3 1 11,6 1 1,4 1 -0,6 1

Tebufenozide 14,2 1 23,1 1 33,8 2 22,5 1 8,1 1 20,8 1

Deltamethrin 73,3 3 98,7 4 98,4 4 99,2 4 97,7 4 95,8 4

Phosalon 95,6 4 98,7 4 99,1 4 98,5 4 96,5 4 97,9 4

Flufenoxuron 16,3 1 34,4 2 50,7 2-3 46,2 2 57,7 3 44,6 2

Table 5. Toxicity classification of the insecticides, tested in Castelo Branco (Trial 2) according to the IOBC Working Group “Pesticides and Beneficial Organisms” (% = reduction in comparison to the control plot, Henderson-Tilton; Cl = Class of toxicity).

T4 T7 T14 T21 T35 Tmean Active ingredient % cl % cl % cl % cl % cl % cl

Bacillus thur. - 46,0 1 33,5 2 14,5 1 14,5 1 20,2 1 11,5 1

Tebufenozide 52,5 3 27,4 2 13,6 1 50,4 2-3 23,2 1 34,5 2

Deltamethrin 100 4 100 4 100 4 100 4 98,7 4 99,5 4

Phosalon 95,8 4 94,4 4 100 4 98,4 4 98,8 4 98,0 4

Flufenoxuron 31,6 2 46,5 2 15,8 1 6,7 1 18,3 1 18,6 1

Bacillus thuringiensis. This bio-insecticide showed practically no toxicity during trial 1. Although in trial 2, the toxicity on phytoseiid mites reached 33.5% seven days after treatment (T7), a recovery was verified in the subsequent assessments. In both trials the mean toxicity of B. thuringiensis was class 1 (harmless). These results confirm a good selectivity of B.

43

thuringiensis on phytoseiid mites, obtained by Giralt & Reyes (1999) in Spanish vineyards and by Rodrigues et al., (2002) in Portuguese’s apple orchards.

Tebufenozide. The action of this insecticide on phytoseiid mites resulted in a slightly higher mean mortality in trial 2 (34.5%) than in trial 1 (20.8%). In both trials, tebufenozid did not show an immediate toxicity. In trial 1 the highest value of mortality was 33.8%, 14 days after the treatment. In trial 2, the mortality of phytoseiid mites reached the highest values four days (52.5%) and 21 days after the treatment (50.4%).

In spite of the mean toxicity of this product that was harmless in trial 1 and slightly harmful in trial 2, these results showed that this insecticide can be used in integrated pest management programs (IPM) because of the good selectivity on phytoseiid mites. Similar results were obtained by Torres et al., (2002) in apple orchard in Northeast region of Portugal.

Deltamethrin and phosalon. These insecticides presented identical results during the two trials. The mean mortality during the trials was always higher than 95% in both cases. As we expected, deltamethrin and phosalon showed a poor selectivity to the phytoseiid mites in both regions, they were harmful. Concerning phosalon, our results corroborate the poor selectivity of this insecticide to the phytoseiid mites verified already in previous researches in apple orchards in Northern region of Portugal by Rodrigues et al. (2002) and Torres et al. (2002).

Flufenoxuron. This insecticide showed a good selectivity to the predatory mites in both trials. In trial 1 the mortality reached 50,7% 14 days after the treatment. However in trial 2, the highest value of toxicity (46.5%) was obtained seven days after the treatment. The mean toxicity for this product was slightly harmful in trial 1 (44,6%) and harmless in trial 2 (18,6). Conclusions These trials were carried out in two different geographical regions, where the dominant phytoseiid species were different, but the results revealed a similar selectivity of the insecticides in both cases.

Although tebufenozide and flufenoxuron performed differently in both trials, these insecticides showed a good selectivity to predatory mites and these products can be used in IPM programs in vineyards.

Deltamethrin and phosalon showed a poor selectivity, they were very harmful to the phytoseiid mites. We consider, that phosalon cannot be recommended for use in IPM programs in these regions, mainly where the species P. plumifer and T. phialatus are dominant. Acknowledgements This study was supported by the Project-Agro Nº 317 entitled: Efeitos Secundários dos pesticidas sobre fitoseídeos (Acari: Phytoseiidae) associados às culturas da macieira e vinha nas regiões de Entre-Douro e Minho e Beira Interior / Side effects of pesticides on predatory mites (Acari: Phytoseiidae) in apple orchards and vineyards in Entre-Douro e Minho and Beira Interior regions”. References

Blümel S., Aldershof S., Bakker F.M., Baier B., Boller E., Brown K., Bylemans D., Candolfi M.

P., Huber B., Linder C., Louis F., Müther J., Nienstedt K.M., Oberwalder C., Schirra K.J,

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Ufer A. & Vogt H. 2000: Guidance document to detect side effects of plant protection products on predatory mites (Acari: Phytoseiidae) under field conditions: vineyards and orchards. – In: Candolfi et al. (Eds.): Guidelines to evaluate side-effects of plant protection products to non target-arthropods. IOBC, BART and EPPO Joint Initiative, IOBC/WPRS Gent: 145-153.

Chant D.A. & Yoshida-Shaul E. 1987: A world review of the pyri species group in the genus Typhlodromus Scheuten (Acari: Phytoseiidae). – Can. J. Zool., 65: 1770-1803.

Costa-Comelles J. 1986: Causas de la proliferación de acaros Panonychus por plaguicidas - Posibilidad de su control biológico en manzano. – Tesis doctoral. Escuela Técnica Superior de Ingenieros Agrónomos / Universidad Politécnica de Valencia: 410 pp.

Ferreira M.A. 1995: Ácaros predadores nas vinhas alentejanas – situação actual. – 3º Simpósio de Vitivinicultura do Alentejo: 181-185.

García-Marí F., Ferragut F., Marzal C., Laborda R., Costa-Comelles J., Coscolla R & Sanchez J. 1987: Contribución al conocimiento de los àcaros fitoseidos en los viñedos valencianos. – Investigación Agrária: Producción y Protección Vegetales, 2(1): 89-95.

García-Marí F., Perez F.F. & Costa-Comelles J. 1994: Curso de acarologia agrícola. – Unidad Docente de Entomologia Agrícola, Dep. De Producción Vegetal – E.T.S. de Ingenieros Agrónomos, Universidad Politécnica de Valencia, Valencia: 278 pp.

Giralt L. & Reyes J. 1999: Efecto de diversos insecticidas y acaricidas sobre poblaciones de Typhlodromus pyri Scheuten en viña. – Phytoma España, 105: 30-35.

Hassan, S.A. 1994: Activities of the IOBC/WRPS Working Group “Pesticides and Beneficial Organisms”. – IOBC/wprs Bull. 17(10): 1-5.

Henderson C.F. & Tilton E.W. 1955: Test with acaricides against brown wheat mite. – J. Econ. Ent. 48: 157-161.

Kreiter S. & Bourdonnaye D. 1993: Les typhlodromes, acariens prédateurs. Clé simplifiée d’identification des principales espéces des cultures de plein champ en France. – Les Cahiers de Phytoma-la défense des végéteaux, 446 (suplément): I-IX.

McMurtry J.A. & Croft B.A. 1997: Life-Styles of phytoseiid mites and their roles in biological control. – Annu. Rev. Entomol. 42: 291-321.

Rodrigues, J.R. 2000: Avaliação da eficácia de vários acaricidas sobre Panonychus ulmi (Koch) e dos seus efeitos secundários sobre ácaros predadores da família Phytoseiidae. – Tese de Mestrado em Horticultura Fruticultura e Viticultura, Utad: 164 pp.

Rodrigues J.R., Miranda N.R.C., Rosas, J.D.F., Maciel C.M. & Torres L.M. 2002: Side effects of fifteen insecticides on predatory mites (Acari: Phytoseiidae) under field conditions in an apple orchard. – IOBC/wprs Bulletin: 25(11): 53-62.

Sabelis M.W., Bakker F.M. 1992: How predatory mites cope with the web of therir tetranychid prey: a functional view on dorsal chaetotaxy in the Phytoseiidae. – Exp. Appl. Acarol. 16: 203-225.

Teixeira L. 1995: O papel da limitação natural no combate aos ácaros fitófagos. – 3º Simpósio de Vitivinicultura do Alentejo: 187-199.

Torres L.M., Carlos C.R. & Espinha I.G. (2002): Efeitos secundários de insecticidas sobre ácaros fitoseídeos associados à macieira. – Revista de Ciências Agrárias, 25 (1,2): 76-87.

Torres L.M. 1999: Apresentação das acções de I&DE desenvolvidas e em curso no âmbito da protecção integrada da macieira contra o aranhiço vermelho, Panonychus ulmi (Koch), pelo gruo de trabalho da UTAD. – Simpósio “Protecção integrada da macieira contra o aranhiço vermelho, Panonychus ulmi (Koch) em condições mediterrânicas. Vila Real, Junho de 1999.


pp. 45 - 57

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Effects of the fungicide zoxamide, alone and in combination with mancozeb, to beneficial arthropods under laboratory and field conditions M. Miles & E. Green Dow AgroSciences, European Development Centre, 2nd Floor, 3 Milton Park, Abingdon, OX14 4RN, UK Abstract: Zoxamide (RH-7281) is a new fungicide for use on potatoes, vines and vegetables for control of oomycete fungi. Zoxamide (240 g/L SC formulation) was tested under worst case laboratory conditions at application rates of 150 and 300 g a.i./ha (equivalent to x1 and x2 the maximum use rate). No effects were noted on predatory mites (Typhlodromus pyri and Amblyseius andersoni) or to a range of predatory arthropods (Chrysoperla carnea, Orius insidiosus, Poecilus cupreus and Pardosa sp.) at either the x1 or x2 rate. On the parasitic wasp Aphidius rhopalosiphi no effects were seen at x1 and only minor effects were observed at x2. Under normal conditions zoxamide is used in combination with mancozeb as the commercial product ELECTIS1 containing 83 g zoxamide and 667g mancozeb/Kg. The effects of Electis were investigated under worst case laboratory conditions at x1 and x2 the maximum field rate (equivalent to 150 + 1200 and 300 + 2400 g a.i./ha). No adverse effects were seen on A. rhopalosiphi, O. insidiosus, P. cupreus or Pardosa sp. however harmful effects were seen at both rates on the predatory mites T. pyri and A. andersoni. Extended laboratory studies on T. pyri indicated that the effects on mites were mainly due to the mancozeb in the formulation. To investigate the effect of repeated applications aged residue extended laboratory tests were conducted on A. rhopalosiphi and C. carnea using a multiple application factor (MAF) to calculate doses. At x3.5 rate of Electis no adverse effects were noted on either species, at a x5 rate Electis was safe to parasitic wasps and only slightly harmful to the lacewing. After one week of ageing the x5 rate was safe to C. carnea. Under field conditions in vines 4 applications of Electis were selective to populations of T. pyri. Six applications caused harmful effects within season but recovery was seen the following year. The effects of zoxamide alone and in combination with mancozeb have been thoroughly investigated allowing the combination to be used in disease management programmes compatible with integrated plant management (IPM). Key-words: Fungicide, Zoxamide, Mancozeb, predatory mite, beneficial arthropod, side-effects, Aphidius rhopalosiphi, Orius insidiosus, Chrysoperla carnea, Poecilus cupreus, Pardosa sp., Typhlodromus pyri, Amblyseius andersoni, Kampimodromus aberrans, Euseius stipulatus. Introduction Zoxamide (RH-7281) is a new fungicide from Dow AgroSciences for use on potatoes, vines and vegetables for control of oomycete fungi. It has a novel mode of action and inhibits cell division by disruption of cellular microtubules due to highly specific binding to the β subunit of tubulin. Due to the single site of action and specificity of effect, zoxamide is currently formulated with mancozeb a broad spectrum multisite contact fungicide. This protects zoxamide from the risk of development of resistance and produces a highly effective product with a wide range of activity.

1 Trademark of Dow AgroSciences

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Zoxamide can be used to control fungal diseases in crops where the conservation of predatory insects and mites is an important component of Integrated Pest Management (IPM). Additionally, there are governmental and environmental pressures to develop and use products safely with minimum impact on non-target arthropods. A range of laboratory and field tests were conducted with zoxamide and Electis on beneficial insects and mites. The objective of this paper was to review the effects of zoxamide alone, and in combination with mancozeb to a range of predatory insect and mite species. Materials and methods Experimental test systems (study type and species) are outlined in Table 1. All studies were carried out using zoxamide formulated as 240 g/L SC formulation or as Electis (83 g zoxamide and 667g mancozeb/Kg WG formulation). Table 1. Study type and beneficial species tested in side-effects studies with zoxamide formulations.

Study type Test item Species

Laboratory (tier I) Zoxamide 240SCElectis

Aphidius rhopalosiphi, Typhlodromus pyri, Amblyseius andersoni, Pardosa spp.,

Poecilus cupreus, Chrysoperla carnea, Orius insidosus

Extended laboratory Electis A. rhopalosiphi, T. pyri, C. carnea

Field (side-effect study) Electis T. pyri

Field (timing and number

of sprays)

Electis T. pyri, Euseius stipulatus, Kampimodromus aberrans

Laboratory (tier I) studies Laboratory tier I studies were conducted to investigate the intrinsic toxicity of zoxamide alone and in combination with mancozeb (Electis) to a range of representative beneficial arthropod species (see Table 1). For each species, Zoxamide (240 g/L SC formulation) was tested at application rates of 150 and 300 g a.i./ha (equivalent to x1 and x2 the maximum use rate) and Electis at 1800 and 3600 g product/ha (150 g zoxamide + 1200 g mancozeb and 300 g zoxamide + 2400 g mancozeb/ha which are equivalent to x1 and x2 the maximum field rate). In all tests a suitable toxic reference and water treated control treatment were also included.

Sensitive life history stages were exposed to artificial substrates (glass plate, quartz sand) according to internationally recognised guidelines. A study with A. rhopalosiphi followed the method of Poglar (1988) with modifications by Mead-Briggs (1992). Adult wasps were exposed to treated glass plates for 48 hours. The parasitism rate of surviving female wasps was assessed using cereal seedlings infested with bird cherry aphids (Rhopalosiphi padi) as a suitable host. In the mortality phase each treatment was replicated three times with ten wasps per test unit and in the parasitism phase, ten individually confined wasps were used per treatment. For the predatory mites, T. pyri and A. andersoni a glass plate, open method based

47

on that of Overmeer (1988) was used. Each test unit consisted of a glass plate with a barrier of damp tissue paper and a sticky non-toxic gel. Twenty protonymph mites were placed onto each test unit and fed with untreated pollen. Each treatment was replicated five times. Mortality was assessed after seven days exposure and the fecundity of the surviving female mites was measured between days seven and fourteen. Adult Pardosa spp. were exposed individually to treatments in test units containing quartz sand (Wehling and Heimbach, 1994). For each treatment, ten male and ten female spiders were exposed for fourteen days and mortality and food consumption were measured throughout. The effect on adult carabid beetles (P. cupreus) was investigated using the method of Heimbach (1992). Test units containing quartz sand, holding three male and three female beetles were prepared with fly pupae as food. Each treatment was replicated six times and mortality and food consumption measured up to fourteen days after treatment. A total of forty larval C. carnea less than 24 hours old were exposed individually to glass plates using the method of Bigler (1988). The fecundity of the surviving adults was measured. Following the method of Stäubli and Pasquier (1988) ten replicates of 10 second instar nymphal O. insidiosus were exposed to test product in glass coffin cells and the fecundity of the adults was assessed ten days after exposure. Extended laboratory studies These tests are characterised by the inclusion of a natural substrate in the test system. The effect of Electis on the predatory mite T. pyri was investigated using the laboratory tier I design of Blümel et al (2000a). Leaf discs were substituted for glass plates and a rate response test was conducted to estimate the rate corresponding to 50% mortality. Seven rates of Electis were tested and each was replicated three times with each test unit housing 10 mites.

To investigate the effect of repeated applications aged residue extended laboratory tests were conducted on A. rhopalosiphi and C. carnea using a multiple application factor (MAF) to calculate doses. Rates equivalent to x3.5 and x5 the single maximum field application rate of Electis (6400 and 9200 g product/ha) were tested to simulate extreme worse case exposure arising from season long spray programmes. Bioassays were conducted on the day of application (when spray deposits had dried) and after one week of ageing following the methods of Mead-Briggs and Longley (2000) for A. rhopalosiphi and Vogt et al. (2000), modified for bean leaf substrate for C. carnea. For A. rhopalosiphi pots of barley seedlings were sprayed and six replicates of five adult female wasps were used in each bioassay. The fecundity of surviving wasps was measured. Lacewing larvae two to three days old were used for the bioassay with forty exposed to each treatment. Fecundity of the surviving adults was assessed for the x3.5 rate of Electis in the first bioassay and for both rates in the seven day bioassay. Appropriate toxic reference and control treatments were included for both species. Field studies Electis was applied to field populations of T. pyri in vines at a site located in a vine growing area of Germany (Pfalz, Ruppertsberg) in 2001 according to BBA guideline VI, 23-2.3.4. (BBA, 1991) Electis was applied at a test concentration of 0.18% and two regimes were investigated; four applications (two pre- and two post-flowering) and six applications (two pre- and four post-flowering). The water volume used to apply the treatments increased throughout the season so that the application volumes used corresponded to, 600, 800, 1000, 1400, 1600, and 1600 L water/ha for applications 1, 2, 3, 4, 5 and 6 respectively. This gave nominal application rates of 1080, 1440, 1800, 2520, 2880 and 2880 g Electis/ha for each of the six application timings. Each plot consisted of one row of fifteen vines; four plots were allocated to each treatment. Dithianon and water were included as soft reference and control treatments respectively, each applied six times. Twenty-five leaves were collected from each

48

replicate at intervals and the number of mites present determined using a washing method. The pre-treatment sample was made fourteen days before the first application. The study was concluded with additional sampling in 2002.

A series of field studies in vines was conducted in 2003 to investigate the effect of number and timing of applications of Electis to predatory mite. Studies were conducted in Germany, Spain and Italy reflecting a range of mite species and growing conditions. The studies followed the guideline of Blümel et al (2000b) and a summary of test information is given in Table 2. Electis was applied either twice or four times per season at different timings at a spray concentration of 0.18%. Methamidathion was applied six times as a toxic reference and a control or soft reference was included. Leaves were sampled at intervals and the number of mites determined using a washing technique. The treatment schedules and timings used in the trials are given Table 3. As different countries not only reflect different mites species, but also different growing and application conditions the water volumes used and hence the actual test application rates vary. These are summarised in for each trial in Table 4. Table 2. Summary of test information for field trials conducted in 2003 on vines with Electis (83 g zoxamide and 667g mancozeb/Kg WG formulation) on predatory mites.

Country Test species No. reps.

Plot size No. leaves

sampled/plot

Untreated reference

Pre-treatment sample

Germany Typhlodromus pyri 4 11 – 12

vines 25 Water 26DBA

Spain Euseius stipulatus 4 17 x 2.2m

= 37.4 m2 50 Dimethomorph Not assessed

Italy Kampimodromus aberrans 4 7 vines 20 Water 6DBA

Note: DBA = days before first application. Table 3. Summary of treatment timings for field trials conducted in 2003 on vines with Electis (83 g zoxamide and 667g mancozeb/Kg WG formulation) on predatory mites.

Application No. / timing Treatment Preflowering Pre- to end of

flowering Fruit set to pea

size Untreated 1+2 3+4 5+6

Electis 1+2 1+2 – – Electis 3+4 – 3+4 – Electis 5+6 – – 5+6

Electis 1+2+3+4 1+2 3+4 – Electis 3+4+5+6 – 3+4 5+6 Methamidathion 1+2 3+4 5+6

49

Table 4. Nominal water volume and rate applied in field trials conducted in 2003 on vines with Electis (83 g zoxamide and 667g mancozeb/Kg WG formulation) on predatory mites.

Country Application No. / volume (L/ha) / rate (g Electis/ha) 1 2 3 4 5 6

Germany L/ha 400 600 800 800 800 800 g Electis/ha 720 1080 1440 1440 1440 1440

Spain L/ha 235 378 420 435 595 595 g Electis/ha 423 680 756 783 1071 1071

Italy L/ha 300 400 400 500 500 500 g Electis/ha 540 720 720 900 900 900

Classification of effects The effect of zoxamide alone or in combination with mancozeb as Electis, was categorised according to the IOBC (International Organisation for Biological and Integrated Control of Noxious Animal and Plants) classifications (Hassan 1992) described in Table 5. Table 5. IOBC (International Organisation for Biological and Integrated Control of Noxious Animal and Plants) classification system for side effects of plant protection products to beneficial and non-target arthropods (Hassan 1992).

% Effect observed Classification Laboratory studies All other studies *

Class 1 Harmless <30% <25% Class 2 Slightly Harmful 30 – 79% 25 – 50% Class 3 Moderately Harmful 80 – 99% 51 – 75% Class 4 Harmful >99% >75%

Note: *Study types are extended laboratory, semi-field and field tests. Results and discussion Laboratory (tier I) study The results from the laboratory tier I studies with zoxamide and Electis conducted with representative beneficial arthropod species are presented in Tables 6 and 7. Both lethal and sub-lethal effects are listed along with the performance of the untreated and toxic reference treatments. The performance of the control and toxic reference treatments indicated valid studies according to the guidelines in force at the time of conduct. For zoxamide all species tested resulted in either a harmless (class 1) or slightly harmful classification (class 2). In terms of mortality zoxamide was always harmless. For T. pyri, the 150 g zoxamide/ha rate indicated a 53.7% reduction in fecundity. However this was based on a low number of eggs per female in all treatments and no effect was seen at the x2 rate (22.8%). It was concluded that the apparent effect at the lower rate was erroneous. These findings indicted a low risk to beneficial arthropods due to applications of zoxamide. When zoxamide was tested in combination with mancozeb, with the excepti on of the predatory mites, all tests returned either a harmless or slightly harmful classification. For both T. pyri and A. andersoni, Electis was classified as harmful (class 4) at either the x1 or x2 rate.

50

Table 6. Effects of zoxamide (240 g/L SC) to a range of beneficial arthropod species in tier I laboratory tests.

Test species

Treatment M (%) Corr M (%)

P/R/F (% reduction)

IOBC Class

Zoxamide 150g/ha 0.0 --- 19.6 (7.1%) 1

Zoxamide 300g/ha 0.0 --- 13.6 (35.8%) 2

Dimethoate EC 100 100 N/A

Aphi

dius

rh

opal

osip

hi

Control 0.0 --- 21.1

Zoxamide 150g/ha 16* 14.9 0.57* (53.7%) 1

Zoxamide 300g/ha 19* 15.6 0.95 (22.8%) 1

Ethyl parathion 64* 62.5 N/A

Typh

lodr

o-m

us p

yri

Control 4.0 N/A 1.23

Zoxamide 150g/ha 10.4* --- 0.97 (2.1%) 1

Zoxamide 300g/ha 5.1 --- 0.95 (-2.1%) 1

Ethyl parathion 100* 100 N/A

Ambl

ysei

us

ande

rson

i

Control 0.0 --- 0.95

Zoxamide 150g/ha 0.0 --- 0.68 (5.5%) 1

Zoxamide 300g/ha 0.0 --- 0.59* (18.1%) 1

λ-cyhalothrin EC 80* 80 0.30* (58.3%)

Pard

osa

sp.

Control 0.0 --- 0.72

Zoxamide 150g/ha 0.0 --- 0.07 (-38.1%) 1

Zoxamide 300g/ha 0.0 --- 0.03 (42.8%) 2

Pyrazophos 97 97 0.0

Poec

ilus

cupr

eus

Control 0.0 --- 0.05

Zoxamide 150g/ha 5.0 2.6 12.6 (79.9%) 1

Zoxamide 300g/ha 5.0 2.6 9.4 (89.4) 1

Dimethoate 40 EC 100 100 N/A

Chr

ysop

erla

ca

rnea

Control 2.5 --- 14.7 (80.7%)

Zoxamide 150g/ha 6.0 4.1 3.7 (-2.8%) 1

Zoxamide 300g/ha 6.0 4.1 3.8 (-5.5%) 1

Ethyl parathion 100 100 N/A Ori

us

insi

dios

us

Control 2.0 --- 3.6

Notes: * Indicates significant difference from control (P = 0.05). M=Mortality, Corr. M=Corrected mortality, P=parasitism as mummies/female, R= fecundity as eggs/female, F=food consumption No. prey/individual/day.

51

Table 7. Effects of Electis (83 g zoxamide and 667g mancozeb/Kg WG formulation) to a range of beneficial arthropod species in tier I laboratory tests.

Test species


P/R/F (% reduction)

IOBC Class

Electis 1800g/ha 3.3 3.3 19.9 (6.1%) 1

Electis 3600g/ha 0.0 --- 22.9 (-8.0%) 1

Dimethoate EC 100 100 N/A

Aphi

dius

rh

opal

osip

hi

Control 0.0 --- 21.2

Electis 1800g/ha 97* 96.9 0.0 (100%) 4

Electis 3600g/ha 99* 99 0.0 (100%) 4

Ethyl parathion 63* 62.2 N/A

Typh

lodr

o-m

us p

yri

Control 2.0 N/A 1.46

Electis 1800g/ha 99 --- 0.0 (100%) 4

Electis 3600g/ha 100 --- 0.0 (100%) 4

Ethyl parathion 100 --- N/A

Ambl

ysei

us

ande

rson

i

Control 0.0 --- 0.93

Electis 1800g/ha 0.0 --- 0.58* (19.1%) 1

Electis 3600g/ha 5.0 --- 0.44* (39.4%) 2

λ-cyhalothrin EC 80* 80 0.30* (58.8%)

Pard

osa

sp.

Control 0.0 --- 0.72

Electis 1800g/ha 0.0 --- 0.04 (-28.6%) 1

Electis 3600g/ha 0.0 --- 0.05 (-4.8%) 1

Pyrazophos 97 97 0.0

Poec

ilus

cupr

eus

Control 0.0 --- 0.05

Electis 1800g/ha 10 7.7 12.1 (80.7%) 1

Electis 3600g/ha 15 12.8 15.9 (85.3%) 1

Dimethoate 40 EC 100 100 N/A

Chr

ysop

erla

ca

rnea

Control 2.5 --- 14.7 (80.7%)

Electis 1800g/ha 19* 17.3 3.7 (-2.8%) 1

Electis 3600g/ha 15* 13.3 4.1 (-13.9%) 1

Ethyl parathion 100 100 N/A Ori

us

insi

dios

us

Control 2.0 --- 3.6

Notes: * Indicates significant difference from control (P = 0.05). M=Mortality, Corr. M=Corrected mortality, P=parasitism as mummies/female, R= fecundity as eggs/female, F=food consumption No. prey/individual/day.

52

Extended laboratory studies The mortality of mites exposed to a range of rates of Electis on bean leaf discs under extended laboratory conditions is presented in Figure 1. All test rates of 0.1 g Electis/ha and above exceeded the 25% threshold for harmlessness. Dimethoate was clearly harmful and control survival was greater than 80%. A flat dose response was observed which had poor fit to a Log-Probit transformation. In terms of a non-statistical evaluation effects above 50% were seen at rates equal or greater than 100 g Electis/ha indicating that the LR50 for product lies between 50 and 100 g Electis/ha. This is lower than the recommended field application rate indicating risk to T. pyri under extended laboratory conditions. By contrast a high level of safety was exhibited to A. rhopalosiphi and C. carnea at x3.5 and x5 the field rate (Table 8). Adult survivorship and parasitism rate was unaffected at both rates for A. rhopalosiphi. In the lacewing test, fresh residues of x3.5 the field rate were harmless and at the x5 rate only slightly harmful. When aged for 7 days the x5 rate was harmless demonstrating short persistence.

0

10

20

30

40

50

60

70

80

90

100

Control

0.1 1 5 50 100

200

300

Dimeth

oate

%M

orta

lity

(cor

rect

ed)

Rate of Electis g product/ha

Figure 1. Effect of Electis on the mortality of the predatory mite Typhlodromus pyri, under extended laboratory conditions. Field studies The effects of Electis to natural populations of the predatory mite T. pyri were investigated in vines in Germany (Figure 2). Two treatment regimes were investigated where four or six applications were made. Initial mite populations were good with greater than 25 mites per leaf in all plots before treatment. During the study predator levels declined in all treatments including the control to around two to three per leaf by August. Four applications of Electis per season caused a maximum reduction of 48% compared to the control and mite population recovered by the end of the season. A higher level of effect was seen with six applications. A maximum reduction of 89% was observed 7 weeks after the final application. However sampling in the following year revealed that this level of residual population was sufficient to

53

give rise to a mite density on the treated vines comparable to the control and no lasting adverse effects were anticipated. Dithianon was applied six times and the safety of this product was confirmed. Table 8. Effects of Electis (83 g zoxamide and 667g mancozeb/Kg WG formulation) to Aphidius rhopalosiphi and Chrysoperla carnea in tier II extended laboratory tests.

Test species


P/R/F (% reduction)

IOBC Class

Electis 6400g/ha 0.0 --- 13.8 (-22.5%) 1

Electis 12600g/ha 6.7 --- 10.7 (4.8%) 1

Dimethoate EC 90* --- N/A

Aphi

dius

rh

opal

osip

hi

0DA

A

Control 0.0 --- 11.3

Electis 6400g/ha 20 13 29.4 (-32%) 1

Electis 12600g/ha 35* 30 Not measured 2

Dimethoate EC 80* 78 N/A 0DA

A

Control 8.0 --- 22.2

Electis 1800g/ha 24 9.0 29.5 (-4.2%) 1

Electis 3600g/ha 18 3.0 25 (11.7%) 1 Chr

ysop

erla

car

nea

7DA

A

Control 16 --- 28.3

Notes: * Indicates significant difference from control (P = 0.05). M=Mortality, Corr. M=Corrected mortality, P=parasitism as mummies/female, R= fecundity as eggs/female.

The changes in mite populations observed in the series of trials to investigate the effect of number and timing of Electis sprays are presented in Figures 3, 4 and 5 for Germany (T. pyri), Spain (Euseius stipulatus) and Italy (K. aberrans) respectively. In the T. pyri trial mite populations were initially between four and six mites per leaf in all treatments and these remained relatively constant throughout the trial period. The toxic reference caused harmful effects (93% Abbott corrected mortality). When Electis was applied twice per season maximum reductions of 41, 34 and 35% were observed for the early, mid and late season sprays. In the four application regime, 22% was seen for the early season combination and 27% for the late season combination. By the end of the study (28 days after the final application) all Electis treatments were below 25% effect.

The number of mites per leaf in the trial on E. stipulatus was low with typically one or fewer mites per leaf in all observations. However during the trial the population demonstrated a steady increase in numbers and mite abundance remained stable allowing for meaningful comparisons to be made. The application of the toxic reference caused a reduction in mite numbers of 96% (7DAA4). The two early season applications of Electis caused no reduction in mites through the study. The maximum effect for Electis applied twice mid-season was 28% (7DAA6) and 8% late season (7DAA6). Similar levels of selectivity were seen in the four spray regimes. The early season combination gave a maximum effect of 11% (7DAA6)

54

and the late season combination a maximum effect of 25% (7DAA6). At the end of the study, all Electis treated plots were below the 25% effect threshold for harmlessness.

K. aberrans numbers increased from an average of 5 mites per leaf to between 17 and 26 per leaf at a mid season peak (8DAA4). From this point mite numbers slowly declined with good numbers throughout the trial. The toxic reference performed as expected with 98% effect noted at 8DAA6. The safe reference, dimethomorph remained similar to the control throughout the test and a maximum reduction of only 18% was recorded at the end of the study. Electis applied twice either early or late season gave a maximum reduction in mite numbers of 22% (7DAA2 and 35DAA6 respectively). Electis applied twice mid-season caused a maximum effect of 35% reduction (35DAA6). Both four spray combinations of Electis caused 41% maximum effect at 8DAA6. At the end of the test Electis applied twice in the early and late timings were below 25% effect. The other Electis treatments were above 25% but below 50%. This level is still considered selective for the predatory mites but suggests that applications made mid-season (during flowering) could be more disruptive than those made either before or after flowering for this species. However these observations should be treated with caution as they are based on the findings of only one study.

0

5

10

15

20

25

30

35

Pre-trt

9DAA1

6DAA2

7DAA3

7DAA4

7DAA5

7DAA6

28DAA6

78DAA6

May yr 2

June yr 2

No.

mite

s/le

af

Control

Electis 4 apps

Electis 6 apps

Dithianon 6 apps

Figure 2. Effects of four and six application of Electis to the predatory mite Typhlodromus pyri under field conditions in vines in Germany. DAA = number of days after application. Conclusions It was concluded that zoxamide was highly selective to all beneficial insect, mite and spider species tested in tier I laboratory studies. When tested under identical conditions Electis (zoxamide + mancozeb) was safe to all species with the exception of the predatory mites T. pyri and A. andersoni. Further studies under extended laboratory conditions confirmed the potential toxicity to mites and revealed safety to parasitic wasps and lacewing larvae. The effect on mites was clearly due to the presence of mancozeb in the formulation. Under field

55

0

1

2

3

4

5

6

7

Pre-trt 7DAA2 7DAA4 7DAA6 28DAA6

No.

mite

s/le

af

Electis 1+2

Electis 3+4

Electis 5+6

Electis 1+2+3+4

Electis 3+4+5+6

Untreated

Methamidathion 40EC

Figure 3. Effects of two and four application of Electis to the predatory mite Typhlodromus pyri under field conditions in vines in Germany. DAA = number of days after application. See text for explanation of rates.

0

0.2

0.4

0.6

0.8

1

1.2

7DAA2 7DAA4 7DAA6 23DAA6 36DAA6

No.

mite

s/le

af

Electis 1+2

Electis 3+4

Electis 5+6

Electis 1+2+3+4

Electis 3+4+5+6

Dimethomorph 150SL

Methamidathion 20EC

Figure 4. Effects of two and four application of Electis to the predatory mite Euseius stipulatus under field conditions in vines in Spain. DAA = number of days after application. See text for explanation of rates. conditions multiple applications of Electis on predatory mites in vines was studied. When applied at field rate, four applications were shown to be suitably selective to T. pyri (less than

56

50% maximum effect over the season). Six applications at field rate caused significant reductions in mite numbers but recovery was observed the following season. Further studies under different climatic and growing conditions with a range of important predatory mite species in key locations in Europe confirmed that up to four applications per season of Electis was suitably selective. Furthermore Electis could be integrated into disease control programmes at a variety of spray timings giving the grower excellent flexibility, disease control and selectivity to predatory mites and other beneficial arthropods.

0

5

10

15

20

25

30

Pre-trt 7DAA2 8DAA4 8DAA6 22DAA6 35DAA6

No.

mite

s/le

af

Electis 1+2

Electis 3+4

Electis 5+6

Electis 1+2+3+4

Electis 3+4+5+6

Dimethomorph500WP

Methamidathion20EC

Untreated

Figure 5. Effects of two and four application of Electis to the predatory mite Kampimodromus aberrans under field conditions in vines in Italy. DAA = number of days after application. See text for explanation of rates. Acknowledgements The authors would like to recognise and thank the following Dow AgroSciences co-workers for their valuable contributions to this manuscript; L. Bacci, J. Becker, A. Duriati and M. Garcia. References Abbott, W.S. 1925: A method of computing the effectiveness of an insecticide. – J. Econ.

Entomol., 18: 265-267. BBA, 1991: BBA Richtlinienvorschlag für die Prüfung von Pflanzenschutzmitteln im

Zulassungsverfahren Teil VI: 23-2.3.4. Auswirkungen von Pflanzenschutzmitteln auf Raubmilben im Weinbau.

Blümel, S; Baier, B; Bakker, F; Brown, K; Candolfi, M; Goßmann, A; Grimm, C; Jäckel, B.; Nienstedt, K; Schirra, K.J; Ufer, A.; Waltersdorfer, A. 2000a: Laboratory residual contact test with the predatory mite Typhlodromus pyri Scheuten (Acari: Phytoseiidae) for regulatory testing of plant protection products. – In: Guidelines to evaluate side-

57

effects of plant protection products to non-target arthropods. IOBC/WPRS; Gent, Belgium: 121-143 (ISBN 92-9067-129-7).

Blümel, S., Aldershof, S., Bakker, F., Baier, B., Boller, E, Brown, K, Bylemans, D., Candolfi, M.P., Huber, B., Linder, C. Louis, F. Müther, J., Nienstedt, K.M., Oberwalder, C., Reber, B., Schirra, K.J., Ufer, A. and & Vogt, H. 2000b: Guidance document to detect side effects of plant protection products on predatory mites (Acari: Phytoseiidae) under field conditions: vineyards and orchards. In: Guidelines to evaluate side-effects of plant protection products to non-target arthropods. IOBC/WPRS; Gent, Belgium: 145-158 (ISBN 92-9067-129-7).

Bigler, F. 1988: A laboratory method for testing side-effects of pesticides on larvae of the green lacewing, Chrysoperla carnea Steph. (Neuroptera, Chrysopidae). – IOBC/wprs Bulletin 11(4): 71-77.

Hassan, S.A. 1992: Guidelines for testing the effects of pesticides on beneficial organisms: Description of test methods. – IOBC/wprs Bulletin 15(3): 1-3.

Heimbach, U. 1992: Laboratory method to test effects of pesticides on Poecilus cupreus (Coleoptera, Carabidae). – IOBC/wprs Bulletin 15(3): 103-109.

Mead-Briggs, M. 1992: A laboratory method for evaluating the side-effects of pesticides on the cereal aphid parasitoid Aphidius rhopalosiphi (DeStephani-Perez). – Aspects of Applied Biology 31: 179-189.

Mead-Briggs, M., Longley, M. 2000: An extended laboratory test for evaluating the effects of plant protection products on the parasitic wasp, Aphidius rhopalosiphi (DeStephani-Perez) (Hymenoptera: Braconidae). – Unpublished draft method, 12th January 2000.

Overmeer, W.P.J. 1988: Laboratory method for testing side-effects of pesticides on the predacious mites Typhlodromus pyri and Amblyseius potentillae (Acarina: Phytoseiidae). – IOBC/wprs Bulletin 11(4): 65-69.

Polgar, L. 1988: Guideline for testing the effects of pesticides on Aphidius matricariae Hal. Hym., Aphidiidae: Laboratory contact tests: 1-on adults, 2-on aphid mummies, semi-field test on adults. – IOBC/wprs Bulletin 11(4): 29-34.

Stäubli, A., Pasquier, D. 1988: Méthode de laboratoire pour tester I’action secondaire des pesticides sur Anthocoris nemoralis F. (Anthocoridae:Heteroptera). – IOBC/wprs Bulletin 11(4): 91-97.

Vogt, H., Bigler, F., Brown, K., Candolfi, M.P., Kemmeter, F., Kühner, Ch., Moll, M., Travis, A., Ufer, A., Viñuela, E., Waldburger, M. and Waltersdorfer, A. 2000: Laboratory method to test effects of plant protection products on larvae of Chrysoperla carnea (Neuroptera: Chrysopidae). – In: Guidelines to evaluate side-effects of plant protection products to non-target arthropods. IOBC/WPRS, Gent, Belgium: 27-44 (ISBN 92-9067-129-7).

Wehling, A., Heimbach, U. 1994: BBA Richtlinienvorschlag für die Prüfung von Pflanzen-schutzmitteln im Zulassungsverfahren Teil VI: 23-2.1.9. Auswirkungen von Pflanzen-schutzmitteln auf Spinnen der Gattung Pardosa (Araneae, Lycoside) im Laboratorium.


pp. 59 - 66

59

Development of an extended-laboratory method to test bait insecticides

Pilar Medina, Isabel Pérez, Flor Budia, Angeles Adán & Elisa Viñuela Unidad de Protección de Cultivos. Escuela Técnica Superior de Ingenieros Agrónomos. Ciudad Universitaria, s/n. 28040. Madrid. Spain. Abstract: The Mediterranean fruit fly, Ceratitis capitata (Wied.) is a major agricultural pest in temperate and subtropical regions worldwide. In order to control the medfly in countries where it is already established, bait spray mixtures, using malathion as insecticide applied by ground and/or air are regularly used. Because of malathion environmental impact, several materials with insecticidal properties in conjunction with a bait attractant have been tested against medfly. Moreover, there is an increasing concern about testing these formulations also on beneficial insects, because the potential impact of large-scale applications of malathion alternatives on the structure of insect communities is not so well known. We have tried to contribute with our experiences to the design of a methodology to evaluate bait insecticides in laboratory, taking into account that the method should be useful to test the effects not only on pests, but also on natural enemies of importance in crops. Initially, several experiments were done to evaluate the suitability of the method and, finally, the novel bait formulation Spinosad GF-120 and a mixture of malathion plus hydrolyzate proteins were tested on the main citrus pest: Ceratitis capitata and one coccinellid of importance in this crop to control Icerya purchasi: Rodolia cardinalis. We conclude that Spinosad GF-120 is a promising alternative to malathion for the control of the medfly and it is not harmful for the studied coccinellid. Concerning the methodology, we stress the importance of adding a source of alternative food to the bait. Key-words: bait formulation, Spinosad GF-120, Ceratitis capitata, Rodolia cardinalis. Introduction The Mediterranean fruit fly, Ceratitis capitata (Wied.) is a major agricultural pest in temperate and subtropical regions worldwide, attacking over 200 varieties of cultivated fruit crops, more than 100 of economic importance (Christenson & Foote, 1960). Although probably originated in the eastern subsahara region (Hancock, 1989), within the last hundred years has spread throughout much of the world, as far away as most of Mediterranean countries where it is established from many years ago, South and Central America, the Hawaiian islands and Australia.

In order to control the medfly in countries where it is already established, bait spray mixtures applied by ground and/or air are regularly used. Bait applications involve the use of an insecticide (malathion) and a bait (commonly, hydrolyzate proteins) to attract male and female adult medflies. However, public concerns over property damage, environmental impact and public health caused by malathion led to the immediate need and acceptability of alternative pesticide/bait combinations, because, up to date, malathion is overused. Even though malathion is considered to be one of the safest organophosphate insecticides, and it has been widely studied in a variety of systems, there is still some controversy over malathion mutagenic and/or genotoxic potential for humans (Pluth et al., 1996). Moreover, malathion has harmful effects on bees and many natural enemies of pest insects (Troetschler, 1983; Gary & Mussen, 1984; Messing et al., 1995).

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Although there is a need to obtain final conclusions about the insecticidal effect of a new compound, field assays are too costly to be carried out as preliminary test. As such, we have tried to design a method to evaluate bait formulations in laboratory, that includes the possibility of assessing jointly pest and natural enemies if necessary. Although bait formulations are reported to have little direct impact on populations of fruit fly enemies the potential environmental consequences of insecticide-based baits should be carefully evaluated since beneficials can consume sugar-protein baits. The natural enemy selected for these experiments is of recognised importance and tradition in biological control of the citrus pest, Icerya purchasi Maskell. Rodolia cardinalis (Mulsant) is a small beetle specific to the cottony cushion scale. The introduction of Rodolia is considered to be the beginning of classical biological control and one example of successful results in this research (Caltagirone & Doutt, 1989).

Several materials with insecticidal properties in conjunction with a bait attractant have been tested against the medfly, being the most promising the photoactive dye phloxine B and the alternative fruit fly bait “GF-120” that utilizes spinosad as an active ingredient (Peck & McQuate, 2000). Spinosad is a combination of spinosyns A and B, which are naturally occurring compounds derived from soil-dwelling bacteria. Initial tests in laboratory and field indicate reasonable efficacy of spinosad (applied with or without bait) against medflies (Adán, 1996; King & Hennessey, 1996; Burns et al.,2001) and GF-120 bait appeared more benign overall to non-target insects (Michaud, 2003). Thus, an experiment to evaluate the novel formulation spinosad GF-120 was carried out using the methodology previously tested. Material and methods Insect rearing The flies were from a colony which has been maintained in our laboratory (25±2 ºC; 75±5% r.h.; l6:8 (L:D) photoperiod) and for years without contact with insecticides. A mixture of sucrose and enzymatic autolyzed brewer´s yeast ((4+1) by weight) was used as adult food. Adults of R. cardinalis were collected in Huelva crops (southern Spain) by Agrofresas, S.A. They were fed on an agar-honey based diet.

Chemicals Spinosad GF-120® (0.02% spinosyns A and D; sugars and attractants, DowAgrosciences) at 20 mg a.i./l; Malafin 50® (malathion 50%, EC, Agrodan) at 2.500 mg a.i./l and Biocebo® (protein hydrolyzates, 30%, SL, Bioibérica) at 1500 mg a.i./l, were used in our case study. Concentrations tested are the currently used in citrus tree in Spain for bait formulations. In the case of Spinosad GF-120 we followed DowAgrosciences recommendations because this compound is still being registered to be used in citrus crops.

Experiments Basic method design. Initially, this method was designed to test bait formulations on the medfly. It was also used to test pesticide effects on the selected beneficial once its validity in C. capitata was confirmed.

Orange tree leaves (cv. Navelina) were collected from small trees grown in a greenhouse in Madrid and taken to the laboratory. Five droplets of 5 µl of the corresponding compound were at random distributed over each leaf with a micropipete. The treated leaf was transferred into a plastic cage (11 cm in diameter by 5 cm high), with a lid having a 5 cm diameter hole on the upper side for ventilation. The petiolo of each leaf was introduced into an eppendorf® tube containing a nutritive solution as described by Moutous & Fos (1973) to keep the leave turgidity during the experiments. Eppendorf® tube was fixed to the bottom of the cage with

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Plasticine®. Once droplets were dry, ten adults were introduced per replicate. Water was always offered ad libitum to adults in small glass vials covered with “Parafilm®” and a piece of “Spontex®” wiper. Water is a indispensable constituent for the utilization of other nutrients. At least, six replicates per concentration were done.

Mortality was evaluated at 24, 48 and 72 hours after exposure to the insecticide. It was considered more than one day of assessment to obtain more conclusions even though C. capitata adults should be killed as soon as possible by an insecticide to become a serious alternative to malathion. Trial 1: Suitability of controls in a no-choice test.

As it was recommended by insecticide suppliers to potentiate bait attraction, not any extra food was added. First, we tested the basic method design described in the former paragraph using only a bait control (droplets only contained 1.500 ppm of bait) that was compared to the so-called rearing control and water control in table 1. Five droplets of distilled water plus the artificial diet normally used to feed flies in laboratory (see insect rearing paragraph) added at the bottom of the cage in a recipient was the rearing control. Water controls only contained the troughs with water as drinking place, without extra food. Trial 2: Investigating possible bait toxicity.

From the results obtained we inferred that C. capitata died either by bait toxicity and/or by starvation. First, we tested the potential bait toxicity, trying a range of concentrations, with or without an alternative food (the same used in the rearing). Food was always added separately from bait, in a small recipient kept at the bottom of the cage as described above for rearing control. Trial 3: The role of sucrose in Ceratitis feeding. As proteins needed by C. capitata can be supplied by the bait, we designed a trial to verify the role of the addition of sucrose in the diet. We compared the cumulative mortality in three days when medflies fed on the common diet used for rearing, only bait and bait plus sugar ad libitum supplied in a small recipient kept at the bottom of the cage. To simplify the assay, bait was applied dissolved in the drinking water at the concentration of 1500 mg a.i./l. Trial 4: How to carry out the assays in extended-laboratory method: Case study.

Five males and five females of Ceratitis capitata <24-h-old (never fed before), and 10 adults of the beneficial insect, Rodolia cardinalis (less than one month old) were used per replicate in experiments. Assays were performed as described in the basic method design, and as concluded from former experiments, enough food for feeding insects three days was provided in a small recipient, not mixed with the bait+insecticide. Food used was the previously described for rearing. Five droplets of bait per replicate were used as a control.

Statistical analysis Results, presented as means±S.D. of untransformed data were analysed by one-way analysis of variance (ANOVA) using Statgraphics (STSC, 1987). Means were separated by a least significant difference (LSD) multiple range test (P<0.05). When premises of ANOVA were violated, even after a transformation to arcSen√x, data were subjected to the non-parametric test Kruskal-Wallis. Results and discussion Adults were observed to feed on the bait droplets at all experiments, with no apparent repellency irrespective of the insecticide used, confirming the bait attractive ability. The phagostimulatory properties of protein hydrolizates as well as the olfatory responses of flies to them (Galun et al., 1985) were conceived by Steiner (1952) who first used them successfully as poisoned baits for fruit fly control.

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In the first trial, mean percentages of adult medflies that were able to survive with different controls are presented in Table 1. Surprisingly, bait control showed a high pattern of mortality, with 75% of flies died at 48h, whereas rearing control behaves as expected. The percentage of flies died with water control was higher than 82% at 48 h, and reached 100% at the third day, as it was shown for the olive tephritid Bactrocera oleae (Gmelin) adults reared with water only (Tsitsipis et Voyatzoglou, unpublished data, cited by Tsitsipis, 1989). From the results obtained we inferred that C. capitata died by bait toxicity and/or starvation. Table 1. Mean percentages (±S.D.) of adult medfly cumulative mortality in a test carried out to establish the suitability of controls.

Cumulative mortality (%) 24 h1 48 h2 72 h3

Bait control (1500 mg a.i./l) 20.0±4.0 b 75.0±5.0 b 100 b Water control 22.5±4.7 b 82.5±4.7 b 100 b

Rearing control 0 a 5.0±2.8 a 10.0±4.0 a Within the same column, data followed by the same letter did not differ significantly. 1F=34.13; df=2,9; P<0.001. 2F=53.55; df=2,9; P<0.001. 3K=10.50; P=0.005.

Although very unlikely, a second trial was carried out to rule out an hypothetical bait toxicity. As shown in Table 2, not any mortality could be detected irrespective of the bait concentration, when an alternative food was added in the same cage, not mixed with the bait. This confirms that bait is only an attractant and, although rich in proteins, cannot be used as a substitute for food. Table 2. Cumulative mortality of adult medflies after ingestion of different concentrations of bait, alone or with an alternative food added, not mixed with the bait.

Cumulative mortality (%) Compounds (active ingredient)

N 24 h1 48 h2 72 h3

Water control 10 0.0±0.0a 31.0±6.0b 94.0±2.7b Rearing control 10 0.0±0.0a 1.0±1.0a 1.0±1.0a 500 ppm bait 6 0.0±0.0a 23.3±4.9b 85.0±4.3b 750 ppm bait 6 1.7±1.7a 28.3±8.3b 85.0±4.3b 1500 ppm bait 6 0.0±0.0a 15.0±5.6b 90.0±5.2cd 500 ppm bait + food 6 0.0±0.0a 0.0±0.0b 0.0±0.0a 750 ppm bait + food 6 0.0±0.0a 0.0±0.0a 1.6±1.6a 1500 ppm bait + food 6 0.0±0.0a 0.0±0.0a 0.0±0.0a Within the same column, data followed by the same letter did not differ significantly 1K=8.33; P=0.30. 2K=40.44; P<0.001. 3K=48.06; P<0.001. N= number of replicates.

Protein hydrolyzates are a good source for vitamins and minerals, beside being rich in amino acids. The main components that are found in diets are: water, a carbohydrate source

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(sucrose) and a protein source, either unhydrolyzed, i.e. brewer´s yeast, or hydrolyzed, or both. The protein source contains also mineral salts and vitamins (Tsitsipis, 1989). In trial 3, we have shown that to assure the survival of C. capitata for more than 24 hours after emergence is essential to supply them sucrose (Table 3). The lack of sugar in diet caused direct mortality from 48 h onwards, affecting males and females in the same way. On the other hand, once sugar is supplied, the influence of the protein concentration (or bait) makes no difference on mortality (unpublished data). As a conclusion from all, C. capitata adults died of starvation in our first trial. In field, they utilize various sources such as fungus, bird droppings, pollen, etc. Because of their inherent needs, fruit flies are higly attracted to high quality protein and sugar baits.

In assays performed with beneficials we have to take into account that baits are developed to provide an attractant to Ceratitis, but we do not know about the toxicity of them in natural enemies. Michaud (2003) found that Nu-lure bait without malathion also caused significant mortality to Chrysoperla rufilabris Burmeister after 24 hours in cage trials and a similar pattern of results where obtained with the syrphid Peudodorus clavatus (F.). No extra food was supplied in both cases. Table 3. Influence of the addition of sucrose in the diet of Ceratitis capitata

Cumulative mortality (%) Compounds (active ingredient)

N 24 h1 48 h2 72 h3

Rearing control 10 0.0±0.0a 1.0±1.0a 5.0±1.6a 1500 ppm bait 10 1.0±1.0a 26.0±3.7b 89.0±4.6b 1500 ppm bait + sucrose 10 1.0±1.0a 8.0±3.2a 10.0±3.3a

Within the same column, data followed by the same letter did not differ significantly 1F=0.50; df=2,27; P=0.61. 2F=24.04; df=2,27 P<0.001. 3F=83.07; df=2,27; P<0.001. N= number of replicates.

A new question to think about was that flies might be more attracted by food than bait and this effect could distort the real toxicity of the insecticide. Insecticides that are very toxic to adults of C. capitata, such as malathion, killed flies in less than 24 hours, so it is irrelevant if alternative food is provided or not. Moreover, an insecticide that does not have a fast knock down effect on flies cannot be an alternative to the currently proposed insecticides, such as organophosphates. However, it is necessary to design a test that gives us information, not only about the pest, but also about natural enemies that can be affected by the treatment. In that particular case, more days of study are needed and an alternative food becomes a question of importance.

The conclusions we obtained from all preliminary series of trials were: – Supplying of food is necessary, even though the real effect of the insecticide is diminished

because the power of attraction of the alternative source of food. This can be very high and the number of times that insect goes to the bait is considerable reduced. In field, there is also different sources of food. As such, the ability of the bait as attractant is nearly as important as the insecticide.

– More than 24 hours of assessment are necessary to evaluate the effect on beneficials. – Positive controls give a good idea of the real efficacy of the novel insecticide tested. – Bait control is always necessary.

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We were able to verify our method design in our last experiment (Table 4). Spinosad GF-120 is very toxic to C. capitata at very low doses in comparison with those used with malathion, 125 times higher. One day after treatment, the organophosphates showed higher toxicity, killing more than 95% of medflies, whereas spinosad only killed 66% at the highest concentration tested. Nevertheless, there is a trend of reaching similar percentages of mortality with the time. King & Hennessey (1996) reported that effective concentration of spinosad in ppm to kill 99% of females was 23.8 and 9.4 at 24 and 48 h respectively. These relative lower values they obtained in comparison with results of Spinosad GF-120 can be explained taking into account that spinosad was combined with a sugar-yeast hydrolysate mixture and tested as a bait spray in a no-choice test. This confirms the importance of the test design to explain the results. Table 4. Mean percentages (±S.D.) of Ceratitis capitata and Rodolia cardinalis killed by Spinosad GF-120 at 20 mg a.i./l in comparison with the positive control malathion bait.

Cumulative mortality (%) Compounds N 24 h1 48 h2 72 h3

Ceratitis capitata Bait control 5 2.0 ± 2.0 a 2.0 ± 2.0 a 4.0 ± 2.4 a Spinosad GF-120 5 66.0 ± 8.1 b 82.0 ± 3.7 b 94.0 ± 2.4 bc Malathion baita 5 96.0 ± 2.4 c 100.0 ± 0.0 c 100.0 ± 0.0 c Rodolia cardinalis Bait control 5 4.0 ± 2.4 a 6.0 ± 4.0 a 14.0 ± 6.8 a Spinosad GF-120 7 7.1 ± 2.8 a 10.0 ± 2.0 a 22.8 ± 6.4 a Malathion baita 4 35.0 ± 8.6 b 47.5 ± 6.2 b 77.5 ± 8.5 b Within the same column and species, data followed by the same letter did not differ significantly. Ceratitis capitata: 1F=78.5; df=2,12 P<0.001. 2F=275.98; df= 2,12; P<0.001. 3F=147,99; df=2,12; P<0.001. Rodolia cardinalis: 1F=8,27; df=2,13; P=0.005. 2F=15.55; df=2,13; P<0.001. 3F=13.50; df=2,13; P<0.001. aMalathion (2500 mg a.i./l) was mixed with Biocebo® at 1.500 mg a.i./l. to be applied as a bait formulation.

Concerning the natural enemy tested, spinosad caused much fewer mortality than in the pest. Twenty three per cent of adults died 72 hours after treatment compared to 94% in C. capitata. This result is consistent with those of Michaud (2003), who reported GF-120 caused no significant mortality to any coccinellid (Curinus coeruleus Mulsant, Cycloneda sanguinea L., Exochomus childreni Mulsant, Harmonia axyridis Pallas, Olla v-nigrum Mulsant, Scymnus sp.) in either choice or no-choice situations, despite considerable consumption of baits. Malathion was very harmful 3 days after treatment. Spinosad, in its first formulation (without bait), when used according to good agricultural-horticultural practices, has been shown to be also compatible with the coccinellids Hippodamia convergens Guerin and Coccinella septempunctata L. evaluated at the stages commercially available for biological control (Miles and Dutton, 2000). However, safety profile of spinosad is not so clear for some predators (Viñuela et al., 1998; Cisneros et al., 2002) and many parasitoids (Elzen et al., 1999; 2000; Pietrantonio and Benedict, 1999; Ruberson and Tillman, 1999, Williams et al., 2003) and an important effort in researching the selectivity of this new formulation of the

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compound has to be done. Pollinators are highly sensitive to spinosad via oral and contact exposure routes, although the new formulation is not so risky (Rendon et al., 2000) because applications can be made when bee activity is low and they are allowed to enter such areas only after spray deposits have dried In addition, the bait formulation contains ammonium acetate, a compound that attracts medfly but repels honey bees. Acknowledgements We thank Roni Parias (Agrofresas S.A.) for collecting Rodolia cardinalis in open fields. Funding for these tests was provided, in part, by Dow Agrosciences. We are grateful to the company for supplying of Spinosad GF-120. References

Adán, A., Del Estal, P., Budia, F., González, M. & Viñuela, E. 1996: Laboratory evaluation of the novel naturally derived compound spinosad against Ceratitis capitata. – Pestic. Science 48: 261-268.

Burns, R.E., Harris, D.L, Moreno, D.S. & Eger, J.E. 2001: Efficacy of sponosad bait sprays to control mediterranean and caribbean fruit flies (Diptera: Tephritidae) in commercial citrus in Florida. – Florida Entomol. 84: 672-678.

Caltagirone, L.E. & Doutt, R.L. 1989: The history of the vedalia beetle importation to California and its impact on the development of biological control. – Annu. Review. Entomol. 34: 1-16.

Christenson, L.D. & Foote, R.D. 1960: Biology of fruit flies. – Annu. Review Entomol. 5: 171-191.

Cisneros, J., Goulson, D., Derwent, L. C., Penagos, D. I., Hernández, O. & Williams, T. 2002. Toxic effect of spinosad on predatory insect. – Biological Control 23:156-163.

Elzen, G.W., Maldonado, S.N. & Rojas, M.G. 2000. Lethal and sublethal effects of selected insecticides and an insect growth regulator on the boll weevil (Coleoptera: Curculi-onidae) ectoparasitoid Catolaccus grandis (Hymenoptera: Pteromalidae). – J. Econ. Entomol. 93: 300-303.

Elzen, G.W, Rojas, M.G., Elzen, P.J., King, E.G. & Barcenas, N.M. 1999. Toxicological responses of the Boll Weevil (Coleoptera: Curculionidae) ectoparasitoid Catolaccus grandis (Hymenoptera: Pteromalidae) to selected insecticides. – J. Econ. Entomol. 92: 309-313.

Galun, R., Gothilf, S., Blondheim, S. Sharp, J.L., Mazor, M. & Lachman, A. 1985: Olfatory and gustatory responses of normal and irradiated fruit flies Ceratitis capitata (Wied.) (Diptera: Tephritidae) and Anastrepha ludens (Loew) to nutrients. – Environ. Entomol. 14: 726-732.

Gary, N.E. & Mussen, E.C. 1984: Impact of mediterranean fruit fly malathion bait sprays on honey bees. – Environ. Entomol. 13: 711-717.

Hancock, D.L. 1989: Pest status. Southern Africa. – In: Robinson & Hooper (eds.). World Crop Pests. Fruit flies. Their biology, natural enemies and control, Vol. 3A: 51-57.

King, J.R. & Hennessey, M.K. 1996: Spinosad bait for the caribbean fruit fly (Diptera:Tephritidae). – Florida Entomol. 79: 526-531.

Messing, R.H., Asquith, A. & Stark, J.D. 1995: Effects of malathion bait sprays on nontarget insects associated with corn in Western Kanai, Hawai. – J. Agric. Entomol. 12: 225-265.

Michaud, J.P. 2003: Toxicity of fruit fly baits to beneficial insects in citrus. – J. Insect Sci. 3(8): 9 pp.

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Miles, M, & Dutton, R. 2000: Spinosad-A naturally derived insect control agent with potential for use in glasshouse integrated pest management systems. – Med. Fac. Landbouww. Univ. Gent 65: 393-400.

Moutous, G. & Fos, A. 1973: Essais de rhizogénèse chez la feuille de vigne isolée. –Revue de Zoologie Agricole et de Pathologie végétale. Premier trimestre. 1973: 27-28.

Peck, S.L. & McQuate, G.T. 2000: Field test of environmentally friendly malathion replacements to supress wild mediterranean fruit fly (Diptera: Tephritidae) populations. – J. Econ. Entomol. 93: 280-289.

Pietrantonio, P.V. & Benedict, J.H. 1999. Effect of new cotton insecticide chemistries, tebufenozide, spinosad and chlorfenapyr, on Orius insidiosus and two Cotesia species. – Southwestern Entomol. 24: 21-29.

Pluth, J., Nicklas, J., O´Neill, P. & Albertini, R. 1996: Increased frequency of specific genomic deletions resulting from malathion exposure. – Cancer Research 65: 2393-2399.

Rendon, P.A., Jeronimo, F., Ibarra, J. & Alvarez, V.C. 2000: Effectiveness of Success 0.02 CBTM for the control of fruit flies and its effect on bees, Apis mellifera L. – USDA, APHIS, PPQ, Methods Development Station, Guatemala.

Ruberson, J.R. & Tillman, P.G. 1999. Effect of selected insecticides on natural enemies in cotton: laboratory studies. – Proceedings of the Beltwide Cotton Conference, Vol. 2. National Cotton Council. Memphis TN: 1210-1213.

Steiner, L.F. 1952. Fruit control in Hawaii with poison-bait sprays containing protein hydro-lyzates. – J. Econ. Entomol. 45: 838-843.

STSC, 1987: User’s Guide Statgraphics. – Graphic software system STSC Inc., Rockville, MD, USA.

Troestschler, R.G. 1983: Effects on nontarget arthropods of malathion bait sprays used in California to eradicate the Mediterranean fruit fly, Ceratitis capitata (Weidemann) (Diptera: Tephritidae). – Environ. Entomol.12: 1813-1822.

Tsitsipis, J.A. 1989: Nutrition. Requirements. – In: Robinson & Hooper (eds.). World Crop Pests. Fruit flies. Their biology, natural enemies and control, Vol. 3A: 103-119.

Viñuela, E., Adán, A., González, M., Budia, F., Smagghe, G., DeClerq, P., Vogt, H.& del Estal, P. 1998. Spinosad y azadiractina: efectos de dos plaguicidas de origen natural en el chinche depredador Podisus maculiventris (Say) (Hemíptera: Pentatomidae). – Bol. San. Veg. Plagas. 24: 57-66.

Williams, T., Valle, J. & Viñuela, E. 2003. Is the naturally derived insecticide Spinosad® compatible with insect natural enemies? – Biocontrol Sci. Tech. 13: 459-475.


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Side effects of surfactants and pesticides on entomopathogenic nematodes assessed using advanced IOBC guidelines Arne Peters1 & Delphine Poullot2

1 E-nema GmbH, Klausdorfer Str. 28-36, D-24223 Raisdorf, Germany 2 Enigma, Hameau de Saint Véran, F-84190 Beaumes de Venise, France Abstract: Some surfactants and pesticides were tested for their side effects on entomopathogenic nematodes (Steinernema feltiae and Heterorhabditis bacteriophora) using the guidelines proposed in 2002. In addition to the effect on viability and infectivity, the tests also considered the fecundity of the nematodes treated with the chemicals. The adjuvants BOND (Spieß-Urania ) and Schaumexx (Sudau Agro) had no side effects on S. feltiae while a minor effect on the infectivity (40% less morality to mealworms) and the fecundity (46% less offspring per infected mealworm) was observed for Adhäsit (Spieß-Urania). The most harmful surfactant was Li700 (Loveland Agrochemicals) resulting in 18.5% loss in viability, no depression of infectivity, but 68% loss in fecundity of S. feltiae when applied at twice the recommended application rate of 0.5%. Lower concentrations of Li700 were tested showing that 0.5% had no detrimental effect on nematode viability and infectivity but 60% less offspring was produced. When the concentration was lowered to 0.04% fecundity was not affected. For H. bacteriophora no effect on viability was observed with BOND and Schaumexx, whereas Li700 caused nematode mortality of 43% at 2-times the maximally recommended concentration of 0.5%. None of the insecticides tested (Imidaclopride, Spinosad, Fipronil and Chlorpyriphos) had a detectable effect on nematode viability. Only one formulation of Spinosad decreased nematode infectivity by 55% but only if added to the substrate not if added to the nematodes before they were applied to the substrate. With assessing fecundity the sensitivity of the test was improved. Fecundity of both nematode species was affected by at least one formulation of each insecticide ingredient. Key-words: Heterorhabditis, Steinernema, side effects, surfactants, insecticides Introduction Side effects of certain chemical pesticides on mortality and infectivity of entomopathogenic nematodes have been assessed by Rovesti et al. (1990). The advanced IOBC guidelines presented during the last IOBC meeting of the working group ”Pesticides and Beneficials”, Peters (2002) exposed a new testing scheme.

Two different levels of exposure are studied considering the mode of nematode use in practice. The first level of exposure corresponds to a short exposure in the spray tank, because nematodes are mostly applied with conventional spraying equipment and may be tank-mixed with pesticides. The second level is exposing nematodes in the soil to a foliar or a soil treatment, which corresponds to an infinite exposure in the substrate. Besides mortality and infectivity nematode fecundity is assessed in order to include all nematodes traits that are essential for successful pest control.

Side effects of four surfactants and four insecticides active ingredients were assessed following the advanced IOBC guidelines.

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Material and methods Four recently registered insecticides active ingredients were selected for this study: Spinosad, Imidacloprid, Fipronil and Chlorpyriphos. Table 1 and Table 2 present the characteristics of the formulated plant protection products and surfactants tested. Table 1: List of insecticides tested

Commercial name or code

Active ingredient a.i. Test Rates *

S01 Spinosad 0.02 % 3 L/ha S02 Spinosad 44.2 % 20 g/ha Schuß ® Fipronil 80 % 125 g/ha Confidor SL200® Imidacloprid 200 g/L 70 g/ha Gaucho ® Imidacloprid n.a. 0.75 g/L substrate SuSCon Green ® Chlorpyriphos n.a. 0.75 g/L substrate

n.a.: not applicable; * based on a water volume of 200 L/ha Table 2: List of surfactants tested

Commercial name or code

Active ingredient a.i. Concentration tested

w/w in water Li700® Propionic acid

Alcylphenyl-hydroxypolyoxy-ethylen

35% 10%

5% ; 1% and 0.04%

Bond® Latex Alkoxylated-Alcohol

45% 10%

0.2%

Adhäsit® Marlopon Methanol

10% 15%

0.2%

Schaumexx Polydisiloxan data not available 0.0024%

Two different species of entomopathogenic nematodes, currently commercially available and supplied by E-Nema, were tested: Steinernema feltiae Filipjev and Heterorhabditis bacteriophora Poinar. Tenebrio molitor was used as a model insect and in order to reduce the treatment variation for the infectivity assay (Vainio, 1992, Peters, 2000).

The protocol used was the same as described in the advanced guidelines proposed by Peters (2003). The insecticides were tested at Enigma whereas the surfactants were investigated at E-Nema. Mortality was assessed after a 24-hours exposure period of the nematodes to the pesticides. Infectivity and propagation assays were performed using nematodes previously exposed to pesticides and nematodes exposed to water from the control units.

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Mortality data were corrected using the Abbott formula:

100%100%%

% ×⎟⎠⎞

⎜⎝⎛

−−

=McMcMtMcorr

Where: Mcorr% = Corrected mortality, Mt% = Mortality in the treated unit, Mc%= Mortality in the control unit.

Infectivity was measured as a percentage of infected Tenebrio molitor larvae. The reduction of infectivity in the treated unit compared with the control was calculated using the following formula:

100%%1%inf ×⎟⎠⎞

⎜⎝⎛ −=

IcItR

Where: Rinf% = Reduction of infectivity of the treatment from the control, It% = Percentage of infectivity in the treated units, Ic% = Percentage of infectivity in the control units.

Propagation was measured as a number of offspring produced per infective juvenile. The reduction of propagation in the treated unit compared with the control was calculated using the following formula:

1001% ×⎟⎠⎞

⎜⎝⎛ −=

FcFtRfec

Where: Rfec% = Reduction of propagation of the treatment from the control, Ft = Production of offspring per insect in the treated units, Fc= Production of offspring per insect in the control units.

The global effect of the treatment is then assessed using the three different values collected and calculated as above:

)100()100()100(100% inf feccorr RRME −×−×−−=

The classes of toxicity levels are:

1 E<30% 2 E<80% 3 E<99% 4 E≥99%

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Results and discussion Table 3 and Table 4 present the results for each nematode species following the two levels of exposure studied:

Table 3: Impact of insecticides on Steinernema feltiae

Level of exposure

Insecticide Mc (%) Rinf (%) Rfec(%) E(%) Toxicity level

S01 2.36 0 39.74 41.16 2 S02 0 1.00 4.69 5.64 1 Schuss 10.78 18.00 37.63 54.37 2

Tank mix

Confidor 0 0 18.26 18.26 1 S01 0 55.44 11.84 60.72 2 S02 0 5.70 51.43 54.20 2 Schuss 0 5.70 47.75 50.73 2 Confidor 0 5.70 -8.70 5.70 1 Gaucho - -29.53 93.43 93.43 3

Substrate

SuSCon Green - 3.63 20.19 23.08 1

Table 4: Impact of insecticides on Heterorhabditis bacteriophora

Level of exposure

Insecticide Mc (%) Rinf (%) Rfec(%) E(%) Toxicity level

S01 0 -25.00 54.16 54.16 2 S02 0 16.00 4.56 19.83 1

Schuss 0 29.00 33.93 53.09 2 Tank mix

Confidor 0 31.00 1.57 32.08 2 S01 0 51.87 36.95 69.66 2 S02 0 -15.42 50.17 50.17 2

Schuss 0 -15.42 67.47 67.47 2 Confidor 0 -15.42 19.41 19.41 1 Gaucho - -63.09 99.55 99.55 3

Substrate

SuSCon Green - -18.23 60.99 60.99 2

No significant effect of the different insecticide tested could be observed on the viability of the nematodes of both species. Infectivity of both species was significantly reduced when nematodes were exposed to S01 in the substrate. This formulation of Spinosad is less concentrated in active ingredient, compared with S02 (44.2% of Spinosad), because it is a bait

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formulation (0.02% of Spinosad). However the rate applied was higher (3 l/ha) than S02 (20 g/ha) which caused no reduction of infectivity of the nematodes. The previous exposure of the nematodes to both Spinosad formulations had no significant effect on their infectivity. The reduction of infectivity observed when the nematodes are exposed to Spinosad once they are on the substrate with Tenebrio molitor can be explained by the perturbation of the searching behaviour of nematodes. This perturbation can be the result either of the action of the Spinosad on the chemo-receptors of the nematodes implicated in prey location or an action of the partially hydrolysed proteins contained in the bait formulation of Spinosad S01 which could saturate the chemo-receptors of the nematodes and prevent or reduce the host location. Some studies of possible effects of biotic and abiotic factors on host and penetration site location were realised and revealed the importance of these factors on the success of host location (Wang and Gaugler, 1999). Another interesting observation was the increase of infectivity of nematodes when they are exposed to Gaucho® (Imidacloprid). Indeed, some studies have demonstrated a synergistic effect of Imidacloprid on nematodes (Koppenhöfer and Kaya, 1998). This phenomenon was only observed for Gaucho® but not for the other formulation used. A nematode free control with insecticides and mealworms was not included in this study and synergetic interactions can therefore not be deducted. The testing scheme should be expanded to include such a nematode free control with the pesticde.

Table 5: Impact of surfactants on Steinernema feltiae and Heterorhabditis bacteriophora.

Surfactant Nematode Mc (%)

Rinf (%)

Rfec (%)

E (%)

Toxicity level

Adhäsit (0.2%) (tank mix) S. feltiae 0 40.22 46.57 68.06 2

Bond (0.2%) (tank mix) S. feltiae 0 0 0 0 1 Schaumexx (tank mix) S. feltiae 0 8.70 n.e. n.e. Adhäsit (0.2%) S. feltiae 0 0 0 0 1 Bond( 0.2%) S. feltiae 0 0 0 0 1 Bond (0.2%) (tank mix) H. bacteriophora 16.37 3.28 n.e. n.e. Shaumexx (tank mix) H. bacteriophora 0 29.51 n.e. n.e. Li-700 (5%) (tank mix) H. bacteriophora 47.28 14.75 n.e. n.e. Li-700 (1%) (tank mix) S. feltiae 8.06 0 68.00 70.58 2 Li-700 (0.04%) (tank mix) S. feltiae 0 0 0 0 1

Li-700 (1%) S. feltiae 8.06 0 55.00 58.63 2 Li-700 (0.04%) S. feltiae 0 0 82.70 82.70 3

n.e.: not evaluated

Fecundity was significantly reduced when nematodes where exposed to insecticides on the substrate. This reduction was observed for both species of nematodes after treatment with Fipronil (Schuss®), Imidacloprid (Gaucho®) or Spinosad (S02). The slow release granule containing Chlorpyriphos (SuSCon Green®) also had a significant effect on fecundity of Heterorhabditis bacteriophora. The effect on fecundity for some of the insecticides is

72

observed only for one of the formulation of the tested insecticide (e.g.: Spinosad, Imidaclopride). The difference in the effect of two formulations of the same insecticide is difficult to explain, but it has to be considered in future testing strategies, choosing not a single formulation of an insecticide for side effects tests. The effect of surfactants on S.feltiae and H.bacteriophora is listed in Table 5. The effect of Adhäsit, Bond and Schaumexx was marginal. For practical purposes, when nematodes are exposed only a few minutes to the chemicals, the impact on infectivity and fecundity will be even smaller. Li700 was more harmful to the nematodes. It is assumed that the propionic acid is responsible for this negative effect. The other component of Li700, Alcylphenyl-hydroxypolyoxy-ethylen has been used successfully for the application of S. feltiae to foliage (Wardlow et al., 2001). The effect was observed in nematode survival at the high concentration (5%) for H. bacteriophora). The lower concentrations had an impact only on the fecundity. Interestingly, even the low concentration (0.04%) still affected fecundity. In this assay the sand was moistened with the Li700 solution instead of water, which gives a higher concentration than in reality, where the spray hits a moistened soil. By including the fecundity in the assessment, the sensitivity of the test is improved. The propagation of nematodes is not always required to obtain good insect control. It will therefore always be necessary to list all single values for nematode mortality, infectivity and fecundity. By testing fecundity, an insight in possible long term effects of treatments on the entomopathogenic nematode populations in the soil is obtained. Entomopathogenic nematodes are common and widespread in many soil types (Sturhan, 1996). Little is known about their impact on insect populations but epizootics on some pest insects have been observed (Peters, 1996). Most interestingly, the granular formulation of imidacloprid had a siginificant effect on nematode fecundity. It remains to be tested, whether the common practice of treating seeds with imidacloprid effects nematode fecundity in the field. References Hara, A.H., Kaya, H.K. 1983. Toxicity of selected organophosphate and carbamate pesticides

to infective juveniles of the entomogenous nematode Neoplectana carpocapsae (Rhabditida: Steinernematidae). – Environmental Entomology. 12: 496-501.

Koppenhöfer, A.M., Kaya, H.K. 1998. Synergism of imidacloprid and on entomopathogenic nematode: a novel approach to white grub (Coleoptera: Scarabaeidae) control in turf-grass. – Journal of Economic Entomology 91(3): 618-623.

Peters, A. 1996. The natural host range of Steinernema and Heterorhabditis spp. and their impact on insect populations. – Biocontr. Sci. Technol. 6: 389-402.

Peters, A. 2000. Insect based assay for entomopathogenic nematode infectiousness: definitions, guidelines, problems. – IOBC/WPRS Bulletin 23(2): 109-114.

Peters, A. 2003. Pesticides and entomopathogenic nematodes – current status and future work. – IOBC/WPRS Bulletin 26(5): 107-110.

Sturhan, D. 1996. Studies on the natural accurence and distribution of entomopathogenic nematodes. – Russian Journal of Nematology 4: 98.

Vainio, A. 1992. Guideline for laboratory testing of the side-effects of pesticides on entomophagous nematodes Steinernema spp. – IOBC/WPRS Bulletin 15: 145-147.

Wang, Y., Gaugler, G. 1999. Host and penetration site location by entomopathogenic nema-todes against Japanese beetle larvae. – Journal of Invertebrate Pathology. 72: 313-318.

Wardlow, L.R., Piggott, S. and Goldsworthy, R. 2001. Foliar application of Steinernema feltiae for the control of flower thrips. – Med. Fac. Landbouww. Univ. Gent 66/2a: 285-291.


pp. 73 - 79

73

Influence of insecticide coated seeds on larvae of Poecilus cupreus (L.) (Coleoptera; Carabidae) using different container sizes and quantities of substrate Julia Heise1, Udo Heimbach1 & Stefan Schrader2

1 Federal Biological Research Centre for Agriculture and Forestry, Institute for Plant Protection in Arable Crops and Grassland, Messeweg 11/12, D-38104 Braunschweig, Germany

2 Federal Agricultural Research Centre, Institute of Agroecology, Bundesallee 50, D-38116 Braunschweig, Germany

Abstract: A laboratory study was conducted to detect lethal and sublethal effects of plant protection products applied as seed coating or granules on larvae of the carabid beetle Poecilus cupreus (L.) (Coleoptera; Carabidae). Larvae of laboratory bred beetles were exposed in a sandy soil to the pesticide carbosulfan coated on rape seeds. The applied rates ranged from 1 to 4 coated seeds in 4 different container sizes. The larvae were exposed to untreated seeds, the test item and a reference item, with at least 20 larvae per treatment group. Mortality effects of one coated seed ranged from 25 to 90 % depending on the surface area of the test containers used. The LC50 value was 172 seeds/m² irrespectively of container size. Control mortality varied between 0 and 10 %. In the case of similar container surfaces, different filling heights of substrate had no influence on the mortality rate. Sublethal effects of coated seeds such as on the hatching weight of adult beetles and their developmental period were detected but highly variable. Key-words: larvae of Poecilus cupreus, Carabidae, laboratory test method, side-effects, plant protection products, coated seeds, carbosulfan, container size Introduction Prior to the placing of plant protection products on the European market, the impact of plant protection products on non-target organisms has to be investigated (Council Directive 91/414/EEC). Carabid beetles occur in high densities with a large variety of species in agricultural crops. Therefore Poecilus cupreus (L.) (Coleoptera; Carabidae), a medium sized carabid beetle, which is encountered frequently on agricultural sites, has been selected as a relevant test species (SETAC guidance document, Barrett et al., 1994).

A laboratory method to evaluate lethal as well as sublethal effects of liquid plant protection products evenly applied to the soil surface on larvae of Poecilus cupreus was described by Heimbach (1998) and validated by an international ring-testing group in different laboratories (Heimbach et al. 2002). The described test method includes the detection of lethal und sublethal effects on larvae of P. cupreus. This method has already been used in several experiments (e.g. Abdelgader 1996, Bayley et al. 1995, Heimbach & Abel 1994, Heimbach & Soverini 1998, Heise et al. 2004, Metge 1996).

In the present study, the method required some adaptations for measuring the impact of pesticides applied as seed dressing on coated seeds. In preliminary tests with one carbosulfan coated seed in standard glass tubes with 2.5 cm inner diameter (5 cm2 surface) 100 % of the larva died (Heimbach, not published). To achieve a more realistic exposure regarding seed densities in field situations, a test with larger containers (106 cm2 surface) was selected and

74

tests were carried out by several laboratories (Heimbach et al., not published). The aim of the experiments reported in this paper was to compare different container sizes and soil heights on effects obtained. Therefore two types of tests have been carried out: experiment 1) using four container sizes with different surfaces and the same filling height of soil (approx. 5 cm), experiment 2) using three different filling heights of soil with the same surface area. Material and methods The method used for testing was published by Heimbach (1998) and modified for the carbosulfan coated rape seeds (14.2 g a.i. carbosulfan/kg seeds, equivalent to 71.1 µg/seed). Experiment 1 Four different container sizes were used to test the influence of the soil surface area on the effect of carbosulfan coated seeds on larvae of Poecilus cupreus (Coleoptera; Carabidae). The smallest container was a glass tube used for the standard test for soil surface applied plant protection products with 2.5 cm diameter, 7 cm height and 5 cm2 surface Heimbach (1998). Each tube contained 25 g dry soil moistened with tap water until 35 % of the maximum water holding capacity was reached. In relation to the amount of soil in these glass tubes adequate quantities of soil were filled into the other containers corresponding to their surface areas of 53, 106 and 163 cm² to get the same filling height (approx. 5 cm) (Table 1). Experiment 2 Containers with a surface area of 55 cm2 and 18 cm height (Table 1) were used to test the influence of the substrate depth in a second test. These containers were filled with three different heights of soil 5 cm, 10 cm, and 15 cm. Table 1: Container size and number of carbosulfan coated seeds per container

Surface [cm2] 5 53 106 163 55

Height [cm] 7 7 8 7 18

Inner Diameter [cm] 2.5 8.2 11.6 14.4 8.4 Dry weight of soil

[g/container] 25 269 539 831 277 / 554 / 831

Number of coated rape seeds 1 1, 2 1, 2, 3 1, 2, 3, 4 2

All test containers were round with transparent walls and bottoms which allowed an observation of the larvae of Poecilus cupreus. Differing from the standard glass tubes all lids had a hole of 1.2 cm diameter 1 cm apart from the centre. The containers were kept dark at 20° C at air humidity of 70 to 80 % during the experiment. Soil humidity was readjusted once during the whole test period. The 40 standard glass tubes were placed together in plastic boxes. The bottoms of the boxes were covered with wet filter paper. For ventilation the lid of the boxes were kept open for about 2 %.

One day before test starting the carbosulfan coated seeds were applied in one row in the middle of the container, 1.5 cm deep into the soil in rates between 1 to 4 coated seeds per container (Exp. 1) and 2 coated seeds (Exp. 2). As control 1 to 2 uncoated seeds were applied with at least 20 replicates per treatment group.

75

One larva of 24 to 48 hours age was released into each test container. Afterwards food was supplied on the soil surface and replaced three times a week. One half deep-frozen pupa of Calliphora spp. from commercial producers per Poecilus larva was offered as food.

A sandy soil was used as substrate (1 % organic C, 92 % sand, pH 4.8, CEC 1.6 mval/l, WhC 32 Vol %).

At each feeding-date the development and behaviour as uncoordinated movement, immobility or mortality of the larvae were documented. Containers in which no larvae had been visible were opened 28 days after release of the larvae and the soil was searched for them. Just before the first beetles hatched from pupae, the glass tubes were checked daily to determine the exact hatching date.

Mortality and sublethal effects such as the developmental time and the hatching weight were determined as endpoints. Newly hatched beetles were weighed before they were fed for the first time.

A probit analysis (Easy assay critical values, Ratte 1992-1995) was used for LD50 calculation. SPSS (Version 11.00) for Windows was used for statistical analysis. The student`s t-test and Mann-Whitney-U-test were carried out to test for statistical significance. The Pearson correlation was used for normal distributed data and the Spearman’s rho-correlation for not normally distributed data. Results and discussion Mortality All test results could be evaluated, because control mortality was between 2.5 and 10 % which is below the maximum natural mortality rate of 20 % accepted for untreated controls (Heimbach 1998).

Experiment 1). Figure 1 presents the mortality rates plotted against the number of coated rape seeds for four container sizes with the same filling height of soil but different surface areas. Control mortality reached 0 – 5 % in this experiment. Regarding the same number of coated seeds (1, 2 and 3) the mortality decreased with increasing surface area. In containers of same surface area (for 106 and 163 cm2) the mortality increased with increasing numbers of coated seeds. Only in containers of 53 cm² a similar mortality for 1 and 2 coated seeds was found (50 %). But the 50 % effect of 2 seeds seems to be too low in comparison with the results of experiment 2 with the same filling height of 5 cm, a container size of 55 cm² and 2 coated seeds resulting in 75 % mortality.

At 5 cm2 only one seed was tested, because in a preliminary investigation 100 % of the larvae died at this rate (Heimbach, not published).

Experiment 2). Figure 2 presents the mortality rates including control mortality plotted against the different filling heights (5, 10 and 15 cm) in containers with a surface of 55 cm². It shows that the mortality rate of 70 % ± 5 % is independent of the filling heights of soil between 5 and 15 cm. The mortality rate is only influenced by the relation between surface area and number of coated seeds.

Experiments 1 and 2). Figure 3 presents the mortality rates plotted against the number of coated rape seeds per m2 for different sizes of the surface area. The value of 2041 seeds/m² (1 seed per 5 cm²) was excluded because of the extreme low surface-seed proportion. The mortality rates of containers with filling heights of 5, 10 and 15 cm and with 55 cm2 surface area are included in Figure 3. Except the value of 380 seeds per m² a clear dose respond relationship is visible. Between the number of coated seeds and the mortality was a high significant correlation (p < 0.001, R2 = 0.776) (Spearman-rho test).

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The 3 values of about 200 seeds per m², each from a different container size, are quite similar which again shows that the number of seeds per m² is most relevant for mortality effects.

A LC50-value was calculated for the number of rape seeds per m2 using all data from Experiment 1 and 2 except the data from the 5 cm² containers. The LC50 reached 172 seeds per m2. The 95 % confidence interval was between 119 and 247 (p = 0.608). For the active ingredient the LC50-value was 12 g a.i./m2 (95 % confidence interval > 8 and < 18, p = 0.512). Sublethal parameters Table 2 shows differences of hatching weights of adult beetles and of the time larvae and pupae needed to complete their development. An influence of food quality (Theiss & Heimbach 1993) and temperature (Theiss & Heimbach 1994) on this sublethal parameters could be excluded because the same batch of food (Calliphora spp.), the same feeding rate and the same temperature (20.1 ± 0.6°C) was used in all treatments. The mean of the control values of each experiment was used for data analysis, because no significant differences were detected between the different control groups. There is a general tendency that additionally to increased mortality sublethal parameters are visible after using carbosulfan treated seeds. The developmental time is prolonged in all cases except one (1 seed in 106 cm² containers), this effect is significant in 7 cases. The hatching weight is reduced in most cases, when larvae were exposed to carbosulfan treated seeds but being significantly different only in 1 case. Sublethal effects like this also were observed when Poecilus larvae were exposed to other active substances (Heimbach & Soverini 1998). All in all, the hatching weight seems to be a less adequate parameter to detect effects of plant protection products. This was also shown by e.g. Heimbach et al. (2002). The filling height of the soil had no influence on the hatching weight and the developmental period. Generally no significant difference between male and female beetles hatched during the experiments were detected.

0

10

20

30

40

50

60

70

80

90

100

1 2 3 4number of coated seeds

mor

talit

y [%

]

5 cm²

53 cm²

106 cm²

163 cm²

X X X X X X

soil surface

Figure 1. Mortality [%] of Poecilus cupreus larvae using different rates of carbosulfan coated rape seeds in four different container sizes [cm2], x = not tested

77

0

10

20

30

40

50

60

70

80

90

100

5 10 15filling height of soil [cm]

mor

talit

y [%

]

controlmortality

2 coatedseeds

Figure 2. Mortality [%] of Poecilus cupreus larvae exposed to 2 carbosulfan coated seeds and control values using 3 different filling heights of soil [cm] in containers of 55 cm² surface area

0

10

20

30

40

50

60

70

80

90

100

0 50 100 150 200 250 300 350 400

coated seeds per m²

mor

talit

y [%

]

Exp.1) different surface area

Exp.2) different filling height variant

Figure 3. Mortality [%] of Poecilus cupreus larvae using different numbers of carbosulfan coated rape seeds per m² in containers with different surface areas (53, 106 and 163 cm²) and different filling height variants (5, 10 and 15 cm) and 55 cm² surface area

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Table 2. Hatching weight and larval developmental time of Poecilus cupreus larvae in 2 different experiments for all treatments using carbosulfan coated seeds and the control. (N = number of surviving beetles used for analysis)

Experiment Surface area Treatment N Developmental time [d]

Hatching weight [mg]

Mean Control 77 33.9 69.5

5 cm² 1 seed 2 40.0 * 58.5 *

1 seed 10 34.0 68.6 53 cm²

2 seeds 10 35.9 * 67.3

1 seed 11 34.4 70.5

2 seeds 9 35.9 * 69.0 106 cm²

3 seeds 4 34.7 70.1

1 seed 15 33.7 69.5

2 seeds 13 35.9 ** 65.3

3 seeds 10 35.5 * 72.5

Exp. 1

163 cm²

4 seeds 9 36.3 * 64.4

Mean Control 57 38.1 70.3

2 seeds 5 40.2 70.3

2 seeds 7 39.3 * 68.0 Exp. 2

55 cm²

2 seeds 6 38.5 69.5

*p ≤ 0.05 significant; ** p ≤ 0.01 high significant to control (t-test) Conclusion The hypothesis that the container size is very important for effects on larvae of Poecilus having the same number of coated seeds exposed was confirmed. The filling height of soil between 5 and 15 cm had no influence on mortality effects. In the test system the container diameter (different soil surface areas) was a very relevant parameter. This means that mobile organisms should not be tested in small test containers when pollutants are not evenly applied to the soil surface but exposed as hot spots in the system. The number of hot spots applied per m² is the main driving force for mortality values.

With respect to mortality, a good dose response relationship was obtained. Hatching weight of adult beetles and developmental time were more variable than mortality. Further research seems to be necessary to ensure reliability and reproducibility of the test results and to figure out if a reduction of variability is possible.

Generally, the use of test containers with a surface of about 53 cm² seems to be suitable for laboratory testing of side effects of coated seeds on larvae of Poecilus cupreus. The size of the test container should be selected carefully to get seeding densities more close to field conditions.

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Acknowledgements We would like to thank all the colleagues involved in carrying out the experiments and doing most of the practical work, especially U. Busch. References Abdelgader, H. 1996: Wirkung von Insektenwachstumsregulatoren auf die Getreideblattlaus-

arten Rhopalosiphum padi (L.) und Sitobion avenae (F.) (Hemiptera; Aphidae) und den polyphagen Räuber Poecilus cupreus (Coleoptera: Carabidae). – Dissertation, TU-Braunschweig.

Barrett, K.L., Grandy, N., Harrison, E.G., Hassan, S. & Oomen, P. 1994: Guidance document on regulatory testing procedures for pesticides with non-target arthropods. – SETAC-Europe: 51 pp.

Bayley, M., Baatrup, E., Heimbach, U. & Bjerregaard, P. 1995: Elevated copper levels during larval development cause altered locomotor behavior in the adult carabid beetle Pterostichus cupreus L. (Coleoptera: Carabidae). – Ecotoxicology and Environmental Safety 32: 166-170.

Heimbach, U. & Abel, C. 1994: Effects of three pesticides on Poecilus cupreus (Coleoptera: Carabidae) at different post-treatment temperatures. – Environmental Toxicology and Chemistry 13: 317-324.

Heimbach, U. 1998: Testing the effects of plant protection products on larvae of the carabid beetle Poecilus cupreus (Coleoptera, Carabidae) in the laboratory, method and results. – IOBC/wprs Bulletin 21 (6): 21-28.

Heimbach, U., Baier, B., Blümel, S., Geuijen, I., Jäckel, B., Maus, C., Nienstein, K., Schmitzer, S., Stäbeler, P., Ufer, A. & Winkelmann, G. 2002: First ring test results of a laboratory method to evaluate effects of plant protection products on larvae of Poecilus cupreus (Coleoptera, Carabidae). – IOBC/wprs Bulletin 25 (11): 19-26.

Heimbach, U. & Soverini, E. 1998: Testing side effects of pesticides on larvae of the carabid beetle Poecilus cupreus (Coleoptera: Carabidae). – IOBC/wprs Bulletin 21 (6): 93-99.

Heise, J., Heimbach, U. & Schrader, S. 2004: Influence of soil organic carbon on acute and chronic toxicity of plant protection products to Poecilus cupreus (Coleoptera, Carabidae) larvae. – J. Soils & Sediments, Online First DOI http://dx.doi.org/10.1065/jss2004.10.118

Metge, K. 1996: Entwicklung von Laborzuchtmethoden und ökotoxikologischen Prüfver-fahren für Kurzflügler, insbesondere Philontus cognatus (Staphylinidae, Coleoptera). – Dissertation, TU-Braunschweig.

Ratte, H. 1992-1995: Easy Assay – Critical Values. – © Spirit; Aachen. Theiss, S. & Heimbach, U. 1993: Fütterungsversuche mit Carabidenlarven als Beitrag zur

Klärung ihrer Biologie. – Mitteilungen der Deutschen Gesellschaft für allgemeine und angewandte Entomologie 8: 841-848.

Theiss, S. & Heimbach, U. 1994: Präimaginale Larvalentwicklung der Laufkäferart Poecilus cupreus in Abhängigkeit von Bodenfeuchte und Temperatur (Coleoptera, Carabidae). – Entomol. Gener. 19: 57-60.


pp. 81 - 86

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Laboratory tests of the impact of insect growth regulators on Anthocoris nemoralis F. Luigi Caroli1 & Edison Pasqualini2 1 CRPV, Via Vicinale Monticino, 1969, 47020 – Diegaro (FC) (Italy) 2 DiSTA (University of Bologna), Via G. Fanin, 42, 40126 – Bologna (Italy) Abstract: Laboratory trials were carried out in order to test the effects of three insect growth regulators (diflubenzuron, tebufenozide, flufenoxuron) on Anthocoris nemoralis F. Neonates and third instar nymphs were exposed to insecticides via residual contact on glass plates at concentrations recommended for agricultural use. The results of biological activity of products on neonates showed that diflubenzuron had no appreciable effect, tebufenozide resulted in slight harmfulness (because of mortality), while flufenoxuron was moderately harmful, reducing survival, fecundity and fertility. As regards toxicity on third instar nymphs, tebufenozide had no appreciable effect, diflubenzuron resulted in moderate harmfulness (because of severe reduction of fecundity), while flufenoxuron was harmful, reducing survival and cancelling out fecundity. Key-words: Anthocoris nemoralis, diflubenzuron, tebufenozide, flufenoxuron, side-effects, laboratory Introduction In pear orchards in Emilia-Romagna, Italy, Anthocoris nemoralis F. (Heteroptera Anthocoridae) usually prevents the pear pest Cacopsylla pyri (L.) (Homoptera Psyllidae) from its increasing to high densities, provided that pesticides applied are not harmful to the predator. When the populations of the antagonist become too low, chemical control of C. pyri is necessary. In such cases production costs rise and farmers are hardly able to comply with Integrated Fruit Production guidelines. Knowledge of impact of pesticides on A. nemoralis is therefore a key issue in pear pest management.

CRPV and University of Bologna have set up a bioassay laboratory in Cesena to test insecticides for efficacy against pear pests (mainly Cydia pomonella) and selectivity versus A. nemoralis. Side effects of insecticides are evaluated under worst case laboratory (exposure on glass plates) and extended laboratory (exposure on treated leaves) conditions.

Insect Growth Regulators (IGRs) are widely used against pear pests (C. pomonella, leafrollers, C. molesta, etc.). Even though they have a higher selectivity than other insectides like e.g. organophosphates, pyrethroids or carbamates, and for this reason are often regarded as harmless to beneficial arthropods, it is known, that they can harm immatures stages. With regard to A. nemoralis field trials have sometimes shown adverse effect (Girolami et al., 2001; Pasqualini et al., 2001). To better understand interactions between IGRs and the antagonist, laboratory tests were carried out to evaluate acute treatment (juvenile mortality) and sub-lethal treatment effects (reproductive performance of survivors).

In this study we present investigations on diflubenzuron, flufenoxuron and tebufenozide formulated as Dimilin, Cascade and Mimic respectively.

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Material and methods Insecticides were applied at their highest recommended commercial application rate (see Table 1) using a Potter spray tower. The amount of solution deposited on glass surface was 1-2 mg/cm2 as recommended by IOBC guidelines. Table 1. Pesticides tested.

Pesticide Formulation Active ingredient

Company % product

% a.i. g/l or ml/l

Dimilin 5 % WP diflubenzuron Du Pont 0,4 0,02 4 g/l

Mimic 240SC tebufenozide Isagro 0,08 0,0192 0,8 ml/l

Cascade 50DC flufenoxuron BASF 0,15 0,0075 1,5 ml/l

The testing procedure described by Stäubli and Pasquier (1988) was modified in order to assay all the juvenile instars of A. nemoralis. Pairs of neonates (acute treatment test 1) and third instar nymphs (acute treatment test 2) were exposed to residues, applied to the inside of ventilated glass cages (50 mm diameter, 15 mm height), for 10 days. 20 predators in ten cages were used for each IGR treatment, including a control treated only with distilled water. Glass dishes as well as green beans were treated; as soon as they dried, a treated green bean, Ephestia kuehniella Keller (Lepidoptera Pyralidae) eggs and neonates/nymphs of A. nemoralis, collected from rearing units, were placed in each cage. Test insects (insectary-bred bugs) were maintained in a climatic chamber at 20±1 °C, 80±5% RU, 18L:6 D for the whole duration of the study. Fresh E. kuehniella eggs were offered every 3 days. Mortality was recorded 10 days after the beginning of the test. Two replications were carried out with a total of 40 bugs for each treatment-life stage combination.

Survivors from each acute treatment test were transferred in 2 groups (exposed as neonates – sub-lethal treatment test 1; exposed as nymphs – sub-lethal treatment test 2) to untreated plastic cages and supplied with E. kuehniella eggs, water and treated green beans wherein the females could deposit eggs. Effects on reproduction (number of deposited eggs/female and hatching rate) were assessed on treated green beans during 3-4 day intervals until all bugs had died. At the start of each interval, fresh treated green beans and E. kuehniella eggs were offered. Sub-lethal treatment tests were not replicated.

Results Results of acute treatment tests of residual toxicity to neonates and third instar nymphs are shown in Figure 1. Dimilin and Mimic had no appreciable effect on the development of neonates to third instar nymphs as well as on the development of third instar nymphs to adults. Cascade affected development of neonates to third instar nymphs with a mortality of 60,2% (P<0,05, Fisher’s exact test). Results of sub-lethal evaluation based on the reproductive performance of survivors exposed to residuals as neonates and as third instar nymphs, respectively, are shown in Figures 2 and 3. Mimic (Figure 2) showed a small negative impact on female fecundity: number of eggs in the treated samples were 84 and 77% of the control after exposure to residuals as neonates or as third instar nymphs, respectively.

83

Dimilin had a similar small impact after exposure to residuals as neonates: number of eggs in the treated sample was 75% of the control. However fecundity after exposure as third instar nymphs was 17% of the control.

Figure 1. Mortality of A. nemoralis after exposure to IGR residues.

Cascade showed a negative impact on female fecundity. Number of eggs in the treated sample was 61% of the control after exposure as neonates. No egg was laid by females exposed as third instar nymphs.

Dimilin and Mimic (Figure 3) did not affect hatching of the eggs laid by adults exposed to IGRs residues when they were either neonates or third instar nymphs. Adults exposed to Cascade when they were neonates showed an egg fertility reduction of 64%. No data were collected on adults exposed when they were third instar nymphs since they laid no eggs.

The overall effects of IGRs tested, expressed as described by Angeli et al. (2000), are given in table 2. Discussion The methodology used was adequate to evaluate acute toxicity of IGRs. However, the procedure used to evaluate sub-lethal effects could be improved. The observation of egg laying for the whole life-time of A. nemoralis females (up to three months) is time consuming. Moreover, such a long experimental period makes environmental bias and operator mistakes. more frequent. This may explain the lower fecundity that we observed in the control (approximately 120 eggs/female) in comparison with that reported for a good laboratory colony (229 eggs/female); fertility of eggs in the control (70-90%) was indeed acceptable (Stäubli and Baillod, 1988). We feel that reproductive performance should be observed for a shorter period as suggested in laboratory tests with Orius laevigatus Fieber (Heteroptera Anthocoridae) (Bakker et al., 2000).

0

20

40

60

80

100

Dimilin Mimic Cascade

Abb

ott M

orta

lity

Exposed as N IExposed as N III

84

Figure 2. Fecundity of A. nemoralis females after exposure to IGR residues.

Figure 3. Fertility of eggs of A. nemoralis after parental exposure to IGR residues.

Our study shows stage-specific effects of IGRs on A. nemoralis when field rates are applied to a glass substrate. Dimilin residues were harmless to neonate bugs. Mimic caused light mortality and fecundity reduction. Cascade strongly reduced survival and fecundity of tested insects; fertility of eggs laid by the survivors was also reduced. When third instar nymphs were exposed to residues, survival generally increased, but effects on fecundity worsened. The overall impact of Mimic was low. On the other hand Dimilin and Cascade strongly reduced, or even cancelled out, egg deposition.

0

20

40

60

80

100

120

140

control Dimilin Mimic Cas cade

n. e

ggs/

fem

ale

Exposed as N I

Exposed as N III

0

20

40

60

80

100

control Dimilin Mimic Cascade

% e

gg h

atch

ing Exposed as N I

Exposed as N III

85

Table 2. Contact toxicity of IGRs on 1st and 3rd instar nymphs of A. nemoralis.

Exposed instar

Insecticides Abbott Mortality

(M%)

Female Fecundity

(R1)

Egg Fertility

(R2)

Coefficient of toxicity

(E%)

Toxicity class (1-4)*

N I Dimilin 0,3 0,75 1,05 21,5 1 Mimic 13,1 0,84 0,95 30,6 2 Cascade 60,2 0,61 0,64 84,5 3 N III Dimilin 5,9 0,17 1,08 82,7 3 Mimic 5,1 0,77 1,12 18,2 1 Cascade 18,2 0 - 100 4

*Class 1 (harmless)= E<30%; Class 2 (slightly harmful)= 30%<E<80%; Class 3 (moderately harmful)= 80%<E<99%; Class 4 (harmful)= E>99%.

Effects of the three insect growth regulators on mortality and fecundity of A. nemoralis (laboratory test type ”a”) were evaluated in the framework of the IOBC/WPRS testing programmes. Tebufenozide was harmless at 0,0288 % a.i. (Schaub, 2003, personal communication). This is in agreement with the results of the present study: Mimic is classified as slightly harmful to neonates only because fertility is also included in the calculation of the overall toxicity and this raises the coefficient value from 27 to 30,6%. Diflubenzuron was harmless at both 0,0125 % a.i. (Hassan et al., 1994) and 0,05 % a.i. (Sauphanor and Stäubli, 1994), while Flufenoxuron was evaluated as slightly harmful at 0,005 % a.i. (Sterk et al., 1999). This study shows different results. Moderate or severe harmfulness shown by the two insecticides to third instar nymphs might be related to the presence of treated green beans in the cages. By feeding on beans, the bugs might have ingested sub-lethal doses of insecticide; phytofagy is indeed an important component to the nutritional ecology of many heteropterans (Hagler et al., 2004).

No short term effect has been observed on field populations of A. nemoralis following application of the three insect growth regulators (Stäubli et al., 1984; Sauphanor and Stäubli, 1994; Pasqualini et al., 1999). These results have been confirmed by long term studies only for diflubenzuron and tebufenozide; a significant reduction of numbers of larvae and nymphs have been observed after application of flufenoxuron (Girolami et al., 2001; Pasqualini et al., 2001). Great care must be paid to field methodology since short term studies might underestimate toxicity of insect growth regulators to A. nemoralis (Sauphanor and Stäubli, 1994).

The sequence of tests proposed by IOBC/WPRS to evaluate pesticide side-effects is based on the assumption that due to the ”worst-case” scenario in the laboratory tests, harmless products can be identified with a high certainty. For products identified as harmful in the laboratory test, tests in higher tiers (semi-field, field) have to be carried out to investigate if or to which extent the harmfulness remains under more practical conditions. The harmfulness found in our modified glass plate tests following Stäubli and Pasquier (1988), i.e. by including a treated green bean, was confirmed for diflubenzuron and tebufenozide in the field tests. The effects of flufenoxuron, however, were underestimated in the glass plate test according to Stäubli and Pasquier (1988). The laboratory method proposed in the present study appears more adequate to evaluate sub-lethal effects of insect growth regulators. References

86

Angeli, G., Forti, D. & Maines, R. 2000: Side-effects of eleven growth regulators on the

predatory bug Orius laevigatus (Fieber) (Heteroptera: Anthocoridae). – IOBC/wprs Bull. 23 (9): 85-92.

Bakker, F.M., Aldershof, S.A., Veire, M.v.d., Candolfi, M.P., Izquierdo, J.I., Kleiner, R., Neumann, Ch., Nienstedt, K.M. & Walker H. 2000: A laboratory test for evaluating the effects of plant protection products on the predatory bug, Orius laevigatus (Fieber) (Heteroptera: Anthocoridae). – In: Guidelines to evaluate side-effects of plant protection products to non-target arthropods, eds. Candolfi, Blümel, Forster, Bakker, Grimm, Hassan, Heimbach, Mead-Briggs, Reber, Schmuck and Vogt: 57-70.

Girolami, V., Berti, M. & Coiutti, C. 2001: Toxicity of new IGR-based insecticides to anthocorid predators of psyllids. – Informatore Agrario 57(30): 63-66.

Hagler, J.R., Jackson, C.G., Isaacs, R. & Machtley, S.A. 2004: Foraging behaviour and prey interactions by a guild of predators on various lifestages of Bemisia tabaci.– Journal of Insect Sciences 4(1): 13 pp. Available online: insectscience.org/4.1.

Hassan, S.A., Bigler, F., Bogenschütz, H., Boller, E., Brun, J., Calis, J.N.M., Coremans-Pelseneer, J., Duso, C., Grove, A., Heimbach, U., Helyer, N., Hokkanen, H., Lewis, G.B., Mansour, F., Moreth, L., Polgar, L., Samsøe-Petersen, L., Sauphanor, B., Stäubli, A., Sterk, G., Vainio, A., van de Veire, M., Viggiani, G. & Vogt, H. 1994: Results of the sixth joint pesticide testing programme of the IOBC/WPRS-working group "Pesticides and beneficial organisms". – Entomophaga 39(1): 107-119.

Pasqualini, E., Civolani, S., Vergnani, S., Cavazza, C. & Ardizzoni, M. 1999: Selettività di alcuni insetticidi su Anthocoris nemoralis. – Informatore Agrario 46: 71-74.

Pasqualini, E., Vergnani, S., Ardizzoni, M., Cavazza, C., Civolani, S. & Ferioli, G. 2001: Effetti nel lungo periodo di insetticidi regolatori della crescita (IGRs) su Anthocoris nemoralis F. – Informatore Fitopatologico 6: 53-54.

Sauphanor, B. & Stäubli, A. 1994: Evaluation au champ des effets secondaires des pesticides sur Forficula auricularia et Anthocoris nemoralis: validation des resultats de laboratoire. Bull. – IOBC/wprs 17(10): 83-88.

Stäubli, A. & Baillod, M. 1988: Les effets secondaires des pesticides sur les auxiliaries: exemples pratiques en arboriculture et en viticulture. – Revue Suisse Vitic. Arboric. Hortic. 20(4): 205-209.

Stäubli, A. & Pasquier, D. 1988: Méthode de laboratoire pour tester l’action secondaire des pesticides sur Antochoris nemoralis F. (Heteroptera: Anthocoridae). – Bull. IOBC/wprs 11(4): 91-97.

Stäubli, A., Hächler, M., Antonin, P. & Mittaz, C. 1984: Tests de nocivité de divers pesticides envers les ennemis naturels des principaux ravageurs des vergers de poiriers en Suisse romande. – Revue Suisse Vitic. Arboric. Hortic. 6(5): 279-286.

Sterk, G., Hassan, S.A., Baillod, M., Bakker, F., Bigler, F., Blumer, 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., Plogar, L., Rovesti, L., Samsøe-Petersen, L., Sauphanor, B., Schaub, L., Stäubli, A., Tuset, J.J., Vainio, A., van de Veire, M., Viggiani, G., Vinuela, E. & Vogt, H. 1999: Results of the seventh joint pesticide testing programme carried out by the IOBC/WPRS-Working Group ”Pesticides and Beneficial Organisms”. – BioControl 44: 99-117.


pp. 87 - 92

87

Laboratory evaluation of four fungicides, two insecticides and an insecticide/acaricide to Agistemus fleschneri Summers (Acari: Stigmaeidae) Noubar J. Bostanian, Gaétan Racette & Nancy Larocque Horticulture Research and Development Centre, Agriculture & Agri-Food Canada, 430 Gouin Blvd, Saint Jean-sur-Richelieu, Qc., Canada, J3B 3E6 Abstract: Extensive laboratory tests showed that trifloxystrobin (Flint® 50 WG), myclobutanil (Nova® 40 WP), flusilazole (Nustar® 50 DF), and kresoxim-methyl (Sovran® 50 WG) were non toxic to Agistemus fleschneri Summers females and did not affect their reproductive capacity. The two insecticides, lambda-cyhalothrin (Warrior® T 12%) and imidacloprid (Admire® 240 g liter-1) were also non toxic. However, the insecticide/acaricide pyridaben (Pyramite® 75 WG) caused 70% mortality to the adults. At present pyridaben can not be used in IPM programs for Quebec apple orchards where A. fleschneri plays an important role to control phytophagous mites. Keywords: fungicides, insecticides, Agistemus fleschneri, biological control Introduction In integrated pest management programs, it is essential that pesticides that are effective against target species, are at the same time relatively innocuous to non-target parasitic and predatory arthropods. In apple orchards, this is most pronounced in summer, because several predators attain maximum abundance at that time (Croft and McGroarty 1977, Bostanian et al. 1984). Consequently, adverse toxic side effects of a pesticide used in summer may disrupt a fragile pest/predator ratio, and delay or completely disrupt the biological control of arthropod pests. In Quebec apple orchards, biological control of phytophagous mites in IPM orchards depends on the abundance of at least one and often two or even three of the following predacious mites: Amblyseius fallacis (Garman), Typhlodromus caudiglans Schuster and Agistemus fleschneri Summers. The first two species are phytoseiids whereas the third is a stigmaeid. Our study reports baseline toxicity data for four fungicides, two insecticides and an insecticide/acaricide to field collected A. fleschneri. The seven pesticides evaluated are currently registered or being developed for use in commercial orchards. Materials and methods Field collection Agistemus fleschneri females were collected from a commercial orchard in South western Quebec from early June to the end of August. Leaves with mites were picked from apples, placed in paper bags and brought in a cooler to the laboratory within 30 minutes. Toxicity to females Thirty females were transferred to a double-sided tape (Cantech®, Montreal, Canada) on microscope slides. Each slide was dipped for 5 seconds in a different concentration of a pesticide (Table 1) and allowed to dry. The slides were then placed in a growth chamber at

88

21°C, 80% relative humidity and 16:8 h light:dark cycle. After 24 hours of exposure, mortality of the females was recorded by touching their abdomens. Death was defined by the absence of appendage movements when the mites were prodded with camel-hair brush. Percent mortality was calculated according to Abbott (1925). Probit analysis (LeOra Software 1987) was carried out on the mortality data. Table 1. Pesticide doses used to determine the LC50 of each compound to A. fleschneri.

Product Trade name Doses tested in g AI /liter

Fungicides Kresoxim-methyl

Sovran 50 WG

0.140 0.070 0.035* 0.018 0.009 0.004

Trifloxy-strobin

Flint 50 WG

0.180 0.088 0.044 0.022 0.011 0.006

Flusilazole Nustar 50 DF

0.140 0.070 0.035 0.018 0.009 0.004

Myclobutanil Nova 40 WP

0.240 0.120 0.060 0.030 0.015 0.008

Insecticides Pyridaben Pyramite

75 WG 0.225 0.113 0.056 0.028 0.014 0.007 0.004

lambda-cyhalothrin

Warrior T 12%

0.0134 0.0067 0.0034 0.0017 0.0009 0.0004

Imidacloprid Admire 240 g litre-1

0.125 0.062 0.031 0.016 0.008 0.004 0.002 0.001

*Values in bold represent the recommended doses. Effects on reproduction An apple leaf was cut in a rectangular form (4 X 3 cm) and its axil was inserted in a small vial containing water. To reduce evaporation the top of the vial was sealed with Parafilm® (American National Can, Chicago, IL, USA). Three A. fleschneri females were added with two-spotted spider mites as food and all were treated with the pesticide at different dosages (Table 1) with a sprayer set at 10.34 kPa. At this pressure the amount of residue on the leaves was 2.00 mg x cm-2. Each treatment was replicated five times. The experimental setting was then placed in a growth chamber at 21°C, 80% relative humidity and 16:8 h light:dark cycle. After 48 hours of exposure, all A. fleschneri females were removed and the eggs were counted under a binocular stereoscope and kept in the growth chamber until hatching. Percent mortality was calculated by probit analysis (LeOra Software 1987). Results and discussion None of the four fungicides evaluated had any adverse effects to A. fleschneri adults (Fig. 1). The slopes are such that the graphs are virtually parallel with the X axis. Walker et al. (1988) had reported no adverse effects of myclobutanil to Typhlodromus pyri in New Zealand. No effects of myclobutanil to Amblyseius fallacis adults and eggs have also been reported by Bostanian et al. (1998).

89

Figure 1. Fungicide effects on A. fleschneri adult females. The dotted line represents 50% mortality on probit scale.

Pyridaben was the only insecticide evaluated which had adverse effects to A. fleschneri adults (Fig. 2). The LC50 calculated by POLO-PC (LeOra Software 1987) was 0.01587 g a.i /liter and the 95% confidence limits were 0.0078-0.0311. In Quebec apple orchards, pyridaben was reported to have no toxic effects to any life stages of the predacious mite A. fallacis (Larocque et al. 2000). Imidacloprid and lambda-cyhalothrin had no adverse effects to A. fleschneri. The low residual toxicity of the latter to A. fallacis had already been reported as an asset to be used in IPM programs in Quebec apple orchards (Bostanian and Racette 1997). No adverse effects on egg hatch were noted with six of the pesticides tested (Figs 3 and 4). As pyridaben was toxic to adult female mites, its effects on egg hatch could not be elucidated. The results of this study show that the four fungicides, kresoxim-methyl, trifloxystrobin, flusilazole and myclobutanil may be incorporated into existing IPM programs for Quebec apple orchards without any restrictions. On the other hand, Pyridaben may be used any time provided that A. fallacis is the principal predator. Its toxic effects to A. fleschneri preclude its recommendation in IPM programs unless field studies show it to be safe. Presently we do not have any toxicity data for pyridaben with T. caudiglans. Important notice: No endorsement of named products or companies is made or implied, nor is any criticism intended of similar products or companies which are not mentioned.

0.04

Myclobutanil

R 2 = 0.0885

123456789

0 0.02

Kresoxim-methyl

R 2 = 0.0172

1 2 3 4 5 6 7 8 9

0 0.1 0.2 Log (1+ dose)

Trifloxystrobin

R2 = 0.3085

123456789

0 0.01 0.02 0.03 0.04 Log (1+ dose)

Flusilazole

R 2 = 0.1908

1 2 3 4 5 6 7 8 9

0 0.005 0.01

P r o b i t

P r o b i t

90

Figure 2. Insecticide effects on A. fleschneri adult females. The dotted line represents 50% mortality on the probit scale.

Pyridaben

R2 = 0.9441

123456789

0 0.05 0.1

Imidacloprid

R 2 = 0.0101

1 2 3 4 5 6 7 8 9

0 0.02 0.04 0.06

Lambda-cyhalothrin

R 2 = 0.0344

1 2 3 4 5 6 7 8 9

0 0.002 0.004 0.006

Log (1+ dose)

P r o b i t

P r o b i t

Log (1+ dose)

91

Figure 3. Fungicide effects on the eggs of A. fleschneri after treatment of females. The dotted line represents 50% mortality on the probit scale.

References

Flusilazole

R 2 = 0.369

1 2 3 4 5 6 7 8 9

0 0.005 0.01

Myclobutanil

R2 = 0.2321

123456789

0 0.0 0.0 0.0 0.0

Kresoxim-methyl

R 2 = 0.098

1 2 3 4 5 6 7 8 9

0 0. 0.

Log (1+ dose)

Trifloxystrobin

R 2 = 0.2301

123456789

0 0.0 0.0

Log (1+ dose)

P r o b i t

P r o b i t

Imidacloprid

R 2 = 0.034

1 2 3 4 5 6 7 8 9

0 0.02 0.04 0.06

Log (1+ dose)

lambda-cyhalothrin

R 2 = 0.2287

123456789

0 0.002 0.004 0.006

P r o b i t

Log (1+ dose)

Figure 4. Insecticide effects on the eclosion of A. fleschneri eggs after treament of females. The dotted line represents 50% mortality on the probit scale.

92

References Abbott, W.S. 1925: A method of computing the effectiveness of an insecticide. – J. Econ.

Entomol. 18: 265-267. Bostanian, N.J. & Racette, G. 1997. Residual toxicity of lambda-cyhalothrin on apple foliage

to Amblyseius fallacis and the tarnished plant bug, Lygus lineolaris. – Phytoparasitica 25: 193-198.

Bostanian, N.J., Dondale, C. Binns, M.R. & Pitre, D. 1984. Effects of pesticides used on spider (Aranea) in Quebec apple orchards. – Can. Entomol. 116: 663-675.

Bostanian, N.J., Thistlewood, H. & Racette, G. 1998. Effects of five fungicides used in Quebec apple orchards on Amblyseius fallacis (Garman) (Phytoseiidae: Acari). – J. Hortic. Sci. Biotechnol. 73: 527-530.

Croft, B.A. & McGroarty, D.L. 1977. The Role of Amblyeius fallacis in Michigan Apple Orchards. – Res. Rep. 33., Mich. Agric. Exp. Stn. 48 p.

Larocque, N., Bostanian, N.J., Racette, G. & Lasnier, J. 2000. Toxicité des pesticides sur les prédateurs d’acariens des vergers de pommiers au Québec. – Journée pomicole provinciale, St. Hyacinthe, Fév.,45-47.

LeOra Software, POLO- PC 1987. Probit and logit analysis. – Berkeley, CA. Walker, J.T.S., Baynon, G.T., Shaw, P.W. & Cassidy, D. 1988. Evaluation of fungicide

programs compatible with integrated control of European red mite. – Proc. N.Z. Weed Pest Control Conf. 41: 193-197.


pp. 93 - 103

93

Variation in response of earthworms and soil microflora to reference test substances M. J. Mallett, J C Hayward & N. A. Davies CEMAS, Glendale Park, Fernbank Road, North Ascot, Berkshire, SL5 8JB, UK Abstract: The variation in response of the earthworm acute test (OECD 207), the earthworm reproduction test (ISO 11268-2) and soil microflora tests (OECD 216 and OECD 217) have been investigated at CEMAS. The earthworm acute (14-day) toxicity study showed good repeatability with a percentage coefficient of variation (%CV) of 10% for the 14-day LC50 of 2-chloroacetamide. Precision associated with the other studies was lower with a %CV for the earthworm reproduction study of 32% (for percentage inhibition of juvenile production at 1.8 mg carbendazim/kg dry soil) and %CVs of 41% and 35% for inhibition of soil respiration and nitrogen transformation respectively at 10 mg dinoseb/kg dry soil. Key-words: Earthworms, Eisenia fetida, acute toxicity, reproduction, soil microflora, soil respiration soil nitrogen transformation, 2-chloroacetamide, carbendazim, dinoseb Introduction Studies of the effects of crop protection products on earthworms and soil microflora (respiration and nitrogen transformation) are required under EC Directive 91/414 for both active ingredients and formulated products. Numerous earthworm acute and reproduction studies, as well as soil microflora respiration and nitrogen transformation studies, have been carried out by CEMAS to satisfy these data requirements. Reference studies conducted at CEMAS following OECD or ISO test guidelines over several years have been analysed to determine the level of repeatability or precision associated with those studies and to determine whether any temporal trend in test sensitivity had occurred. Data from seven soil microflora studies using the reference substance dinoseb, five earthworm reproduction studies using carbendazim and five earthworm acute studies using the reference substance 2-chloro-acetamide were used in this assessment. Materials and methods for earthworm acute studies Five 14-day acute toxicity studies with Eisenia fetida, based on test guideline OECD 207 (1984), were carried out with the reference toxicant 2-chloroacetamide over the period from March 1998 until May 2002. Four or five concentrations of 2-chloroacetamide together with an untreated control were tested in an artificial soil substrate consisting of a mixture of sand, peat and clay as described in OECD 207 (1984). Each treatment and control was replicated four times. The test vessels were glass beakers (1000 mL) containing approximately 750 g wet weight of test substrate and covered with polyethylene adhesive film. The vessels were continuously illuminated inside a test cabinet in which the air temperature ranged from 20.0°C to 21.5°C.

94

The worms were obtained from cultures maintained at CEMAS in artificial soil substrate and fed with cow manure from an organic farm. When used for testing they were 7 to 10 months old and ranged in wet weight from 0.31 g to 0.54 g at the start of the studies. They were examined for mortality after 7 and 14 days exposure. On days 0 and 14 the live-weights of the worms were measured and the effect of exposure on body weight was assessed.

Results and discussion for earthworm acute studies

The results are presented in Table 1 and are compared with previously published values from Yeardley et al (1995) for the reference test substance 2-chloroacetamide. The results in terms of effects on survival are summarised in Table 1 where means and coefficients of variation are shown. Table 1. LC50’s for 2-chloroacetamide to E. fetida

Date tested

End point Statistical method(s)

LC50 expressed as: mg 2-chloroacetamide/kg dry

soil

March 1998 Feb. 1999 Feb. 2000 Jan. 2001

7/14 day* 7/14 day* 7/14 day*

7 day 14 day

Geometric mean Probit analysis

Trimmed Spearman-Karber Trimmed Spearman-Karber Trimmed Spearman-Karber

40.0 (95% c.l. not calculable) 39.4 (95% c.l. = 36.0 - 43.1)

49.1 (95% c.l. = 44.5 - 54.2) 47.5 (95% c.l. = 43.4 to 52.0) 46.8 (95% c.l. = 42.9 to 50.9)

May 2002

7 day 14 day

Probit analysis Probit analysis

48.5 (95% c.l. = 44.3 to 52.6) 47.9 (95% c.l. = 43.8 to 51.9)

Summary of

results given above for 7 and 14

days

7 day

As given above

mean = 44.9 (cv = 10.7%, n =

5) (sd = 4.79, ± 2 sd = 35.3 to 54.5)

14 day

As given above

mean = 44.6 (cv = 10.2%, n =

5) (sd = 4.59, ± 2 sd = 35.5 to 53.8)

Yeardley et al (1995)

7 day

Trimmed Spearman-Karber and graphical

method

mean = 33.3 (cv = 36.3%, n =6) (sd = 12.1, ± 2 sd = 9.1 to 57.5)

14 day

Trimmed Spearman-Karber and graphical

method

mean = 33.1 (cv = 35.3%, n =

6) (sd = 11.7, ± 2 sd = 9.7 to 56.5)

* both 7 and 14 day LC50 values were the same since no further mortalities occurred between days 7 and 14.

95

The 14-day LC50 values are plotted against time in the form of a quality control chart in Figure 1 where a warning limit has been defined as the mean ± 1 times the standard deviation and the action limit as the mean ± 2 time the standard deviation.

0

10

20

30

40

50

60

Sep 97 Feb 98 Jul 98 Dez 98 Mai 99 Okt 99 Mrz 00 Aug 00 Jan 01 Jun 01 Nov 01 Apr 02 Sep 02

Time (month/year)

14 D

ay L

C 50 v

alue

s (m

g 2-

chlo

roac

etam

ide/

kg

dry

soil)

with

95%

con

fiden

ce li

mits

Figure 1. 14-day LC50’s for 2-chloroacetamide to E. fetida. Quality control chart where dashed lines represent warning limits (±1 sd from the mean) and the dotted lines are action limits (±2 sd from the mean).

Length of time in culture appeared to have had no effect on the sensitivity of earthworms since no consistent trend over time (from March 1998 to May 2002) was seen in terms of sensitivity of the worms to 2-chloroacetamide. Although the data are very few, with only five studies being compared. The response over time was consistent, with 14-day LC50’s ranging from 39.4 mg/kg to 49.1 mg/kg and the coefficient of variation was relatively low at 10.7%.

The 7-day LC50 was very similar to the 14-day LC50 suggesting little benefit in extending the duration of exposure for seven to 14 days, this was also found by Yeardley et al (1995). This will not hold true for other test substances however (Spurgeon and Weeks, 1998). The toxicity of 2-chloroacetamide to E. fetida found at CEMAS was slightly lower, but not significantly different from that found by Yeardley et al (1995). The 14-day LC50s all fall within the acceptable range of 20 mg/kg to 80 mg/kg given in ISO 11268-1 (1993).

Body weight always decreased during the studies (since the worms were not fed). The control weight loss ranged from 4.9% to 17.0% of that at the start. The Dunnetts test was used to compare the weight loss between the controls and the treated groups and in all except one study no differences were seen up to those concentrations where significant mortalities were seen (56 mg/kg) indicating that, for 2-chloroacetamide, weight change was a poor indicator of toxic effect compared with mortality. However, experience gained at CEMAS and elsewhere (for example, Kula, 1998) indicates that this will not be the case for all chemicals.

96

Materials and methods for earthworm reproduction studies Five 56-day reproduction studies with Eisenia fetida based on test guideline ISO 11268-2 (1998) were carried out using the toxic reference substance carbendazim over the period March 2000 to April 2004. Five concentrations were tested in the first study and two in the second, third and fourth and one in the fifth study. Each treatment (concentration and control) was replicated four times. The test vessels consisted of rectangular plastic boxes of 1.2 L capacity. To each was added 600 g (by dry weight) of artificial soil supplemented with food consisting of pasteurised, macerated, air dried and re-moisturised cow manure. Carbendazim concen-trations were prepared by dissolving in HPLC grade acetone which was mixed with acid washed silica sand, placed on an unheated sample concentrator and evaporated to dryness, the sand aliquots were rolled for 30 minutes to mix before incorporation into the soil. After incorporation ten adult worms of known age from cultures maintained at CEMAS were placed on the surface of the soil to start the study. Adult worms were removed after 28 days and weighed, leaving the juveniles to develop in the treated soil for a further 28 days. The numbers of juveniles and cocoons present in each vessel were then counted. The treated groups were compared for numbers of juveniles, numbers of unhatched cocoons, and change in biomass and mortality of adult worms with untreated controls which had also received the solvent-treated sand. Results and discussion for earthworm reproduction studies The numbers of juveniles present at the end of the studies are compared in Table 2. The results for the other test end-points (adult survival, weight change of adults and cocoon production are shown in Table 3. Of the end-points examined inhibition of juvenile production was the most sensitive, adult survival was least sensitive followed by weight change among adults. Higher numbers of unhatched cocoons were found at 1.8 mg/kg indicating an adverse effect on hatching success at that concentration. The no observed effect concentration (NOEC) was 1.0 mg/kg and the lowest effect concentration was 1.8 mg/kg (statistically determined at P = 0.05) for inhibition of juvenile production. This is in line with Annex F of ISO 11268-2 where a ring-test resulted in 81% inhibition at 1.68 mg carbendazim/kg compared with the controls. The rate of inhibition of juvenile production can be compared at 1.0 mg/kg (for four studies) and 1.8 mg/kg (for all studies). At 1.0 mg/kg the respective percentage inhibition was -16%, -15%, +0.5% and -12%. At 1.8 mg/kg the respective percentage inhibition was -92%, -40%, -51%, 66% and 81% with a mean value of -66% and a % coefficient of variation (%CV) of 32%. A considerably lower rate of juvenile production was seen for the first study (mean control juvenile number per surviving adult = 4.4) compared with the subsequent studies, although all studies satisfied the test guideline criteria (ISO 112698-2) that 30 juveniles should be produced per test container. The shorter length of time in culture and the lack of experience in husbandry may have explained the low juvenile numbers seen in the first study. Although the data are few, the sensitivity of the test in terms juvenile production inhibition relative to the controls has not been markedly affected by either the large variability in fecundity among the control worms between the studies, or the length of time the worms were kept in culture.

97

Table 2. Earthworm reproduction studies - numbers of juvenile worms present at end of studies (day 56).

Number of juveniles present per surviving adult worm

Summary Statistics

Carben-dazim

concentra-tion

(mg/kg)

Replicate

1

Replicate

2

Replicate

3

Replicate

4

Average number of juveniles per surviving adult

worm

Coefficient of variance

(%)

Inhibition relative to controls

(%)

First Study – March 2000 to May 2000

0 (control) 0.6 1.0 1.8 3.1 5.5

3.3 3.7 5.0 0.7 0 0

4.3 1.9 3.9 0.2 0 0

5.5 4.3 5.4 0.5 0 0

4.4 6.2 5.4 0 0 0

4.4 4.0 5.1

0.4 * 0 0

21 44 17 89 – –

– 8 -16 92 100 100

Second study – September 2000 to December 2000

0 (control) 1.0 1.8

17.8 18.6 14.3

20.1 21.2 14.3

26.6 19.6 12.2

22.1 14.3 11.2

21.7 18.4

13.0*

17.3 16.0 12.0

– 15 40

Third study – May 2002 to July 2002

0 (control) 1.0 1.8

15.7 23.0 11.0

23.9 22.4 9.6

18.5 19.0 11.3

21.8 15.9 7.2

20.0 20.1

9.78*

18.1 17.5 19.0

– -0.5 51

Fourth study – November 2003 to January 2004

0 (control) 1.0 1.8

13.7 13.4 4.7

10.6 13.0 1.8

18.1 10.7 5.3

17.2 15.2 8.6

14.9 13.1

5.1*

23.1 14.1 54.7

– 12 66

Fifth study – February 2004 to April 2004

0 (control) 1.8

44.9 11.5

36.4 4.5

36.7 5.0

37.2 8.2

38.8 7.3*

10.5 44.4

– 81

(*) = significant (Dunnetts test or t-test, one-tailed, P = 0.05) difference from controls

98

Table 3. Earthworm reproduction studies – other end-points - results for adult mortality, adult weight change and cocoon production.

Carbendazim concentration

(mg/kg)

Percent mortality of adults

(after 28 days)

Percent weight change for

adults (0 to 28 days)

Mean numbers of cocoons at day 56

per adult worm

First Study – March 2000 to May 2000

0 (control) 0.6 1.0 1.8 3.1 5.5

0 0

2.5 0 0

37.5

+0.5 +1 +4 +6

-21* -45*

0.6 0.6 0.7

1.8* 0.7 0.0

Second study – September 2000 to December 2000

0 (control) Solvent control

1.0 1.8

0 0 0 0

-3 -8 -2

-0.2

0.3 0.2 0.3 0.4

Third study – May 2002 to July 2002

0 (control) 1.0 1.8

0 7.5 0

-15 -15 -15

0.1 0.2

1.2*

Fourth study – November 2003 to January 2004

0 (control) 1.0 1.8

0 0 0

+47 +55

+32*

0.0 0.1

0.8*

Fifth study – February 2004 to April 2004

0 (control) 1.8

0 0

+3.9 +13.2

0.3 3.4*

(*) = significant (P = 0.05) difference from controls Materials and methods for soil microflora studies Seven soil microflora studies examining the effects of the reference substance dinoseb on soil respiration and nitrogen transformations were carried out over the period April 1999 to January 2003. The studies followed test guidelines OECD 216 (2000), for nitrogen transformations and OECD 217 (2000), for soil respiration. The studies were carried out using a sandy soil, as recommended by OECD 216 (2000) and OECD 217 (2000). The soil had a sand content between 50% to 75%, a pH (H2O) of 5.5 to 7.5, and a low (0.5% to 1.5%) organic carbon content (but with a microbial biomass of not

99

less than 1% of the total organic carbon). This represents a ‘worst-case scenario’, so that tests of effects in other soils are unnecessary. For the first three studies the soil was taken from a site at Bredfield, Suffolk, UK, for the subsequent four studies the soil was taken from a site at White Waltham, Berkshire, UK. Information from the site managers indicated that neither site had received soil fertilisers for six months or crop protection products for one year prior to soil collection. The soils were acclimated to the test conditions for at least two days for each study. At the start of each study the soil samples for nitrogen analysis only were amended with 0.5% w/w ground lucerne to act as a nitrogen source. For the dinoseb treatments dinoseb was dissolved in acetone and dried onto quartz sand for incorporation into the soil at the desired treatment rates. The control replicates received acetone-treated sand only. Six samples of soil were treated at each dinoseb concentration and six samples were left untreated to act as controls. For four of the studies a single concentration of 10 mg dinoseb/kg dry soil was used together with un-treated controls. For two of the studies concentrations of 0 (control), 1 mg and 10 mg dinoseb/kg dry soil were used and for the remaining study three concentrations were used: 0 (control), 5 mg, 15 mg and 45 mg dinoseb/kg dry soil. A concentration of 10 mg/kg was chosen for the majority of studies because that concentration has previously been found to distinctly affect (reduce or stimulate) the activity of soil microflora (C. Kula pers. comm.). All concentrations are expressed as mg/kg dry soil. The dry weight of the soil from each study was determined by drying soil samples to constant weights at 105°C. The soil samples for both soil nitrogen transformation and soil respiration were incubated for a period of 28 days at a temperature of 20°C ± 2°C in the dark. Soil respiration measurements and extractions for nitrogen were made within six hours of dosing the soil with dinoseb. Further measurements of soil respiration and extractions for nitrogen determinations were carried out 7 (or 6), 14 and 28 days after dosing the soil. Soil respiration measurements were performed on 100 g sub-samples from each replicate. The samples were placed in separate chambers and CO2-free air was passed through each chamber. The CO2 content of the air exiting each chamber was measured once per hour for 12 hours at each measurement time-point using an ADC 2250 infra-red gas analyser. Soil nitrate concentrations were measured in 2M KCl extracts of 50 g sub-samples of soil taken at each time-point using an Alpkem RFA analyser. The mean values for nitrate concentrations and respiration rates for the controls and the dinoseb-treated soils were tested for significant differences (Ho control=treatment at P = 0.05) using the Dunnetts test. Results and discussion for soil microflora studies The response of the soil to dinoseb at 10 mg/kg (dry soil equivalent) has been compared with the controls for the first six studies and the response is summarised in Table 4. The effect of dinoseb at 10 mg/kg on soil respiration for the six studies is plotted in Figure 2 and similarly for nitrogen transformations in Figure 3. The mean values for nitrate concentrations and soil respiration at 10 mg/kg (but not at 1 mg/kg for tests 1 and 2) were significantly different (P=0.05) from the controls after 28 days. Indicating that dinoseb at 10 mg/kg was a suitable choice of concentration for use as a toxic reference, or positive control treatment. It can be seen that dinoseb at 10 mg/kg always caused an increase in nitrate formation and a decrease in soil respiration rate. Comparing soil respiration for the six studies at 10 mg/kg dinoseb it can be seen that the respiration rates for the soil from studies 3 to 6 (soil from White Waltham) was lower than that for the soil from

100

Bredfield (studies 1 to 3) but this has not had a significant effect on the sensitivity of the test. Comparison of the nitrate concentrations does not show significant differences between the two sites (Bredfield and White Waltham). The %CV for difference between control and dinoseb treated soil for nitrate formation was 41%, and for soil respiration was 35%. Table 4. Soil microflora studies – summary of effects of dinoseb at 10 mg/kg on soil nitrogen transformations and soil respiration after an exposure period of 28 days.

Nitrate concentrations (mg N/kg) Soil respiration (mg CO2/kg/hour)Date, soil source and study number

Control dinoseb Difference (%)

control dinoseb Difference (%)

April 1999, Bredfield, 1

33.4 65.4 +96 17.2 12.3 -29

Jan. 2001, Bredfield, 2

48.9 66.4 +36 23.0 16.0 -30

May 2001, Bredfield, 3

40.8 63.9 +57 26.4 16.9 -36

Oct. 2001, W.W. 4

40.5 65.5 +62 12.2 5.8 -53

April 2002 W.W. 5

39.2 65.0 +66 13.2 8.9 -32

June 2002 W.W. 6

50.2 65.1 +30 15.3 12.6 -17

Mean 42.2 65.2 +58 17.9 12.1 -33 Standard deviation

6.3 0.8 23.6 5.7 4.2 11.6

% CV 15.0 1.25 41 31.6 34.8 35 In study number 7 three concentrations of dinoseb were tested, the results are shown in Table 5. In this study a marked inhibition of nitrate formation was seen at 45 mg/kg but an increase was seen at 5 mg/kg and 15 mg/kg, an increase was also seen in studies 1 to 6 at 10 mg/kg. However, strong interference with nitrogen-transformation at both 15 and 45 mg dinoseb/kg was apparent because high ammonium-nitrogen concentrations of 7.28 and 57.5 mg ammonium-N/kg respectively, were found compared with <0.3 mg/kg in the control and 5 mg/kg dinoseb treated soil. This indicates the importance of reporting soil ammonium-N as well as soil nitrate-N for soil nitrogen transformation studies. Soil respiration was inhibited at 5 mg/kg and 15 mg/kg, but the apparent inhibition was reduced at 45 mg/kg. This was because soil respiration rate was taken as the mean value for 12 consecutive hours of measurement (following the procedure described in OECD 217). At 45 mg/kg the soil was initially inhibited during the 12-hour soil respiration measurement (at the day 28 time-point) but showed recovery to levels well above those in the controls from 9 to 12 hours. This can be seen in Figure 4 where mg CO2/kg/hour measured each hour is plotted against time in hours over the 12-hour period of measurement. This demonstrates the

101

importance of examination of the shape of the respiration curves when reporting effects on soil respiration. Table 5. Summary of effects of three concentrations of dinoseb on soil microflora (study number 7).

Dinoseb concentration

(mg/kg)

Time-point (day

number)

Nitrate concentration (mg N/kg)

% difference from control

Soil respiration (mg CO2/kg/h)

% difference

from control

0 (control)

0 14 28

10.3 22.1 36.3

– – –

30.9 25.7 25.7

– – –

5 mg/kg

0 14 28

10.0 41.2 51.7

-3% +86% +42%

25.0 20.9 20.0

-19% -19% -22%

15 mg/kg

0 14 28

9.9 14.9 60.6

-4% -33% +67%

22.3 20.7 16.9

-28% -20% -35%

45 mg/kg

0 14 28

10.1 8.8 21.6

-2% -60% -41%

18.0 30.1 23.0

-42% +17% -11%

Figure 2. Effects of dinoseb on respiration in soil (OECD-217) showing means and 95% confidence intervals for control soil and soil treated with dinoseb at 10 mg/kg.

0

5

10

15

20

25

30

1 2 3 4 5 6Study Number

mg

CO

2/100

g dr

y so

il

control10 mg/kg

102

Figure 3. Effect of dinoseb on N-transformation in soil (OECD-216) showing means and 95% confidence intervals for control and dinoseb treated soil at 10 mg/kg dry soil.

Figure 4. Dinoseb: effects on soil respiration after 28 days exposure. Test 7 (January 2003). References Council Directive of 15 July 1991 concerning the placing of plant protection products on the

market (91/414/EEC). – Official Journal of the European Communities L 230, 19 August 1991.

0

10

20

30

40

50

60

70

80

1 2 3 4 5 6

Study Number

Nitr

ate

(mg

N/k

g so

il)

control10 mg/kg

0,00

10,00

20,00

30,00

40,00

50,00

60,00

1 2 3 4 5 6 7 8 9 10 11 12

hours of respiration measurement

mg

CO

2/kg/

hour

control5 mg dinoseb15 mg dinoseb45 mg dinoseb

103

ISO 11268-1 (E). 1993: Soil Quality - Effects of pollutants on earthworms (Eisenia fetida): Part 1: Determination of acute toxicity using artificial soil substrate.

ISO 11268-2 (E). 1998: Soil Quality - Effects of pollutants on earthworms (Eisenia fetida): Part 2: Determination of effects on reproduction.

Kula, C. 1998: in Sheppard Sheppard, S.C., Bembridge, J.D., Holmstrup, M., Posthuma, L.: Advances in earthworm ecotoxicology. Proceedings from the Second International Workshop on Earthworm Ecotoxicology. 2-5 April 1997. Amsterdam, The Netherlands. Pensacola FL: Society of Environmental Toxicology and Chemistry (SETAC): 472 pp.

OECD Guidelines for the Testing of Chemicals – 207: Earthworm, acute toxicity test: Adopted 04 April 1984.

OECD Guidelines for the Testing of Chemicals – 216: Soil Microorganisms: Nitrogen Transformation Test: Adopted 21 January 2000.

OECD Guidelines for the Testing of Chemicals – 217: Soil Microorganisms: Carbon Transformation Test: Adopted 21 January 2000.

Spurgeon, D.J. and Weeks, J.M. 1998: in Sheppard, S.C., Bembridge, J.D., Holmstrup, M., Posthuma, L.: Advances in earthworm ecotoxicology. Proceedings from the Second International Workshop on Earthworm Ecotoxicology. 2-5 April 1997. Amsterdam, The Netherlands. Pensacola FL: Society of Environmental Toxicology and Chemistry (SETAC): 472 pp.

Yeardly, R.B., Lazorchak, J.M. and Pence, M.A. 1995: Evaluation of Alternative Reference Toxicants for use in the Earthworm Toxicity Test. – Environmental Toxicology and Chemistry: 14 (7): 1189-1194.


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Influence of some pesticides on fecundity and longevity of Coccinella septempunctata and Adalia bipunctata (Col., Coccinellidae) under laboratory conditions Remigiusz W.Olszak1, Piotr Ceryngier2, Wojciech Warabieda 1

1 Institute of Pomology and Floriculture, Pomologiczna 18, 96-100 Skierniewice, Poland 2 Institute of Ecology, Dziekanów Lešny, 05-150 Lomianki, Poland The influence of 7 pesticides (6 insecticides and 1 acaricide) on different stages (adults, larvae, eggs) of C. septempunctata and adults of A. bipunctata was evaluated under laboratory conditions by: 1 immersing the adult individuals for 5 s in the pesticide solution; 2 immersing the egg clusters (one, two and three days old) of C. septempunctata for 5 s in

the pesticide solution; 3 placing the second and fourth instar larvae on leaves picked from trees treated with the

pesticide; 4 feeding adults of C. septempunctata and A. bipunctata with aphids contaminated by a

recommended concentration of the pesticide. It was found that food (aphids) contaminated with such chemicals as pirimicarb,

novaluron, pyriproxyfen and fenpyroximate did not decrease neither the longevity nor the fecundity of females of both tested species. The much worse results were obtained with acetamiprid in two differing formulations and the worst with fenitrothion. In the case of larval stages L2 and L4 the worst results were obtained with novaluron and fenitrothion, however the mortality varied with stage and mode of treatment. The tested preparations influenced also the eclosion of larvae from eggs immersed in their solutions. The lowest number of larvae has hatched from the two-days-old egg clusters immersed on the second day after having been laid.


p. 107

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Non-target arthropod field studies: asking the right questions for their purpose Kevin Brown Ecotox Limited, Tavistock, Devon, England, PL19 0YU Large-scale field studies with non-target arthropods are expensive and complex. Despite very large inputs their outputs are often surprisingly simplistic. Such studies typically generate information about the range of taxa affected (proportion of the sampled fauna showing a treatment related reduction in numbers), the magnitude of those effects (often expressed as a percentage reduction in numbers sampled) and the duration of such effects (time taken for recovery to occur). For registration purposes recovery is taken as being the critical end-point for acceptability of effects. However, there is no accepted definition of recovery. Moreover, where do these "recoverers" come from and would they reappear in the same way under commercial agriculture.

Our experience has shown that for information on effects and recovery to be meaningful it has to be generated and interpreted at the species level. Pitfall trap data for carabid and staphylinid species from arable studies over the past ten years are widely open to misinterpretation. Taken at the family level, the presence of large numbers of robust individuals (e.g. Pterostichus melanarius) could lead to a conclusion that a product is harmless to carabid beetles whereas less abundant but more sensitive species from the same treatments show marked effects. Hedgerow dwelling species such as Nebria brevicollis could be perceived to be unaffected, whereas in fact they may have moved into the crop each night from untreated field margins. With hedge margins to each replicate the true effects of individual treatments were masked by a reservoir of fresh immigrants. Interpretation of field studies data has to be related to the ecology of the organisms being collected in the samples. This sounds fine in practice but is limited by two factors. Firstly there is a practical difficulty in identifying specimens to species level. This results in data skewed for those groups such as Carabidae with relatively good keys or obvious markings. Secondly, there is surprisingly limited information about the ecology of many non-target arthropod groups.

Whilst it is not worthwhile to identify every specimen to species level there is much to be gained from identifying a wider range of species than is currently done and from recognising that some species are more useful than others as indicators of pesticide effects within experimental systems.


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Sampling methods in orchard trials: Assessment of treatment effects through beating and inventory sampling Jutta Müther1 & Heidrun Vogt2 1 GAB Biotechnologie GmbH, Eutinger Str. 24, D-75223 Niefern-Öschelbronn, Germany 2 BBA, Institute for Plant Protection in Fruit Crops, Schwabenheimerstr. 101,

D-69221 Dossenheim, Germany Two methods to assess leaf dwelling arthropods in the tree canopy of orchards were compared: beating sampling and inventory sampling. Both methods were applied at the same time in the same apple orchard in Southern Germany during summer 2002. Besides the arthropods collected, supplementary information concerning time, manpower and necessary plot sizes was assessed. At last years meeting of the IOBC WG „Pesticides and Beneficial Organisms“ the results from the pre-treatment assessment in the control plot were presented and are published in the proceedings of the meeting (Müther & Vogt, 2003). Now we present the results about the treatment effects. Pre-sampling was carried out on 23rd July, treatment with ME 605 Spritzpulver (a.i. 405g/kg parathion-methyl) (500g/ha in 1000 l water) took place on 29rd July and the post-treatment sampling was made on 7th August. There were 5 replicates for each sampling type, each comprising the catches of 1 tree per inventory sample and 20 branches per beating sample, respectively.

Excluding spot wise occurring arthropods the variability between replicates was comparable for both sampling methods, with few exceptions. The mean number of arthropods in the inventory samples was 3 times higher than in the beating samples in the pre-treatment sampling, and 2 times higher in the post-treatment samples. Both methods resulted in similar and highest effect sizes (Henderson & Tilton) for Opiliones (> 80 %), Dermaptera (F. auriculuria) (>90 %), and Heteroptera (>76 %). With regard to Araneae, Homoptera (mainly Cicadellidae) and Neuroptera (mainly Chrysopidae), the inventory samples revealed higher effects than the beating samples. References Müther, J. & H. Vogt (2003): Sampling methods in orchard trials: A comparison between

beating and inventory sampling. – IOBC/wprs Bulletin 26 (5): 67-72.


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Comparison between two different collecting methods of Anthocoris nemoralis F. in investigations about side-effects of pesticides in field tests Vergnani S.1, Melandri M.2, Manucci F.2, Civolani S.3, Pasqualini E.3

1 CRPV, Via Vicinale Monticino, 1969 - 47020 - Diegaro (FO) (Italy) 2 Research and Development Terremerse, Via Cà del Vento, 21 - Bagnacavallo (RA) (Italy) 3 DiSTA (University of Bologna), Via G. Fanin, 42 - 40126 - Bologna (Italy) Anthocoris nemoralis is the main predator of Cacopsylla pyri L. on pear orchards of Emilia-Romagna region. The control strategy against C. pyri is considered of basic importance for the Integrated Pest Management programs (IPM). The insecticides applied for the defence strategies against other pest species (i.e. Cydia pomonella, C. molesta, leafrollers, etc.) may interfere with the development of predator’s populations obstructing their activity and the natural control of C. pyri. The correct evaluation about the selectivity of the insecticides used is then a strategic importance acknowledge factor. A difficulty about the evaluation of the selectivity of the insecticide products in the field is the high variability of the collected data, probably due to the distribution of the predator and prey’s populations, but also to the area involved and to the number of sampling instruments. So, to reduce the variability of the data, in the inventory samples, two different collecting systems of A. nemoralis are compared: sheets and funnels. Here are presented the results of the investigation carried out on three different populations of A. nemoralis: faults and merits of the two compared sampling systems are analyzed. A qualitative evaluation about the two systems is also reported considering time and applicative techniques.

working group „pesticides and beneficial organisms“ · bouqut, etienne; dow agrosciences,...

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