working group „pesticides and beneficial organisms“ · preface the iobc/wprs working group...
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IOBC / WPRS
Working Group „Pesticides and Beneficial Organisms“
OILB / SROP
Groupe de Travail „Pesticides et Organismes Utiles“
Proceedings of the meeting
at
San Michele All‘Adige, Trento, Italy
3 – 6 October, 2001
editors:
Heidrun Vogt & Udo Heimbach
IOBC wprs Bulletin Bulletin OILB srop Vol. 25 (11) 2002
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 2002
The Publication Commission of the IOBC/WPRS: Horst Bathon Federal Biological Research Center for Agriculture and Forestry (BBA) Institute for Biological Control Heinrichstr. 243 D-64287 Darmstadt (Germany) Tel +49 6151 407-225, Fax +49 6151 407-290 e-mail: [email protected]
Luc Tirry University of Gent Laboratory of Agrozoology Department of Crop Protection Coupure Links 653 B-9000 Gent (Belgium) Tel +32-9-2646152, Fax +32-9-2646239 e-mail: luc.tirry@ rug.ac.be
Address General Secretariat: INRA – Centre de Recherches de Dijon Laboratoire de recherches sur la Flore Pathogène dans le Sol 17, Rue Sully, BV 1540 F-21034 DIJON CEDEX France ISBN 92-9067-148-X Web: http://www.iobc-wprs.org
Preface The IOBC/WPRS Working Group „Pesticides and Beneficial Organisms“ held its annual meeting from 3rd to 6th October 2001 at the “Istituto Agrario San Michele,“ San Michele All‘Adige, Trento, Italy. The first day of the meeting was reserved for subgroup activities, focusing on improvement, harmonization and validation of test methods. The full meeting was attended by 74 participants from 10 countries. The presentations covered diverse topics in side-effect research, focusing on different beneficial organisms, different testing tiers (from laboratory to field) and methodical aspects. One session was devoted to the risk of effects to non-target arthropods in off-field areas. A survey with regard to future activities of the WG carried out during the meeting, confirmed that there is a need in further development of higher tier test methodology. Especially questions about test design, evaluation parameters and statistics in field tests need further discussion and elaboration. A lot of thanks are due to the local organizer Dr. Diego Forti and his team, for the excellent organisation. They made sure that everything went smoothly. The participants enjoyed the warm welcome and the comfortable conference site at San Michele Institute. An excursion to the Val di Non with a visit of an apple farm and an apple growers association gave the opportunity to learn details about the Integrated Apple Production in this area as well as to get acquainted with the fascinating scenery of this region. It was also a good opportunity to discuss and exchange experience between the particpants. Heidrun Vogt (Convenor) Dossenheim, 1. September 2002
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List of Participants 1. ABDELGADER, Hayder, Dr., BBA, Institut für Biologischen Pflanzenschutz, Heinrichstr.
243, D-64287 Darmstadt, e-mail: [email protected]
2. ALDERSHOF, Saskia, Universiteit van Amsterdam, Dept. of Pure & Applied Ecology, Sect. Population Biology / MITOX, Kruislaan 320, NL-01098 SM Amsterdam, Niederlande, e-mail: [email protected]
3. ANGELI, Gino, Dr., Istituto Agrario San Michele, All‘Adige, Via E. Mach n° 1, I-38010 San Michele a/A Trento, Italy, e-mail: [email protected]
4. BAIER, Barbara, Dr., Biologische Bundesanstalt für Land- und Forstwirtschaft, Institut für Ökotoxikologie im Pflanzenschutz, Stahnsdorfer Damm 81, D-14532 Kleinmachnow, Germany, e-mail: [email protected]
5. BAKKER, F.M., Dr., Universiteit van Amsterdam, Dept. of Pure & Applied Ecology, Sect. Population Biology/MITOX, Kruislaan 320, NL-1098 SM Amsterdam, Niederlande, e-mail: [email protected]
6. BARGEN, Holger, Dr., GAB, Biotechnologie GmbH, Eutinger Str. 24, D-75223 Niefern-Öschelbronn, Germany, e-mail: [email protected]
7. BARTELS, Anja, Mag., Umweltbundesamt Wien, Abt. Pflanzenschutzmittel und Biozide, Spittelauer Lände 5, A-1090 Wien, Österreich, e-mail: [email protected]
8. BARTH, Markus, BioChem agrar GmbH, Kupferstr. 6, D-04827 Gerichshain, Germany, e-mail: [email protected]
9. BERNARDO, Umberto, Dr., Centro Studi CNR, Technice di Lotta Biologica, Via Università 100, I-80055 Portici (NA), Italy, e-mail: [email protected]
10. BLÜMEL, Sylvia, Dr., AGES, Österreichische Agentur für Gesundheit und Ernährungs-sicherheit, Institut für Phytomedizin, Spargelfeldstr. 191, Postfach 400, A-1226 Wien, Austria, e-mail: [email protected]
11. BOCKSCH, Siegrun, GAB, Biotechnologie GmbH, Eutinger Str. 24, D-75223 Niefern-Öschelbronn, Germany, e-mail: [email protected]
12. BUTTURINI, Alda, Servicio Fitosanitario Regione Emilia-Romagna, Via Corticellia 133, I-40129 Bologna, Italien, e-mail: [email protected]
13. BYLEMANS, Dany, Dr., Royal Research Station of Gorsem, Brede Akker 13, B-3800 Sint-Truiden, Belgium, e-mail: [email protected]
14. CARLI, Guido, Dr., CRPV -, Centro Ricerche Produzioni VegetalI-Via Vicinale Monticino, 1969, I-47020 Diegaro di Cesena (FC), Italy, e-mail: [email protected]
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15. CAROLI, Luigi, Dr., CRPV - Centro Ricerche Produzioni Vegetali-Via Vicinale Monticino, 1969, I-47020 Diegaro di Cesena (FC), Italy, e-mail: [email protected]
16. CHIANELLA, Massimo, Dr., SPP-GAB Italia S.N.C., Via dell´ Artigiannato 11, I-44028 Poggio Renatico, Italien, e-mail: [email protected]
17. CIVOLANI, Stefano, Dr., Universitá degli Studi di Bologna, DiSTA (Diapartimento di Scienze e Tecnologie Agroambientali), Via Filippo Re 6, I-40126 Bologna, Italy, c/o e-mail: [email protected]
18. DRIJVER, Cora, Plant Protection Service, Postbus 9102, NL-6700 Wageningen, The Netherlands, e-mail: [email protected]
19. DUSO, Carlo, Dr., Dipartimento di Agronomia ambientale e Produzioni vegetali, Universita di Padova, Via Romea 16, I-35020 Legnaro - Padova, Italien, e-mail: [email protected]
20. Fayel, Olivier, ENIGMA , Serres, F - 26570 Montbrun, France
21. FORSTER, Rolf, Dr., BBA, Department for Plant Protection, Biology Division, Messeweg 11/12, D-38104 Braunschweig, Germany, e-mail: [email protected]
22. FORTI, Diego, Dr., Istituto Agrario San Michele All‘ Adige, I-38010 San Michele a/A Trento, Italien, e-mail: [email protected]
23. FREIER, Bernd, Dr., Biologische Bundesanstalt für Land- und Forstwirtschaft, Institut für integrierten Pflanzenschutz, Stahnsdorfer Damm 81, D-14532 Kleinmachnow, e-mail: [email protected]
24. GAGNIARRE, Marion, CEREXAGRI-1 rue des frères Lumière, BP 9, F- 78373 Plaisir Cedex, France, e-mail: [email protected]
25. GALASSI, Tiziano, Servicio Fitosanitario Regione Emilia-Romagna, Via Corticella 133, I-40129 Bologna, Italien, e-mail: [email protected]
26. GEUIJEN, Ine, NOTOX B.V., P.O. Box 3476, NL-5203 DL´s-Hertogenbosch, e-mail: [email protected]
27. GOßMANN, Angela, IBACON GmbH, Institut für Biologische Analytik und Consulting GmbH, Arheilger Weg 17, D-64380 Rossdorf, 0049(0)6154-6995-371, e-mail: [email protected]
28. GRAY, Jane, Huntingdon Life Sciences Ltd., Woolley Road, Alconbury Huntingdon, Cambridge PG 28 4HS, Großbritannien, e-mail: [email protected]
29. GRIMM, Christoph, Dr., Syngenta AG, R-1058.3.66, CH-4002 Basel, Schweiz, e-mail: [email protected]
30. HALSALL, Nigel, Dr., Mambo-Tox Ltd., Biomedical Sciences Building, Bassett Crescent East, P.O. Box 2, Southampton SO16 7PX, Großbritannien, e-mail: [email protected]
31. HARGREAVES, Natalie, Syngenta, Jealott's Hill International Research Centre, Bracknell, Berkshire RG42 6EY, Großbritannien, e-mail: [email protected]
32. HEIMBACH, Udo, Dr., BBA, Institute for Plant Protection of, Field Crops and Grassland, Messeweg 11/12, D-38104 Braunschweig, Germany, e-mail: [email protected]
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33. HERRMANN, Petra, Dr., GAB, D-75223, Niefern-Öschelbronn, Germany, e-mail: [email protected]
34. HODGE, Simon, Dr., Institute of Arable Crops Research, Rothamsted Experimental Station, Biological and Ecological Chemistry Department, Harpenden, Hertfordshire AL5 2JQ, Großbritannien, e-mail: [email protected]
35. HUGHES, Jaqueline, Dr., Ecotoxicolgy, Dept. of Environmental Sciences, Inveresk Research, Tranent EH33 2NE, Großbritannien, e-mail: [email protected]
36. JÄCKEL, Barbara, Dr., Pflanzenschutzamt Berlin, Mohriner Allee 137, D - 12347 Berlin, Germany, e-mail: [email protected]
37. KAISER-ARNAULD, Laure, Dr., INRA, Laboratoire de Neurobiologie, Comparée des Invertébrés la Guyonnerie, BP 23, F, 91440, Bures-sur-Yvette, Frankreich, e-mail: [email protected]
38. KENNEDY, Peter, J., Dr., Syngenta AG, Jealott's Hill International Research Centre, Bracknell, Berkshire RG42 6EY, Großbritannien, e-mail: [email protected]
39. KNÄBE, Silvio, Dr., GAB, Biotechnologie GmbH, Eutinger Str. 24, D-75223 Niefern-Öschelbronn, Germany, e-mail: [email protected]
40. KOCH, Heribert, Dr., Landesanstalt für Pflanzenschutz, und Pflanzenbau, Essenheimer Str. 144, D-55128 Mainz, Germany, e-mail: [email protected]
41. KOLLMANN, Stefanie, Dipl.-Ing. Agr., Springborn Laboratories (Europe) AG, Seestr. 21, CH-9826 Horn, Switzerland, e-mail: [email protected]
42. LAUDONIA, Stefania, Dr., Universita' di Napoli Federico II-Dipartimento di Entomologia E Zoologia Agraria, Via Università 100, I-80055 Portici (NA), Italy, e-mail: [email protected]
43. LÜCKMANN, Johannes, IBACON GmbH, Institut für Biologische Analytik und Consulting GmbH, Arheilger Weg 17, D-64380 Rossdorf, e-mail: [email protected]
44. MARTIN, Sabine, Dr., Umweltbundesamt, Postfach 33 00 22, D-14191 Berlin, Germany, e-mail: [email protected]
45. MAUS, Christian, Dr., BAYER AG, Landwirtschaftszentrum Monheim, Gebäude 6620, D-51368 Leverkusen, Germany, e-mail: [email protected]
46. MÉGEVAND, Benoit, Dr., IMPACTEST, Aevnida Almirante Reis, 204, 7° Dto, 100-056 Lissabon, Portugal, e-mail: [email protected]
47. MOLL, Monika, Dr., IBACON, Institut für Biologische Analytik und Consulting GmbH, Arheilger Weg 17, D-64380 Rossdorf, Germany, e-mail: [email protected]
48. MORESCHI, Ivana, Dr., Istituto di Entomologia agraria, Università degli Studi di Milano, Via Celoria, 2, I-20133 Milano, Italien, e-mail: [email protected]
49. MOULONGUET, Gilles, PROMO-VERT S.A., Z.I. du Haut Ossau, Rue d'Aste Béon, BP 27, F-64121 Serres Castet, Frankreich, e-mail: [email protected]
50. MÜTHER, Jutta, Dr., GAB, Biotechnologie GmbH, Eutinger Str. 24, D-75223 Niefern-Öschelbronn, Germany, e-mail: [email protected]
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51. NIENSTEDT, Karin, Dr., Springborn Laboratories (Europe) AG, Seestr. 21, CH-9326 Horn, Switzerland, e-mail: [email protected]
52. PASQUALINI, Edison, Dr., Universitá degli Studi di Bologna, DiSTA (Diapartimento di Scienze e Tecnologie Agroambientali), Via Filippo Re, 6, I-40126 Bologna, Italy, e-mail: [email protected]
53. PEAR, Nicholas, FMC Chemical, sprl, 480 Avenue Louise, B-1050 Brussels, Belgien
54. PETTO, Ralf, Dr., IBACON GmbH, Institut für Biologische Analytik und Consulting, Arheilger Weg 17, D-64380 Roßdorf, e-mail: [email protected]
55. PHILLIPS, David, Covance Laboratories LT D-Otley Road Harrogate, North Yorkshire HG3 1PY, Großbritannien, e-mail: [email protected]
56. POULLOT, Delphine, ENIGMA, Hameau de St. Véran, F-84190 Beaumes de Venise, France, e-mail: [email protected]
57. RODRIGUES, J. Raul, Dep. Ciéncias da Planta e do Ambienta, Escola Superior Agrária de Ponte de Lima, Convento do Refóios, P-49909-706 Ponte de Lima, Portugal, e-mail: [email protected]
58. RÖHLIG, Uta, BioChem agrar GmbH, Kupferstr. 6, D-04827 Gerichshain, Germany, e-mail: [email protected]
59. SCHMIDT, Thomas, Dr., Pflanzenschutzzentrum der Bayer AG, Institut für Ökobiologie, D-40789 Monheim, Germany, e-mail: [email protected]
60. SCHULD, Michael, GAB, Biotechnologie GmbH, Eutinger Str. 24, D-75223 Niefern-Öschelbronn, Germany, e-mail: [email protected]
61. SECHSER, Burkhard, Dr., Bodenacker 73, CH-3065 Bolligen, Switzerland, [email protected], Privat: e-mail: [email protected]
62. TEDESCHI, R., Di.Va.P.R.A. Entomologia e Zoologia applicate all'Ambiente, via Leonardo da Vinci 44, I-10095 Grugliasco (Torino), e-mail: [email protected]
63. TESSIER, Céline, PROMO-VERT S.A., Z.I. du Haut Ossau, Rue d'Aste Béon, BP 27, F-64121 Serres Castet, France, e-mail: [email protected]
64. TISO, Rocchina, Servicio Fitosanitario Regione Emilia-Romagna, Via Corticella 133, I-40129 Bologna, Italien, e-mail: [email protected]
65. TORNIER, Ingo, Dr., GAB, Biotechnologie GmbH, Eutinger Str. 24, D-75223 Niefern-Öschelbronn, Germany, e-mail: [email protected]
66. UFER, A., Dr., BASF AG, Agrarzentrum Limburgerhof, APD/RL-LI 426, Postfach 120, D-67114 Limburgerhof, Germany, e-mail: [email protected]
67. VALS, Salvatore, ICAS Tulcea, Str. IsacceI-25, 8800 Tucea, Rumänien, e-mail: [email protected] or [email protected]
68. VAN DE VEIRE, Marc, Dr., University of Gent, Laboratory of Agrozoology, Coupure Links 653, B-9000 Ghent, Belgien, e-mail: [email protected]
69. VIGGIANI, G., Prof. Dr., Universita' di Napoli Federico II-Dipartimento di Entomologia E Zoologia Agraria, Via Università 100, I-80055 Portici (NA), Italy, e-mail: [email protected]
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70. VERGNET, Christine, Dr., Structure Scientifique Mixte (SSM) - INRA/DGAL, Route de Saint-Cyr, F - 78026 Versailles Cedex, France, e-Mail: [email protected]
71. VINALL, Stephen, Mambo-Tox Ltd., Biomedical Sciences Building, Bassett Crescent East, P.O. Box 2, Southampton SO16 7PX, Großbritannien, e-mail: [email protected]
72. VOGT, Heidrun, Dr., Federal Biological Research Centre for Agriculture and Forestry (BBA), Institute for Plant Protection in Fruit Crops, Schwabenheimerstr. 101, D-69221 Dossenheim, Germany, e-mail: [email protected]
73. WAINWRIGHT, Melanie, Miss, Huntingdon Life Sciences Ltd. Woolley Road, Alconbury Huntingdon, Cambridge PG 28 4HS, Großbritannien, e-mail: [email protected]
74. WALTERSDORFER, Anna, Dr., Aventis CropScience GmbH, Industriepark Höchst, H872, D-65926 Frankfurt, Germany, e-mail: [email protected]
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Contents Preface......................................................................................................................................... i List of participants..................................................................................................................... iii PRESENTATIONS MAINLY RELATED TO METHODICAL ASPECTS
Side-effects of plant protection products on mortality and reproductive capacity of the aphid parasitoid Aphidius rhopalosiphi (DeStefani-Perez) (Hymenoptera, Braconidae) under semi-field conditions Schuld, M. & Moll, M. ........................................................................................................ 1
Adaptation of standard regulatory non-target arthropod test methods for testing of plant protection products with special modes of action Grimm, C., Reber, B., Gray, A. & Candolfi, M.P. ............................................................. 7
First ring test results of a laboratory method to evaluate effects of plant protection products on larvae of Poecilus cupreus (Coleoptera: Carabidae) Heimbach, U. ................................................................................................................... 19
Extended laboratory investigations for evaluating the effect of Karate® on females of the predatory mite species Typhlodromus pyri Scheuten (Acari: Phytoseiidae) Baier, B. & Moll, E. ......................................................................................................... 27
Data variability in carabid field studies (Coleoptera: Carabidae) and how to deal with small-scaled inhomogeneities of environmental conditions Schmidt, Th., Schmuck, R. & Maus, Ch. .......................................................................... 37
PRESENTATIONS MAINLY RELATED TO RESULTS OF TESTING SIDE-EFFECTS OF PESTICIDES – on predatory mites
Results of the 8th and 9th IOBC Joint Pesticides Testing Programme: Persistence test with Phytoseiulus persimilis Athias Henriot (Acari: Phytoseiidae) Blümel, S. & Hausdorf, H. ............................................................................................... 43
Side-effects of fifteen insecticides on predatory mites (Acari: Phytoseiidae) in apple orchards Rodrigues, J. R., Miranda, N. R. C., Rosas, J.D. F., Maciel, C.M. &. Torres, L.M ......... 53
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– on Trichogramma
Side effects of plant protection products on Trichogramma cacoeciae Marchal (Hym. Trichogrammatidae) Abdelgader, H. & Hassan, S. A. ....................................................................................... 63
– on Macrolophus
Toxicity of different pesticides to the predatory bug Macrolophus caliginosus (Heteroptera: Miridae) under laboratory conditions Tedeschi, R., Tirry, L., Van de Veire, M. & de Clercq, P. ............................................... 71
– on Rodolia
Effects of Oikos (a. i. Azadirachtin A) on the vedalia ladybeetle Rodolia cardinalis (Mulsant) (Coleoptera: Coccinellidae) Bernardo, U. & Viggiani, G. ............................................................................................ 81
– on Apis
Side-effects of the microencapsulated Fenitrocap/IPM-400 (fenitrothion) and Pyrinex (chlorpyrifos ethyl) on Apis mellifera L. (Hymenoptera: Apidae) G. Angeli, Berti, M., Gottardini, E., Cristofolini, F.& Forti, D. ...................................... 89
– on pear fauna
Mimic-Confirm (a. i. Tebufenozide): a tool for a soft and ecologically sound pest control in pear orchards Pasqualini, E. & Civolani, S. ........................................................................................... 97
PRESENTATIONS MAINLY RELATED TO RISK FOR NON-TARGET ORGANISMS IN OFF-FIELD AREAS New restrictions for the use of pesticides to protect off-field
non-target organisms Forster, R., Kula, C., Gutsche, V. & Enzian, S. ............................................................. 107
Three-year study of the effects of Karate® applications in wheat on arthropod communities in a field margin – Results and the problem of small numbers Freier, B., Kühne, S., Kaul, P., Baier, B., Moll, E., Jüttersonke, B. & Forster, R. ........ 115
Off-crop pesticide drift deposition Koch, H. ......................................................................................................................... 121
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Side effects of plant protection products on mortality and reproductive capacity of the aphid parasitoid Aphidius rhopalosiphi (DeStefani-Perez) (Hymenoptera, Braconidae) under semi-field conditions M. Schuld(1) & M. Moll(2)
(1) GAB-Biotechnologie, Eutingerstr. 24, D-75223 Niefern-Öschelbronn, Germany (2) IBACON GmbH, Arheilger Weg 17, D-64380 Rossdorf, Germany Abstract: A semi-field test with Aphidius rhopalosiphi was carried out at two independent test facilities (GAB-Biotechnologie and IBACON) to investigate the effects on mortality and reproductive capacity under outdoor climatic conditions. For each, the mortality and reproductive capacity part, pots with barely planted in the centre area were sprayed with 5 rates of Perfekthion (400 g Dimethoate/L) and with deionized water as control. Each treatment group of both experimental parts included 3 replicates. 15 female parasitoids per replicate were introduced into the exposure units. To assess the mortality one sticky yellow trap per replicate was placed inside the exposure units after 24 hours of exposure to recapture the survivors. After 48, 72 and 96 hours of exposure the captured wasps on the traps were counted. To assess the reproductive capacity after 24, 48 and 72 hours of exposure barley seedlings grown in pots and infested with aphids (Rhopalosiphum padi) were placed around the treated plants (5 pots per replicate). All pots were replaced after a time of 24 hours. Ten to twelve days after the parasitation period the developed mummies on the barely were counted for each replicate separately. Key words: higher tier study, A. rhopalosiphi, semi-field, dose response relationship, mortality,
reproductive capacity Introduction The semi-field test is a possibility as the following step if an extended laboratory test showed adverse effects. The test design was based on the extended laboratory method developed by Mead-Briggs (1997). The study was carried out twice at IBACON and GAB, two independent testing facilities, working on side effect testing of plant protection products (PPP), at the same time with test and host organisms from the same origin. Based on the results evaluated in 2000 the semi-field test with A. rhopalosiphi was repeated with the same design to assess sublethal effects on reproduction (Moll & Schuld 2001). Additionally the semi-field test was enlarged by a second experimental part to evaluate direct toxic effects on the introduced parasitoids under outdoor climatic conditions. Material and methods To evaluate the side effects of PPP on mortality and fecundity of the aphid parasitoid A. rhopalosiphi under semi-field conditions a test with five different rates of Perfekthion (400 g Dimethoate/L) and a water treated control was performed. The study contained two experimental parts: Assessment of mortality with yellow traps and assessment of reproduction with aphid infested barley seedlings.
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The exposure units were placed under a UV permeable roof, in order to protect them against rain. Air temperature and relative humidity were measured with a data logger during the exposure phase. Approximately 50 - 60 barley seedlings were planted in pots of 32 cm diameter in a central area of approximately 20 cm in diameter. The seedlings were sprayed with 5 rates of Perfekthion. The application rates were 5, 10, 20, 30 and 40 mL per ha in a water volume of 400 L per ha. In addition, a tap water treated control was prepared with the same water volume. Each treatment group consisted of 6 replicates (3 replicates for mortality assessment and 3 replicates for reproduction assessment). Before application the plants were sprayed with a 25 % fructose solution, to increase the attractiveness of the treated plants for the parasitoids. After the spray residue had tried, at latest within one hour after the treatment, the pots were covered with a fine mesh gauze. Then 15 female parasitoids (obtained by PK Nützlingszuchten, Dr. Peter Katz, Industriestr. 38, D-73642 Welzheim) per replicate, less than 48 hours old, were introduced into the exposure units. For the assessment of mortality and reproduction the experiment was divided into two parts, each with 3 replicates. For the mortality part one sticky yellow trap (Fa. Neudorff, Germany “Gelbtafeln Hobby”, 7.5 x 20 cm, both sides sticky) was placed approximately 10 cm above the treated plants into each exposure unit 24 hours after introduction of the parasitoids. The yellow traps were checked 48, 72 and 96 hours after application and the number of caught wasps was recorded. In order to test the fecundity, reproduction units with potted barley seedlings, infested with Rhopalosiphum padi (obtained by PK Nützlingszuchten, Dr. Peter Katz, Industriestr. 38, D-73642 Welzheim), were placed around the treated plants in each exposure unit after an exposure period of 24 hours. These reproduction units were replaced by new ones after 24 hours and this procedure was repeated once. In total, 15 reproduction units per replicate were obtained. The reproduction units were transferred into the laboratory in a climatic chamber with a temperature of 20 °C ± 2 °C and relative humidity of 70 ± 20 %, under long day conditions (16 h light, 8 h dark, with more than 2000 lux light intensity). Before the test units were transferred to the laboratory, the plants were checked for female parasitoids. Detected parasitoids were transferred back to the exposure units. Ten to twelve days after the parasitation period the developed mummies on the barley seedlings were counted for each replicate separately. Some aphids migrated from the reproduction units onto the treated plants during the parasitization phases. Since they could have been parasitised as well, the exposure units were checked for mummies too . Statistical analysis was performed with SAS 6.12. Results and discussion In 2001 the parasitation rates were very low in comparison to the study in the year before. In 2000 16.7 and 10.8 mummies per female were obtained at GAB and IBACON, respectively, compared to 3.3 and 3.4 mummies per female at GAB and IBACON, respectively, in 2001. Only at GAB the test results obtained last year were reproduced and a dose response relationship could be calculated (Figure 1). The difference between LR50 in 2000 and 2001 is a factor of 2. The ER50 was 6.016 mL Perfekthion/ha which is between the ER50 values evaluated last year at IBACON and GAB (10.5 mL Perfekthion/ha at GAB and 2.4 mL Perfekthion/ha at IBACON).
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01020304050607080
5 10 20 30 40
Perfekthion [mL/ha]
IBACONGAB
Para
sita
tion
Rat
e [%
]
Figure 1: Mean parasitation rate relative to the control No relation between the percentage of caught wasps per 15 females on the sticky yellow traps and the rate of Perfekthion could be detected (Figure 2).
0
5
10
15
20
25
30
0 5 10 20 30 40
Perfekthion [mL/ha]
Cap
ture
d W
asps
[%]
IBACONGAB
Figure 2: Mean percentage of caught wasps
Mean temperature during exposure of the semi-field study at IBACON in Darmstadt, which is approximately in the middle west of Germany and at GAB in Pforzheim which is a bit more in the south of Germany seemed to be nearly the same, but differences in the maximum temperature are obvious (Figure 3). Mean relative humidity during exposure of the semi-field study seemed to be also nearly the same at GAB and IBACON, but differences in minimum relative humidity are obvious too (Figure 4).
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10152025303540
30.07.01 31.07.01 01.08.01 02.08.01 03.08.01
Date
Tem
pera
ture
[°C
]
IBACON
GAB
Figure 3: Mean air temperature during exposure and parasitization phase of the semi-field
study (thin lines on the bars indicate the maximum temperature)
01020304050607080
30.07.01 31.07.01 01.08.01 02.08.01 03.08.01
Date
IBACON
GAB
Rel
ativ
e H
umid
ity [%
]
Figure 4: Mean relative humidity during exposure and parasitation phase of the semi-field
study (thin lines in the bars indicate the minimum relative humidity)
To discuss the different results obtained in 2000 and 2001 the similarities are summarised in the following: – Time: July the 30th to August the 3rd. – Test organisms and host organisms from the same origin and with the same age.
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– Same food, (barley seedlings were sprayed over before treatment with a 25 % fructose solution until the point of running off).
– Exposure units: same gauze, size, number of plants, age of plants. – Reproduction units: same size, number of plant, number and origin of host aphids – Handling of reproduction units during their change (controlling of wasps under a glass
cage).
A possible reason for the observed differences in the studies carried out in 2000 and 2001 could be the higher temperature and the lower relative humidity in 2001 compared to 2000 (Figure 5 and 6). The mean temperature was about 5 °C higher in the year 2001. Concerning the mean relative humidity the differences were about 30 % at IBACON and about10 % at GAB compared to 2000.
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10152025303540
Mean Temp.IBACON
Mean Temp. GAB
Tem
pera
ture
[° C
]
20002001
Figure 5: Mean temperature 2000 versus 2001 (thin lines on the bars indicate the maximum
temperature)
0
20
40
60
80
100
Mean Rel. Hum.IBACON
Mean Rel. Hum.GAB
rela
tive
hum
idity
[%]
20002001
Figure 6: Mean relative humidity 2000 versus 2001 (thin lines in the bars indicate the
minimum relative humidity)
6
The differences in the results obtained in 2001 between GAB and IBACON were possibly due to the lower relative humidity in 2001. According to the experience of the ring-test group mortality of the parasitoids was found to increase where the relative humidity was maintained below 50 %. The unusable results from the yellow traps were maybe due to high temperatures. According to these results the best time for performing such a kind of test seems to be spring or autumn and not summer. Acknowledgements The authors thank the technical personnel who were mostly responsible for the successful practical performance of the studies. References Mead-Briggs M. 1997: A standard ‘extended laboratory’ test to evaluate the effects of plant
protection products on adults of the parasitoid, Aphidius rhopalosiphi (Hymenoptera, Braconidae), (unpubl.).
Moll M. & Schuld M. 2001: A semi-field test for evaluating the side-effects of plant protection products on the aphid parasitoid Aphidius rhopalosiphi (DeStefani-Perez) (Hymenoptera, Braconidae) – First results. IOBC/WPRS Bulletin 24 (4), 67-70.
Pesticides and Beneficial Organisms IOBC/wprs Bulletin Vol. 25 (11) 2002
pp. 7 -17
7
Adaptation of standard regulatory non-target arthropod test methods for testing of plant protection products with special modes of action C. Grimm, B. Reber, A. Gray and M.P. Candolfi Syngenta Crop Protection AG, CH-4002 Basel, Switzerland Abstract: Standard methods for testing the effects of plant protection products on non-target arthropods for regulatory approval have been recently published by the IOBC, BART and EPPO Joint Initiative. However, most of the published methods only partially cover the testing requirements for compounds with special modes of action (e.g. insect growth regulators). For this type of product, sensitive species should be tested at the potentially most susceptible life stage taking into account the appropriate route of uptake. Standard designs for extended laboratory tests using natural substrates (leaves, whole plants or soil) were adapted for bioassays with the following species and life stages: Typhlodromus pyri egg test, T. pyri protonymph test with contaminated food, Aphidius rhopalosiphi mummy test with reproduction phase, A. rhopalosiphi adult test with contaminated food, Chrysoperla carnea egg test, C. carnea larval test with contaminated food, Coccinella septempunctata egg test, C. septempunctata larval test with contaminated food, Orius laevigatus egg test, O. laevigatus larval test with contaminated food, Poecilus cupreus larval test, and P. cupreus test on overwintered adults with contaminated food and a reproduction phase. All tests were set up as dose-response tests for estimating the LR50. For the later life stages of each species, fertility was assessed and the <50%OER (less than 50 % observable adverse effect rate) on the reproductive capacity was determined. Key words: Aphidius rhopalosiphi, Chrysoperla carnea, Coccinella septempunctata, extended laboratory test, insect growth regulators, non-target arthropods, Orius laevigatus, plant protection products, Poecilus cupreus, Typhlodromus pyri Introduction The ESCORT 2 Workshop at Wageningen produced a guidance document on the testing of plant protection products with non-target arthropods and risk assessment procedures for registering plant protection products in the European Union (Candolfi et al., 2001). This system consists of a sequential testing scheme in which indicator species are used to establish the dose-response relationship between the tested products and the toxicity to the test organisms, expressed as the LR50, the application rate causing 50 % mortality (Grimm et al., 2001).
The actual testing is carried out according to the technical guidelines set up by the IOBC, BART and EPPO Joint Initiative for the evaluation of potential side-effects of plant protection products to non-target arthropods (Candolfi et al., 2000). However, some plant protection products with special modes of action, as for instance insect growth regulators and insect feeding inhibitors, are exempt from the standard procedures. It is suggested that testing of such products should be conducted with Typhlodromus pyri and one additional species that is likely to be sensitive. Proposed species are Chrysoperla carnea, Coccinella septempunctata or Orius laevigatus (Candolfi et al., 2001).
Testing should focus on those stages that are likely to be affected, e.g. juvenile stages in the case of insect growth regulators, and should take into account the appropriate routes of uptake. In order to be consistent with the hazard quotient approach proposed for the risk
8
assessment for products with a conventional mode of action, the primary toxic endpoint of the studies should be mortality expressed as an LR50 value. If effects on reproduction are expected, appropriate sublethal parameters should also be assessed. As an indicator for the hazard potential of the tested product, the toxicity data are compared to the relevant exposure rates expected within and outside of the crop area. A 50 % trigger value is suggested for both lethal and sublethal endpoints.
In order to obtain the data required for such a risk assessment, the testing guidelines recommended by the IOBC, BART and EPPO Joint Initiative need to be subjected to certain adaptations concerning the life stage of the tested arthropods and the mode of uptake. In order to demonstrate the feasibility of such an approach, examples of methods adapted for testing an insect growth regulator with contact and stomach action are presented for two life stages each of six different beneficial arthropod species: Typhlodromus pyri Scheuten (Acari: Phytoseiidae), Aphidius rhopalosiphi (DeStefani-Perez) (Hymenoptera: Braconidae), Chrysoperla carnea Stephens (Neuroptera: Chrysopidae), Coccinella septempunctata L. (Coleoptera: Coccinellidae), Orius laevigatus (Fieber) (Heteroptera: Anthocoridae) and Poecilus cupreus L. (Coleoptera: Carabidae).
The methods described below are to be considered as proposals. The exact methods used for any product will depend on the mode of action of the tested compound. Material and methods All tests were carried out as extended laboratory tests using soil, leaves or whole plants as substrate. The tests were carried out with a non-neurotoxic insect growth regulator with contact and stomach action. They were dose-response tests with five to six different application rates in a geometric progression. The rates were based on results of range finder tests, but did not exceed the two-fold maximum recommended field rate. All applications were carried out with a track sprayer calibrated to an output of 200 L /ha for the tests on plants or excised leaves and 400 L /ha on soil. The environmental conditions (temperature, light, humidity) were as given in the guidelines for standard tests with the same species (Candolfi et al., 2000). The endpoints were mortality expressed as an LR50 and reproductive capacity expressed as the <50%OER, the highest tested application rate resulting in less than 50 % observable adverse effect. For the egg tests the LR50 reflected both the egg hatch and the development to the adult stage. In the larval tests the LR50 was based on adult development, and for tests carried out at the adult stage the LR50 was based on mortality during a predefined assessment period. Reproduction assessments were carried out in the tests with the later of the two tested life-stages, with the exception of A. rhopalosiphi, where reproductive capacity was established for both tested stages.
The LR50-values were calculated with standard Probit or generalised Probit models according to the maximum likelihood method (Finney, 1971) wherever possible. If the data did not fit the models, the moving average-angle method was used (Harris, 1957) as suggested by Grimm et al. (2001). Dichotomous data were analysed for significance at the 5 % level with 2 x 2 contingency tables using one way χ2- or Fisher exact tests (Zar, 1984). Continuous data were tested for normality and for homoscedasticity and if these assumptions were met, the Dunnett test was used to determine significant differences at the 5 % level (Zar, 1984). Typhlodromus pyri egg test The toxicity to eggs was tested on treated bean (Phaseolus vulgaris) leaves in a test based on the method described by Oomen (1988).
Ten T. pyri eggs that had been laid within a 24-hour period prior to the test were placed into each test unit, consisting of a bean leaf disc (3.8 cm diameter) placed with the upper side
9
facing upwards on damp cotton wool in a small, plastic Petri dish. The outer rim of the leaf disc was limited by a barrier of "Tangle-Trap" insect glue. The mites had continuous access to water from the cotton wool through several holes punctured into the leaf disc with a needle. The eggs and the leaf discs in the assembled test units were spray treated. The hatching mites were fed with untreated pollen. The Petri dishes were closed with a lid with a central hole covered with fine mesh netting.
Six exposure units, each containing 10 eggs, were established for each treatment group. The reference substance consisted of 1 kg Ultracid 40® WP 40 /ha (containing 400 g methidathion /ha).
Egg hatch and mortality were assessed every two to three days until each mite had developed to the adult stage. Mites that escaped from the test units were considered dead. Typhlodromus pyri protonymph test The toxicity to protonymphs was tested on treated bean (P. vulgaris) leaves in a test based on the methods described by Blümel et al. (2000) and Oomen (1988).
The test units and test design were the same as in the T. pyri egg test. Protonymphs were placed onto the bean leaves after treatment application. Pollen was spread thinly in a plastic Petri dish and treated separately. The treated pollen was used for initial feeding and untreated pollen was added during assessments. The positive reference consisted of 25 mL Perfekthion®
EC 400 /ha (containing 10 g dimethoate /ha). Mortality was assessed three and seven days after treatment. Mites that escaped from the
test units were considered dead. The number of eggs laid per female was assessed during the second week of the test. Aphidius rhopalosiphi mummy test The test was adapted from the guidelines by Mead-Briggs et al. (2000) according to an unpublished test design by Mead-Briggs (2000).
Mummies were used in the test. Mummies are the parasitoid pupae, which have developed within the host insects, the bird cherry-oat aphid Rhopalosiphum padi (Linnaeus) (Homoptera: Aphididae) (Jan et al., 1996). The age of the mummies was approximately 8 days from the laying of the parasitoid eggs, which is the earliest stage at which parasitised aphids are easily distinguished from not parasitised aphids without damaging them.
The exposure test units consisted of a 9 cm diameter plastic Petri dish, a black plastic funnel of the same diameter and a clear acrylic cylinder sealed with fine-mesh nylon netting at the apex. The mummies were cut from the barley plants so that they remained attached to a small piece of leaf, which was stuck to the Petri dishes with adhesive tape taking care that the mummies themselves were not in contact with the tape.
For each treatment three replicates with 20 mummies each were set up. The reference substance consisted of an application of 1.2 L Basudin® Extra /ha (containing 600 g diazinon /ha).
After the application, fine quartz sand was strewn into the Petri dishes to cover the treated surfaces in order to avoid contact of the hatched wasps with the dried residues. To prevent a build-up of pesticide vapour in the test units, the black plastic funnel was placed onto the Petri dish over the mummies and the acrylic cylinder was placed over the funnel two days after application. As the emerging wasps were drawn to the light entering through the hole in the funnel, they were trapped in the acrylic cylinder above. The wasps had access to honey and water moistened paper tissue, which were placed on the netting covering the cylinders.
Wasp fecundity was tested as described in the guidelines (Mead-Briggs et al., 2000). Fifteen surviving female wasps per treatment were placed individually into fecundity test
10
units three days after the first wasps had emerged. Each fecundity test unit consisted of 10 potted barley plants enclosed within a clear acrylic cylinder and infested with approximately 100 aphids of the bird cherry-oat aphid R. padi. The wasps remained there for 24 hours before being removed. After approximately 10 days the number of parasitised aphids was assessed by counting the number of mummies per test unit.
The adult emergence rate from the treated mummies and the mortality rate at the end of the holding period were assessed as well as the number of aphid mummies per female wasp that had developed during the second phase of the study. Aphidius rhopalosiphi adult test The toxicity to adults was tested on whole plants according to the description by Mead-Briggs & Longley (1997) and the assessments were made according to Mead-Briggs et al. (2000).
The exposure test units consisted of approximately 10 barley plants, which were planted five days before test initiation into 10 cm plastic pots containing gardening soil. On the morning of the test, a light coating of sucrose solution (25 % w/v in water) was applied to the barley plants using a hand sprayer. The sugar provided a foraging stimulus for the wasps and was subsequently sprayed providing a contaminated food source. The plants were left to dry on the laboratory bench. Before the application of the test substances, the soil in the pots was covered with a layer of dry quartz sand to create a uniform surface. The sugar-covered plants were treated and enclosed in clear acrylic cylinders, which were sealed on the apex with fine nylon netting. Six test units with five wasps each were set up per treatment rate. The positive control was treated with 10 mL Perfekthion® /ha corresponding to 4 g dimethoate /ha. After introducing the wasps, the test units were placed into a well-ventilated environmental chamber.
After 48 hours the mortality was assessed and 15 surviving females were taken and placed into fecundity test units as described in the mummy test above.
Chrysoperla carnea egg test The toxicity to eggs was tested on treated bean (P. vulgaris) leaves in a test based on the guidelines by Vogt et al. (2000). The exposure test units consisted of a glass plate covered with filter paper, which was kept permanently moist. Bean leaves treated on the upper side were placed onto the filter paper. Hollow glass cylinders (diameter approximately 5 cm) were then stuck to the leaves with 1 % agar thus filling any space between the leaf and the cylinder through which the very small larvae may escape. At the apex the glass cylinders were closed with a lid of fine mesh gauze to prevent emerging adults from escaping. The inner walls of the cylinders were coated with talcum to prevent the larvae from climbing.
Eggs that had been laid within 24 hours before the test, and the bean leaves were treated. During treatment, eggs remained attached to the egg laying substrate, a fine mesh cotton cloth. Then the eggs were cut at the stalks and laid onto the treated leaf surface in the test units. For each treatment level 30 replicates with one egg each were set up. The positive reference consisted of 400 mL Basudin® Extra /ha (200 g diazinon /ha). During the test the developing larvae were fed with Ephestia sp. eggs. Egg hatch and mortality were assessed until each insect had developed to the adult stage.
Chrysoperla carnea larval test The toxicity to larvae was tested on treated bean (P. vulgaris) leaves in the same test system as described above for the eggs.
Bean leaves and the initial food for the larvae were treated separately. Untreated first instars (less than three days old) were placed onto the bean leaves in the test units and initially
11
fed with Ephestia sp. eggs that had been treated by overspray at the same rate as the substrate. During the test untreated food was added every two to four days. The reference substance was 200 mL Perfekthion® /ha (80 g dimethoate /ha). Mortality was assessed until each insect had developed to the adult stage.
The surviving adults were transferred to untreated reproduction test units, consisting of 1 L clear plastic containers covered with fine gauze as egg laying substrate. Oviposition and egg viability were assessed as described by Vogt et al. (2000). Egg counting and sampling were performed twice over egg-laying periods of 24 hours and hatching of the larvae was assessed six days later.
Coccinella septempunctata egg test The toxicity to eggs was tested on treated bean (P. vulgaris) leaves in a test based on the guidelines by Schmuck et al. (2000). Hatching test units consisted of small plastic Petri dishes covered with a tightly fitting cotton cloth. Agar was filled into the test units and an excised bean leaf disc was placed onto the agar. Batches of 10 eggs each that had been laid less than 24 hours before the test were placed in hatching test units. The entire hatching units with the eggs were treated. Upon hatch each larva was transferred to a treated exposure test unit similar to those used in the C. carnea tests, where they were held individually until adult development. The ladybird larvae were fed ad libitum with green peach aphids Myzus persicae Sulzer (Homoptera: Aphididae).
The reference substance consisted of 400 mL Basudin® Extra /ha (200 g diazinon /ha). Egg hatch and mortality were assessed until each insect had developed to the adult stage.
Coccinella septempunctata larval test The toxicity to larvae was tested on treated bean (P. vulgaris) leaves according to Schmuck et al. (2000). The test is similar to that described above for C. carnea.
Bean leaves and the initial food, green peach aphids M. persicae, for the larvae were treated. Untreated, 3 to 5 days old second instars were placed onto the bean leaves in the test units and initially fed with M. persicae that had been treated separately at the same rate. During the test untreated aphids was added every two to four days. The reference substance was 200 mL Afugan® 30 EC /ha corresponding to 60 g pyrazophos /ha. Mortality was assessed until each insect had developed to the adult stage.
The surviving adults were transferred to untreated reproduction test units to assess oviposition and egg viability as described by Schmuck et al. (2000). These units consisted of clear plastic containers closed with gauze. The egg laying substrate consisted of paper tissues placed inside the containers. Egg laying and larval hatch was assessed daily over two periods of 5 days.
Orius laevigatus egg test The test units consisted of small plastic Petri dishes covered with a tightly fitting cotton cloth. Agar was filled into the test units and an excised bean (P. vulgaris) leaf disc was placed onto the agar with the lower side facing upwards. On the day before the test, individual female bugs were placed into the test units so that they could embed their eggs into the leaf tissue. The females were removed when they had laid between 5 and 14 eggs per unit. As the number of eggs per test unit varied the test had an unbalanced design. Eight test units were randomly assigned to each treatment. The test units with the eggs embedded in the leaves were treated. Ephestia sp. eggs were provided as food supply. The reference substance consisted of an application of 1.2 L Basudin® Extra /ha (600 g diazinon /ha). Egg hatch and mortality were assessed until the insects had reached the adult stage.
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Orius laevigatus larval test The toxicity to larvae was tested on treated bean (P. vulgaris) leaves in a test design adapted from Bakker et al. (2000).
The test units used to assess pre-imaginal mortality consisted of small plastic Petri dishes covered with a tightly fitting cotton cloth. Agar was filled into the test units and an excised bean leaf disc was placed onto the agar with the lower leaf surface facing upwards. The test system was assembled after the bean leaves were sprayed and had dried. Five second instars that were 1 to 2 days old were placed into each of the ten replicates set up per treatment level. Initially Ephestia sp. eggs that had been treated by overspray at the same rate as the substrate were used as food. Later untreated eggs were added. The reference substance was 125 mL Perfekthion® /ha (50 g dimethoate /ha). Mortality was assessed until 70 to 80 % of the insects had developed to the adult stage.
After a four-day mating period, 15 surviving females were transferred to untreated reproduction test units to assess oviposition and egg viability. These units were similar to the exposure test units, but untreated. Each female was left individually in a unit for two days, before being transferred to a fresh unit. Egg laying and larval hatch was assessed over two such 48-hour periods. Poecilus cupreus larval test The toxicity to larvae was tested in treated soil columns as described by Heimbach (1998).
The test units consisted of glass tubes (2.2 cm diameter and 7 cm height) filled with 30 g (wet weight) LUFA 2.1 soil moistened to one third of its maximum water holding capacity. The soil was spray treated in flat trays and, after spraying, the glass tubes were pressed into the soil and carefully taken from the box together with the soil. Each treatment consisted of 40 glass tubes kept together in two larger acrylic test boxes the bottom of which were covered with damp filter paper to keep the soil moist. The test boxes were closed with lids, which were provided with small holes to allow ventilation. Calliphora sp. (Diptera: Calliphoridae) fly pupae were spray treated separately on filter paper in Petri dishes. The toxic reference consisted of 50 g formulated Methylparathion WP 40 /ha corresponding to 20 g active ingredient /ha. The spray volume was 400 L /ha. Fly pupae were cut in half and into each glass tube a pupa was placed on the soil surface and one 12–48 hours old P. cupreus larva was introduced. The test boxes were closed and placed in a climatic chamber. Two to three times per week the fly pupae were replaced with untreated food and water was added after two weeks. The insects remained in the test units until adults emerged.
Poecilus cupreus adult test with reproduction assessments The test design for the adult test was adapted from the guidelines by Heimbach et al. (2000).
The exposure test units consisted of plastic boxes (surface area 175 cm2) closed with plastic lids with a gauze-covered hole. Each box contained 250 g LUFA 2.1 soil, which had been moistened to one third of the maximum water holding capacity.
The test organisms were 6 months old, overwintered adults of P. cupreus. Three male and three female beetles were placed into each of the five test units per treatment rate together with one Calliphora sp. fly pupa per beetle. The test units were spray treated at a volume of 400 L /ha with the beetles and fly pupae on the soil surface. The same positive reference treatment was made as in the P. cupreus larval test. Food was replaced with untreated pupae and water was added four times during the 14-day mortality assessment period.
After this period all surviving females were removed to be held individually in a reproduction test unit filled with 3 ml black agar and containing a small plastic shelter. The
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eggs laid into the agar during four 24-hour periods were collected and transferred to wet black filter paper in small Petri dishes where egg hatch was assessed.
Results and discussion An overview of the results in the water control and reference substance treatments is given in Table 1. Typhlodromus pyri tests The egg hatch in the water control treatment was 93.3 %. The mortality-escape of the hatched mites was 5.4 %. In the reference substance treatment the egg hatch was significantly lower at 33.3 % and the mortality of the hatched mites was 100 %. The ovicidal effect of the reference substance calculated according to Abbott’s formula (Abbott, 1925) was 64.7 %. The total pre-imaginal mortality in the insect growth regulator treatment groups ranged from 11.7 to 63.3 % and enabled the calculation of an LR50 with a standard Probit analysis.
In the protonymph test the pre-imaginal mortality-escape in the control was 8.3 %. In the positive reference treatment the mortality-escape was significantly higher at 91.7 %. The number of eggs laid per female during the seven-day egg laying period was 11.0 in the control. No LR50 value was calculated as the highest treatment rate tested of the insect growth regulator resulted in less than 50 % mortality-escape. Aphidius rhopalosiphi tests The percentage adult emergence from the mummies in the water control treatment was 100 %. Some emerged adults escaped from the control test units so that the escape rate was 16.7 %. No mortality was observed in the control. In the reference substance treatment the adult emergence was also 100 % but all adults died within the mortality assessment period and no reproduction test was set up. The reference substance therefore lacked pupicidal effect. The total pre-imaginal mortality in the insect growth regulator treatment groups was below 50 % at all application rates and no LR50 was calculated. The number of mummies per surviving female was 43.8 in the water control group. None of the insect growth regulator treatment groups differed significantly from the control.
In the adult test no dead, moribund or missing wasps were observed after the 48-hour exposure phase in the water control. In the reference substance treatment the mortality was 80.0 %. In the insect growth regulator treatments only one dead wasp was observed, so no LR50 was calculated. The number of mummies per surviving female was 31.4 in the control. None of the insect growth regulator treatments differed significantly from this value.
Chrysoperla carnea tests The percentage egg hatch was 80 % in the water control treatment. 5.4 % of the hatched larvae died before reaching the adult stage. The egg hatch in the reference substance treatment was 26.7 % and significantly lower than in the control showing ovicidal effect of the selected compound. No larva pupated or reached the adult stage in the positive reference. In the insect growth regulator treatments corrected pre-imaginal mortality ranged from below zero to 100 % and an LR50 value could be calculated with a standard Probit analysis.
In the larval test control mortality was 3.3 and reference substance mortality 100 %. In the insect growth regulator treatments corrected pre-imaginal mortality ranged from 3.5 to 100 % and an LR50 value could be calculated with a standard Probit analysis. The number of eggs laid per female and day was 27.1 in the control and the hatching rate was 97.7 %.
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Table 1. Results from water control and reference substance treatments.
Control Reference substance
T. pyri egg test egg hatch [%] pre-imaginal mortality [%]
93.3 ± 8.16 5.4 ± 5.9
33.3 ± 22.5 100 ± 0.0
T. pyri protonymph test 7-day mortality [%] eggs /female (days 7 - 14)
8.3 ± 11.7 11.0 ± 1.9
91.7 ± 13.3
n.a.
A. rhopalosiphi mummy test adult emergence [%] mortality-escape [%] mummies /female
100 ± 0.0
16.7 ± 10.4 43.8 ± 23.8
100 ± 0.0 100 ± 0.0
n.a.
A. rhopalosiphi adult test mortality [%] mummies /female
0.0 ± 0.0
31.4 ± 15.5
80.0 ± 21.9
n.a.
C. carnea egg test egg hatch [%] pre-imaginal mortality [%]
80.0 5.4
26.7 100
C. carnea larval test pre-imaginal mortality [%] eggs /female /day egg hatch [%]
3.3
27.1 ± 3.7 97.7 ± 1.4
100 n.a. n.a.
C. septempunctata egg test egg hatch [%] pre-imaginal mortality [%]
93.3 25
0.0 n.a.
C. septempunctata larval test pre-imaginal mortality [%] eggs /female /day egg hatch [%]
10.0
30.6 ± 22.5 94.5 ± 5.45
100 n.a. n.a.
O. laevigatus egg test egg hatch [%] pre-imaginal mortality [%]
89.8 ± 13.5 43.0 ± 23.4
0.0 n.a.
O. laevigatus larval test pre-imaginal mortality [%] eggs /female /day egg hatch [%]
6.0 ± 9.7 8.3 ± 3.9
85.8 ± 12.2
100 n.a. n.a.
P. cupreus larval test pre-imaginal mortality [%]
17.5
100
P. cupreus adult test pre-imaginal mortality [%] eggs /female (4 days) egg hatch [%]
0.0
12.5 ± 10.2 64.7 ± 23.0
100 n.a. n.a.
Values ± standard deviation. n.a. = not applicable.
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Coccinella septempunctata tests The percentage egg hatch was 93.3 % in the water control treatment. 25 % of the hatched larvae died before reaching the adult stage. No eggs hatched in the reference substance treatment showing the ovicidal effect of the selected compound. In the insect growth regulator treatments corrected pre-imaginal mortality ranged from below zero to 100 %. An LR50 value could not be calculated with a standard Probit analysis due to the variability of the data. Therefore the moving average-angle method was used.
In the larval test control mortality was 10.0 % and reference substance mortality 100 %. In the insect growth regulator treatments corrected pre-imaginal mortality ranged from 29.6 % to 100 % and an LR50 value could be calculated with a standard Probit analysis. The number of eggs laid per female and day was 30.6 in the control and the hatching rate was 94.5 %. Orius laevigatus tests The egg hatch in the water control treatment was 89.8 %. The mortality-escape of the hatched nymphs was 43.0 %. Most of this mortality occurred in the first larval stage, possibly due to cannibalism. This would indicate that a lower number of eggs per test unit might be more appropriate. The positive reference was ovicidal as no eggs hatched. The corrected pre-imaginal mortality in the insect growth regulator treatment groups ranged from below zero to 67.1 % and enabled the calculation of an LR50 with a standard Probit analysis.
In the larval test the pre-imaginal mortality-escape in the control was 6.0 %. All nymphs died in the reference substance treatment before reaching the adult stage. The average number of eggs laid per female and day was 8.3 in the control and the hatching rate was 85.8 %. In the insect growth regulator treatments corrected pre-imaginal mortality ranged from 14.9 % to 78.7 %. Due to the variability of the results the LR50 value was calculated with the moving-average angle method. Poecilus cupreus tests The percentage pre-imaginal mortality in the larval test was 17.5 % in the water control treatment and 100 % in the reference substance treatment. In the insect growth regulator treatments corrected pre-imaginal mortality ranged from 30.3 to 81.8 % and an LR50 value could be calculated with a generalised Probit analysis.
In the adult test no control mortality was observed, whereas all beetles died in the reference substance treatment group. No mortality occurred in the insect growth regulator treatments and therefore no LR50 value was calculated. The number of eggs laid per female over the four-day assessment period was 12.5 in the control and the hatching rate was 64.7 %. Conclusions The framework given in the ESCORT 2 document for testing side-effects of plant protection products with special modes of action can be used as guidance for developing appropriate test systems for the most susceptible life stages and additional modes of uptake, based on the technical guidelines published by the IOBC, BART and EPPO Joint Initiative. Where mortality above 50 % was observed, dose-response relationships could be established and LR50 values calculated. It also proved possible to establish the <50%OER, the highest tested application rate resulting in less than 50 % observable adverse effect on reproduction, so that the requirements for a formal risk assessment following the principles of ESCORT 2 were fulfilled. However, in this respect it should be noted that currently for some species (e.g. C. septempunctata and C. carnea) only qualitative assessments of reproduction are described in the guidelines from which these tests for products with special modes of action were adapted (Schmuck et al., 2000; Vogt et al., 2000).
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Validity criteria laid down in the guidelines for the standard tests could usually be met in the adapted test systems as well, if some flexibility is allowed for the longer duration of the tests or for the inclusion of stages that are more sensitive to handling, e.g. 1st instars of O. laevigatus or detached eggs of C. carnea. The publication of suitable positive reference substances with ovicidal or pupicidal effects would greatly ease the design of such tests and contribute to the stipulation of validity criteria. The results given in this paper can be considered a starting point for such an endeavour. Acknowledgements We thank Dr. M. Mead-Briggs for the help in designing the Aphidius rhopalosiphi mummy test. References Abbott, W.S. 1925. A method of computing the effectiveness of an insecticide. Journal of
Economic Entomology 18: 265-267. Bakker, F.M., S.A. Aldershof, M.v.d. Veire, M.P. Candolfi, J.J. Izquierdo, R. Kleiner,
C. Neumann, K.M. Nienstedt and H. Walker. 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, M.P. Candolfi, S. Blümel and R. Forster (Eds.). IOBC, pp.57-70.
Blümel, S., F.M. Bakker, B. Baier, K. Brown, M.P. Candolfi, A. Goßmann, C. Grimm, B. Jäckel, K. Nienstedt, K.J. Schirra, A. Ufer and A. Waltersdorfer. 2000. 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-effects of Plant Protection Products to Non-Target Arthropods, M.P. Candolfi, S. Blümel and R. Forster (Eds.). IOBC, pp.121-143.
Candolfi, M.P., S. Blümel, R. Forster, F.M. Bakker, C. Grimm, S.A. Hassan, U. Heimbach, M.A. Mead-Briggs, B. Reber, R. Schmuck & H. Vogt (Eds.). 2000. Guidelines to Evaluate Side-Effects of Plant Protection Products to Non-Target Arthropods. IOBC, BART and EPPO Joint Initiative. IOBC/WPRS Publisher. 158 pp.
Candolfi, M.P., K.L. Barrett, P.J. Campbell, R. Forster, N. Grandy, M.-C. Huet, G. Lewis, P.A. Oomen, R. Schmuck & H. Vogt. 2001. Guidance Document on Regulatory Testing and Risk Assessment Procedures for Plant Protection Products with Non-Target Arthropods. From the ESCORT 2 workshop (European Standard Characteristics Of non-target arthropods Regulatory Testing), a joint BART, EPPO/CoE, OECD and IOBC Workshop organized in conjunction with SETAC-Europe and EC. SETAC Office, Pensacola FL. 46 pp.
Finney, D.J., 1971: Probit Analysis. 3rd edition, London, Cambridge University Press. Grimm, C., H. Schmidli, F. Bakker, K. Brown, P. Campbell, M. Candolfi, P. Chapman,
E.G. Harrison, M. Mead-Briggs, R. Schmuck & A. Ufer. 2001. Use of standard toxicity tests with Typhlodromus pyri and Aphidius rhopalosiphi to establish a dose-response relationship. Journal of Pest Science 74: 72-84.
Harris, E.K. 1959. Confidence limits for the LD50 using the moving average-angle method. Biometrics 15: 424-432.
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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., P. Dohmen, K.L. Barrett, K. Brown, P.J. Kennedy, R. Kleiner, J. Römbke, S. Schmitzer, R. Schmuck, A. Ufer & H. Wilhelmy. 2000. A method for testing effects of plant protection products on the carabid beetle Poecilus cupreus (Coleoptera, Carabidae) under laboratory and semi-field conditions. In: Guidelines to Evaluate Side-effects of Plant Protection Products to Non-Target Arthropods, M.P. Candolfi, S. Blümel and R. Forster (Eds.). IOBC, pp. 87-106.
Jan, S., H.F. van Emden and Z. Ülya Nurullahoğlu. 1996. What sex is your mummy? (Homoptera: Aphididae). The Entomologist 115: 185-190.
Mead-Briggs, M.A. 2000. Personal communication. Mead-Briggs, M.A., K. Brown, M.P. Candolfi, M.J.M. Coulson, M. Miles, M. Moll,
K. Nienstedt, M. Schuld, A. Ufer and E. McIndoe. 2000. A laboratory test for evaluating the effects of plant protection products on the parasitic wasp, Aphidius rhopalosiphi (DeStefani-Perez) (Hymenoptera: Braconidae). In: Guidelines to Evaluate Side-effects of Plant Protection Products to Non-Target Arthropods, M.P. Candolfi, S. Blümel and R. Forster (Eds.). IOBC, pp.13-25.
Mead-Briggs, M. and M. Longley. 1997. A standard 'extended laboratory' test to evaluate the effects of plant protection products on adults of the parasitoid, Aphidius rhopalosiphi (Hymenoptera, Braconidae). Unpublished draft guideline (March 1997) produced on behalf of the Aphidius Ring-Test group, as part of the Joint Initiative of IOBC, BART and EPPO.
Oomen, P.A. 1988. Guideline for the evaluation of side-effects of pesticides Phytoseiulus persimilis A.-H. IOBC wprs Bulletin 11 (4): 51-63.
Schmuck, R., M. Candolfi, R. Kleiner, M. Moll, S. Nengel, B. Thompson, D. Tobias, A. Walterdorfer and H. Wilhelmy. 2000. Laboratory test system using the plant dwelling non-target insect Coccinella septempunctata (Coleoptera: Coccinellidae) to generate data for registration of plant protection products. In: Guidelines to Evaluate Side-effects of Plant Protection Products to Non-target Arthropods, M.P. Candolfi, S. Blümel and R. Forster (Eds.). IOBC, Gent, pp.45-56.
Vogt, H., F. Bigler, K. Brown, M. Candolfi, F. Kemmeter, C. Kühner, M. Moll, A. Travis, A. Ufer, E. Viñuela, M. Waldburger, A. Waltersdorfer. 2000. Laboratory method to test effects of plant protection products on larvae of Chrysoperla carnea (Neuroptera: Chrysopidae). In: Guidelines to Evaluate Side-effects of Plant Protection Products to Non-target Arthropods, M.P. Candolfi, S. Blümel and R. Forster (Eds.). IOBC, pp.27-44.
Zar, J.H. 1984. Biostatistical Analysis. Prentice Hall Int. New Jersey. 718 p.
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Pesticides and Beneficial Organisms IOBC/wprs Bulletin Vol. 25 (11) 2002
pp. 19 - 26
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First ring test results of a laboratory method to evaluate effects of plant protection products on larvae of Poecilus cupreus (Coleoptera: Carabidae) Udo Heimbach 1, Barbara Baier 2, Markus Barth 3, Sylvia Blümel 4, Ine Geuijen 5, Barbara Jäckel 6, Christian Maus 7, Karin M. Nienstedt 8, Stephan Schmitzer 9, Petra Stäbler 10, Andreas Ufer 11, Gunda Winkelmann 12 1 BBA, Institute for Plant Protection in Arable Crops and Grassland, Messeweg 11/12, D-38104 Braunschweig, Germany; 2 BBA, Institute for Ecotoxicology and Ecochemistry in Plant Protection, Berlin (Dahlem) and Kleinmachnow, Germany; 3 Biochem, Cunnersdorf, Germany; 4 BFL, Institut für Phytomedizin, Wien, Austria; 5 Notox, Hertogenbosch, Netherlands; 6 Pflanzenschutzamt, Berlin, Germany; 7 Bayer AG, Monheim, Germany; 8 Springborn Smithers Laboratories (Europe) AG, Horn, Switzerland; 9 IBACON, Darmstadt, Germany; 10 GAB Biotechnologie GmbH, Niefern-Öschelbronn, Germany; 11 BASF Agrarzentrum Limburgerhof, Germany; 12 Dr. Noack Laboratories, Sarstedt, Germany Abstract: First results of an international ring testing of a laboratory method designed to evaluate effects of plant protection products on larvae of the carabid beetle Poecilus cupreus exposed in a standardised sandy soil are presented. Dimethoate applied to the soil surface was tested by 12 laboratories in 2001. A total of 9 from 12 experiments were considered successful. The applied rates ranged from 15 to 130 g a.i./ha. Mortality effects at 45 g a.i. ranged from 35 to 100% mortality. An average mortality of about 50% was observed at 40 g a.i./ha. Control mortalities varied between 5 and 25%. Results of four experiments from previous years using the same method were in accordance with the ring test data of 2001. LD50 values could be calculated for five experiments and ranged between 26.3 and 54.2 g dimethoate a.i./ha. Results of sublethal parameters such as the hatching weight of adult beetles and their developmental period were highly variable. More research on this aspect has to be carried out as well as on the suitability of the method for soil incorporated forms of application like seed dressings and granules. Key words: laboratory test method, larvae of Poecilus cupreus, validation, plant protection products, dimethoate Introduction Adults of the carabid beetle Poecilus cupreus (L.) (Coleoptera: Carabidae) were proposed as soil dwelling non target test organisms in a guidance document for regulatory testing of pesticides in the EU (Barrett et al., 1994). Reliable results for registration purposes can only be achieved using validated methods which show that the data produced are reproducible. Therefore, validation of test methods is an international demand for acceptance of test results (Barrett et al., 1994). Such validation testing has been carried out for several test methods and organisms during the last years (Candolfi et al., 2000), including a guideline for testing adult Poecilus cupreus (Heimbach et al, 2000). However, validated testing methods for ground dwelling insects and related application types of plant protection products are still scarce. It was decided to ring-test a laboratory method for larvae of P. cupreus, which • was designed to detect lethal as well as sublethal effects of pesticides on larvae of P.
cupreus (Heimbach, 1998),
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• has already been used in several experiments (e.g. Heimbach & Abel, 1991, Abdelgader & Heimbach, 1992, Metge, 1996, Heimbach & Soverini, 1998),
• allows to expose larvae of carabid beetles to sprayed pesticides, pesticides incorporated in the soil, or to seed dressings,
• has shown in most of the laboratory experiments, that larvae of P. cupreus are more sensitive towards pesticides than adults.
Dimethoate was selected as test item, because there were already some dose-response data available for the test system (Heimbach & Soverini, 1998) and dimethoate was frequently included as standard reference item in validation tests with other terrestrial non-target organisms (Candolfi et al., 2000). Material and methods Test method The test method used for the validation testing was published by Heimbach (1998). 24 to 48 hours old larvae were released into small glass tubes (2.5 cm diameter, 7 cm height), each containing 25 g of dry soil and moistened with tap water until achieving 35% of the maximum water holding capacity. One test laboratory used slightly smaller and higher glass tubes (Inst. 3). The test product was applied to the soil surface in 100 µl water per glass tube using a pipette. After 10 to 40 minutes, one Poecilus larva was released into each test tube. Afterwards, food was supplied and replaced 2 or 3 times a week. For at least 28 days after application the glass tubes were checked visually from the outside for any larvae visible without disturbing the soil, rating them (if the larvae were seen) as alive, affected or dead. Not earlier than 28 days after release of the larvae, those glass tubes in which no larvae had been visible for a longer period were checked by searching the soil. Just before first beetles hatched from pupae, the glass tubes were checked daily by most test laboratories to determine the exact hatching date. Freshly hatched beetles were weighed before they were fed for the first time. Thus all laboratories assessed mortality values and most of them also hatching date and weight of adult beetles.
Target test conditions were 20 + 2 °C, high air humidity and little or no light. The soil humidity should have been readjusted once or twice during the whole test period.
As substrate all laboratories used the same batch of LUFA 2.1 soil, with the exception of Inst. 7 which used another batch of the same soil. Main characteristics of LUFA 2.1 soil were: 0.6% of organic C, 4 mval/100g soil cationic exchange capacity. As test item all laboratories used the same batch of dimethoate (EC formulation with 417 g a.i./l). Different rates of dimethoate were used ranging between 15 and 130 g a.i./ha with 35 and 45 g a.i. used by most laboratories. The number of dimethoate rates tested per laboratory ranged from two to six. In two laboratories only quite high rates (75 g a.i./ha and above) were tested due to a mistake in the calculation of the doses. Usually 20 replicates per dose were tested except of Inst. 4, which used 40 replicates for the control and the rate of 35 g a.i./ha.
The test organisms of those working groups, where results were considered for the analysis, were of three different origins (see Table 2). Five laboratories used larvae from a commercial producer (BTL Sagerheide). In three laboratories the larvae derived from one population of beetles, but were further reared for the trials within two of the laboratories. One laboratory used larvae from an own rearing which had been kept for several years. All laboratories used 24 to 48 hours old larvae, except Inst. 3 which used slightly older larvae (up to 72 hours old). As food for the larvae all laboratories used deep frozen pupae of Calliphora spp. from commercial producers, except Inst. 7 where deep frozen larvae of Tenebrio molitor were used.
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For the LD50 calculation the PROBIT procedure of SAS 8.1 was used taking into account the control mortality values (i.e. correction for natural mortality was performed).
Test Laboratories Altogether working groups of twelve different laboratories from four countries carried out tests in 2001 using the same method. Only Inst. 1, Inst. 3 and Inst. 5 had experiences with the test method for larvae of P. cupreus. Data which were generated between 1996 and 2000 by Inst. 1 and Inst. 5 with the same method were also considered.
It is worth while to mention that the names of the institutes are anonymised and the numbers of the institutes given in this paper do not correspond with the numbers given in the authors list.
Results and discussion Mortality The mortality rates from 9 out of 12 participating laboratories which conducted experiments in 2001 could be used for analysis. The test results of two laboratories were excluded from further processing of the ring test data because of high control mortality (> 45%). In one case the reason for this high control mortality might have been, that water was applied to the test containers 8 times providing an overmoistured substrate. In the other case high mortality occurred about 20 days after test begin; the reason is not clear, but it could be due to problems in the climatisation of the test chamber. One additional data set was excluded, because about 50% of the young larvae died shortly before they were intended to be used for the test. The larvae had been kept on very wet filter paper before the test. Death of the larvae might have been caused by keeping them too long on very wet filter paper together with the food. This laboratory achieved 100% mortality already at the lowest dose of 25 g dimethoate a.i./ha. This data set was excluded though control mortality was only 10%, because it was considered that only larvae from a vital batch of larvae should be used in experiments.
Figure 1 presents the mortality rates plotted against the test rates of dimethoate of the 9 valid trials from the ring test in 2001. At 25 g a.i./ha in 1 out of 5 experiments mortality induced by dimethoate is clearly visible. At 45 g a.i./ha in all test laboratories mortality rates of more than 30% up to 100% were achieved. At 75 g a.i./ha there are still 3 data points below 100%. In Figure 2 some data produced in 1996, 1999 and 2000 using the same method (but different batches of soil and dimethoate) were added to the data obtained during the 2001-ring-testing. The effects observed in these experiments were quite similar to those obtained during the 2001 testing.
Control mortality of the 13 experiments varied between 5 and 25% with an average of 11.7% (Table 1, 3). A 50% effect is reached at about 40 g a.i. of dimethoate per ha. Thus a rate of 45 g a.i. dimethoate seems to be reliable to be used as reference item (toxic standard) in future testing. At this dose the effects ranged from 36 to 100% mortality between different laboratories. Compared to the other laboratories Inst. 3 and 9 observed the lowest effects, whereas effects in Inst. 2 and 4 were comparably high. Low mortality figures in Inst. 3 might be explained by the slightly older larvae used by this working group. However, also temperature might have had influence (see discussion below).
It was possible to calculate a dose response relationship from the data of four laboratories in 2001 and one experiment in 1996 (Table 2). In the other laboratories either not enough rates were tested or results achieved did not allow a reliable LD50 analysis. The medium LD50 value is about 40 g a.i./ha with the lowest value achieved being 35% lower than the medium one and the highest one 34% higher.
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Table 1. Average mortality [%] (no correction for natural mortality) of Poecilus cupreus larvae from 13 experiments of different laboratories (4 trials 1996 - 2000 and ring test 2001) after treatment with dimethoate to the soil surface.
g a.i./ha mean mortality Sd no of tests
0 11.7 6.8 13 15 17.7 6.8 6 25 21.2 23.2 5 30 34.0 15.6 2 35 43.3 28.0 6 45 70.9 27.3 8 50 50 - 1 55 67.5 24.6 4 60 90.0 - 1 65 80.0 - 1 70 89.5 - 1 75 75.0 - 1 78 95.0 - 1 80 85.0 - 1 90 100 - 1 100 100 0 2 110 95 - 1 117 100 - 1 125 100 1 130 100 - 1
Sd: standard deviation
Table 2: LD50 values (g a.i. dimethoate/ha, corrected for control mortality) of Poecilus cupreus larvae tested in different laboratories in 1996 and 2001.
Laboratory LD50 95% confidence interval g a.i./ha lower limit upper limit Inst. 1 47.92 37.70 55.24 Inst. 1 1996 26.27 9.65 38.13 Inst. 2 29.3 18.89 35.26 Inst. 6 44.54 1.04 58.99 Inst. 9 54.19 48.28 68.43
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Figure 1. Mortality [ %] of Poecilus cupreus larvae after application of different rates dimethoate during the validation testing (9 tests) of 9 laboratories in 2001. Figure 2. Mortality [ %] of Poecilus cupreus larvae after application of different rates of dimethoate during tests (13 tests, including validation testing in 2001) of 9 laboratories in 1996 – 2001.
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Sublethal parameters Clear differences between the results of the different laboratories are shown in the hatching weight of the adult beetles and in the time larvae needed to complete their development (Table 3). These differences between working groups together with different ranges of dimethoate rates used by the 9 laboratories (e.g. 35 and 55 g a.i. were tested not by the same group of laboratories) makes an analysis of these sublethal parameters impossible. The origin of the larvae had neither detectable influence on mortality figures (Figures 1, 2) nor on hatching weight or developmental time.
Compared to the results of the other laboratories especially in Inst. 3, 5, 7 and 9 hatching weight was higher whereas the developmental period was longer in Inst. 2, 4 and 6. It is known that hatching weight is influenced by food quality (Theiss & Heimbach, 1993, 1994a). Therefore future testing needs better standardised food quality and an accurate reporting of the food used.
The developmental period is mainly influenced by temperature - with a change from 17 to 20 °C resulting in 15 days difference - and to a lower degree also by the food quality (Theiss & Heimbach, 1994b). Small temperature differences might, therefore, have been the reason for differences in developmental time between the laboratories involved. As a consequence, temperature has to be standardised and reported more accurately in future. This seems to be of special importance, because two laboratories with high sensitivity in mortality values also had longer developmental periods. A higher sensitivity with 100% mortality of P. cupreus larvae due to aged dimethoate residues in soil at 15 °C compared to about 70% mortality only at 20 °C was also shown by Heimbach et al. (1995).
Table 3. Control mortality, hatching weight, and larval and pupal developmental period of Poecilus cupreus larvae in untreated soil in different laboratories in the validation ring test 2001.
Laboratory origin of larvae control mortality
[%] hatching weight
[mg] developmental period
[d] Inst. 1 Inst. 1 25 50.8 35.4 Inst. 2 BTL 25 52.0 46.3 Inst. 3 BTL 10 73.8 34.4 Inst. 4 BTL 5.1 50.0 43.6 Inst. 5 Inst. 11 15 62.9 40.9 Inst. 6 Inst. 11 5.3 51.6 46.1 Inst. 7 Inst. 7 10 67.1 38.6 Inst. 8 BTL 5 61.6 35.2 Inst. 9 BTL 10 62.2 -
1 from the same population of beetles as Inst. 1 Conclusions Results of the first ring test for the validation of the test method to evaluate pesticide effects on larvae of P. cupreus were quite promising. With respect to mortality as endpoint, a good dose response relationship was obtained considering data from 2001 together with data obtained in 1996, 1999 and 2000. However, further research seems to be necessary to ensure reliability and reproducibility of the test results and to figure out if a reduction in variability
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will be possible. It seems to be especially important to know more about the influence of larval age, of the batch of soil that is used and of the temperature during the trial, to define further requirements for standardisation.
Hatching weight of adult beetles and developmental time were more variable than mortality. With regard to these sublethal parameters food quality and temperature need more standardisation in order to allow comparisons and analysis of effects between different experiments in the future.
Additionally experiments with other types of application should be carried out, such as seed dressing or granular application. Further testing with regard to these aspects is in progress and results obtained will be published later.
Nevertheless, according to the ring test results presented above the test method principally seems to be suitable for testing of pesticide side effects on larvae of P. cupreus. Acknowledgements We thank all the colleagues involved in carrying out the experiments and doing most of the practical work especially U. Busch (BBA, Braunschweig); A. Bühler (BASF); D. Przygoda, M. Römelt, N. Schürmann (BAYER); H. Hausdorf, B. Walestin (BFL); F. Kemmeter, E. Korzeniewska (GAB); S. Scazzari (IBACON); S. Becker, C. Bruhnke (Dr. Noack Laboratories); Maria Teresa Ambros (Springborn Laboratories AG), as well as those colleagues giving scientific input especially P. Neumann, R. Schmuck (BAYER) and E. Moll (BBA, Central Data Processing Group, Kleinmachnow) for statistical advise and carrying out the LD50 calculations. Thanks also to U. Hoffmann (BTL Bio-Test Labor GmbH Sagerheide) for supporting the experiments with a reduced price for the larvae. References Abdelgader, H. & Heimbach, U. 1992: The effect of some insect growth regulators (IGR) on
first instar larvae of the carabid beetle Poecilus cupreus (Coleoptera: Carabidae) using different application methods. Asp. Appl. Biol. 31: 171-7.
Barrett, K.L., Grandy, N., Harrison, E.G., Hassan, S. & Oomen, P. (Eds.) 1994: Guidance document on regulatory testing procedures for pesticides with non-target arthropods. SETAC-Europe: 51 pp.
Candolfi, M.P.; Blümel, S.; Forster, R.; Bakker, F.M.; Grimm, C.; Hassan, S.A.; Heimbach, U.; Mead-Briggs, M.A.; Reber, B.; Schmuck, R. & Vogt, H. 2000: Guidelines to evaluate side-effects of plant protection products to non-target arthropods. IOBC/WPRS Gent,: 158 pp.
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. & Abel, C. 1991: Nebenwirkungen von Bodeninsektiziden in verschiedenen Applikationsformen auf einige Nutzarthropoden. Verh. Ges. Ökol. (Osnabrück 1989), XIX(3): 163-70.
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.
Heimbach, U., Dohmen, P., Barrett, K.L., Brown, K., Kennedy, P.J., Kleiner, R., Römbke, J., Schmitzer, S., Schmuck, R., Ufer, A. & Wilhelmy, H. 2000: A method for testing effects of plant protection products on the carabid beetle Poecilus cupreus (Coleoptera: Carabidae) under laboratory and semifield conditions. In: Guidelines to evaluate side-
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effects of plant protection products to non-target arthropods. IOBC/WPRS Gent, Eds. Candolfi, Blümel, Forster et al., p. 87-106.
Heimbach, U., Wehling, A., Metge, K., Siebers, J. & Kula, H. 1995: Untersuchungen zur Bioverfügbarkeit von Pflanzenschutzmitteln in verschiedenen Böden für Laufkäfer, Spinnen und Regenwürmer. Gesunde Pflanzen 47: 64-69 + 245.
Metge, K. 1996: Entwicklung von Laborzuchtmethoden und ökotoxikologischen Prüfverfahren für Kurzflügelkäfer, insbesondere Philonthus cognatus (Staphylinidae, Coleoptera). PHD thesis, Technische Universität Braunschweig: 118 pp.
Theiss S. & Heimbach, U. 1993: Fütterungsversuche an Carabidenlarven als Beitrag zur Klärung ihrer Biologie. Mitt. Dtsch. Ges. allg. angew. Ent. 8: 841-847.
Theiss S. & Heimbach, U. 1994a: Verwendung chemisch konservierter Nahrung für die Laborzucht der Laufkäfer-Art Poecilus cupreus (Coleoptera: Carabidae). Entomol. Gener. 18: 273-278.
Theiss S. & Heimbach, U. 1994b: Präimaginale Entwicklung der Laufkäfer-Art Poecilus cupreus in Abhängigkeit von Bodenfeuchte und Temperatur (Coleoptera: Carabidae). Entomol. Gener. 19: 57-60.
Pesticides and Beneficial Organisms IOBC/wprs Bulletin Vol. 25 (11) 2002
pp. 27 - 35
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Extended laboratory investigations for evaluating the effect of Karate® on females of the predatory mite species Typhlodromus pyri Scheuten (Acari: Phytoseiidae) Barbara Baier and Eckard Moll BBA, Institute for Ecotoxicology and Ecochemistry in Plant Protection and Central Data Processing Group, Stahnsdorfer Damm 81, D-14532 Kleinmachnow, Germany Abstract: The effect of Karate® (a.i. 50 g/l lambda-Cyhalothrin) on females of Typhlodromus pyri was determined in extended laboratory tests. Investigations were first conducted on bean leaf disks which were surrounded by a glue barrier to prevent escaping of the mites. The adult females were placed on the leaf disks before treatment with pesticide. Altogether 22 application rates between 0.0001 and 2.0 µg Karate/cm2 leaf area were tested. The portion of females which had escaped and been trapped in the glue was > 30 % in the application rates 0.00018 to 0.0020 µg Karate/cm2 leaf area on day 7. Therefore two LR50 were calculated, one for the dead females only and one for the dead and escaped females together. The difference between the two values was large (by a factor of 68). The question was which LR50 value should be used for the assessment of the insecticide. Was it right to count the females which were trapped in the glue barrier as dead? This question could not be answered by this leaf disk test method. The test was therefore redesigned and carried out with complete leaves of which only one part was treated with insecticide. The application rates 2.0, 0.2, 0.02 and 0.002 µg Karate/cm2 were investigated with the new method. The first results with the complete bean leaf method showed that the number of escaped females can be reduced rapidly in comparison to the leaf disk method. Key words: predatory mites, Typhlodromus pyri, side-effects, extended laboratory experiments, Karate Introduction The insecticide Karate showed very strong effects on the predatory mite species Typhlodromus pyri Scheuten in laboratory investigations on glass. Extended laboratory investigations, i.e. tests on a natural substance such as leaves were required as a next step (Hassan, 1992). The first extended tests were dose-response tests with leaf disks surrounded by a glue barrier to prevent escaping of the mites. The problem of high escaping rates, known from the investigations with Karate on glass by the open method (Louis & Ufer, 1995), did occur, however with many tested application rates here. The test was therefore redesigned and carried out with complete leaves of which only one part was treated with insecticide. The results obtained by this test design are compared with the results of the leaf disk method. Materials and methods Tests on leaf disks Investigations were made on leaf disks cut from cotyledons of Phaseulus vulgaris and measuring 5 cm diameter. These were the actual test arenas. Each leaf disk was placed on a round wet piece of filter paper in a petri dish and surrounded by a glue barrier.
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Treatments were applied with a Potter Tower at a rate of 2 mg of wet deposit per cm2. When the spray film had dried, the leaf disks with the glue barrier were removed from the wet filter paper and placed on a cotton pad in a petri dish filled with water. Adult females which were reared in the laboratory (Bakker et al., 1992) and at the stage of oviposition were placed on the leaf disks and then sprayed with Karate. In all tests the control was treated with deionised water. Twenty test animals were placed on each leaf disk. Five replicates were included. The test animals were fed with pollen of Betula and Pinus as in the rearing. The petri dishes with the test animals were kept in a controlled environment room (temperature 24 to 25 °C, relative humidity 75 to 85 %, and exposure to light for 16 h).
Living and dead mites and those which had escaped and been trapped in the glue barrier were counted 1 day, 3 days and 7 days after application. The dead and glue-trapped test animals were removed on day 1 and 3. Altogether, 22 application rates between 0.0001 and 2 µg Karate/cm2 leaf area were tested.
Mortality rates for the seventh day after application in the test substance arenas were corrected for control mortality (Abbott, 1925). For that the glue-trapped females were counted as dead.
The LR50 in µg Karate/cm2 leaf area (Candolfi et al., 2001) was calculated on the basis of the test results on day 7, using the ‘Probit’ analyse (SAS 8.1, logarithmic normal distribution). The first calculation was made using the number of all escaped and dead females together, and the second using the dead females only. Tests with complete leaves Whole cotyledons from Phaseolus vulgaris were used for these investigations. An area of 5 cm diameter was marked in the centre of every bean leaf by a borer and the bean leaf was placed upside down on a round wet piece of filter paper (18 cm diameter). Then a round, dry filter paper (15 cm diameter, with a cut-out circular area of 5 cm diameter in the centre, was placed on the bean leaf so that the marked area in the middle of the leaf was to be seen and the rest of the leaf was covered up throughout the treatment. In this way, only the area of 5 cm in diameter in the centre of the leaf was treated with the pesticide. For treatment, we used the Potter Tower and a rate of 2 mg of wet deposit per cm2 as in the tests with leaf disks. The upper filter paper was removed from the leaf immediately after treatment. When the spray film on the marked area in the leaf middle had dried, the leaf was removed from the wet filter paper and placed on a cotton pad in a petri dish. Afterwards the petri dish was carefully filled with water, so that the whole leaf was surrounded by water to prevent the escape of test animals. The females were now placed in the centre of the treated area. The food, pollen of Betula and Pinus spp., were also placed in the centre of this leaf area. All other test conditions were the same as in the leaf disk test.
Living and dead mites were counted on the treated and untreated leaf part 1 day, 3 days and 7 days after application separately. The number of test animals which had escaped and been trapped in the water barrier were counted, too. The dead and water-trapped test animals were removed on days 1 and 3. The four application rates 0.002, 0.02, 0.2 and 2.0 µg Karate/cm2 leaf area have been tried with this test method. Mortality rates on the seventh day after application in the test substance arenas were corrected by control mortality (Abbott, 1925). For that purpose, the water-trapped females and those missing were counted as dead.
In further investigations the treated and the untreated leaf part were exchanged, i.e., the untreated leaf part was now the marked area of 5 cm diameter in the centre of the leaf, and the rest of the leaf was treated. When the spray film had dried, the females and pollen as food were placed in the centre of the untreated leaf part. All other test conditions including the four application rates and evaluations were the same as described above.
29
Table 1. Effect of selected application rates of Karate on females of Typhlodromus pyri Treatment Application Number of females 7 days after treatment Mortality rate
rate in living dead escaped (Abbott) in %
µg/cm2 (mean ± std) (mean ± std) (mean ± std) on day 7
Control - 16.6 ± 1.1 1.6 ± 1.1 1.8 ± 1.3 -
Karate 2.00000 0 19.4 ± 0.9 0.6 ± 0.5 100
Karate 1.00000 0 16.4 ± 0.5 3.6 ± 0.5 100
Karate 0.20000 0 16.4 ± 0.9 3.6 ± 0.9 100
Karate 0.10000 0 14.6 ± 0.5 5.4 ± 0.5 100
Karate 0.02000 0 13.0 ± 0.7 7.0 ± 0.7 100
Karate 0.01000 0 11.6 ± 1.5 8.4 ± 1.5 100
Control - 18.0 ± 1.4 0.6 ± 0.9 1.4 ± 1.1 -
Karate 0.00800 0 9.0 ± 3.4 11.0 ± 3.4 100
Karate 0.00600 0 6.6 ± 2.6 13.4 ± 2.6 100
Karate 0.00400 0.8 ± 0.8 2.6 ± 1.9 16.6 ± 1.7 96
Karate 0.00300 0.6 ± 0.5 2.0 ± 1.4 17.4 ± 1.1 97
Karate 0.00200 1.0 ± 0.7 1.4 ± 0.5 17.6 ± 0.9 94
Control - 17.2 ± 1.5 1.4 ± 0.9 1.4 ± 0.9 -
Karate 0.00160 1.2 ± 1.6 0.4 ± 0.9 18.4 ± 1.5 93
Karate 0.00100 1.8 ± 1.6 0.8 ± 0.4 17.4 ± 1.8 90
Karate 0.00080 2.2 ± 0.4 0.2 ± 0.4 17.6 ± 0.5 87
Karate 0.00060 5.0 ± 1.6 1.0 ± 1.2 14.0 ± 2.4 71
Karate 0.00040 7.8 ± 1.8 1.2 ± 0.8 11.0 ± 2.0 55
Control - 17.4 ± 0.9 0.2 ± 0.4 2.4 ± 0.5 -
Karate 0.00020 12.4 ± 2.3 0.6 ± 0.9 7.0 ± 2.1 29
Karate 0.00018 12.6 ± 0.9 0.8 ± 0.4 6.6 ± 1.1 28
Karate 0.00016 14.8 ± 0.8 1.2 ± 0.8 4.0 ± 0.0 15
Karate 0.00014 15.2 ± 1.6 1.0 ± 0.7 3.8 ± 2.2 13
Karate 0.00012 15.8 ± 2.5 1.0 ± 0.7 3.2 ± 2.2 9
Karate 0.00010 16.2 ± 0.8 1.4 ± 0.5 2.4 ± 1.1 7
30
Results and discussion Tests on leaf disks Table 1 shows the results counted on day 7. The mortality-escaping rate was ≤ 17 % of ≤ 20 % in the four control samples, which means that the relevant validity criterion was fulfilled, following the residual contact test method for T. pyri juveniles on glass (Blümel et al., 2000). No living females were observed with the application rates of 2.0 to 0.006 µg Karate/cm2 leaf area. All females were found dead on the leaf surface or in the glue barrier. The first surviving test animals were seen with an application rate of 0.004 µg Karate/cm2. A further reduction of the application rate down to 0.00012 µg Karate/cm2 led to an increase of the number of living females. The portion of dead females was very high at 2.0 µg Karate/cm2. A reduction of the application rate down to 0.002 µg Karate/cm2 produced a decrease of the number of dead animals. The portion of dead females remained roughly at the same low level when the application rate was further reduced. In comparison to the dead females, the number of mites which had escaped and been trapped in the glue barrier was very small with 2.0 µg Karate/cm2, but increased when the application rate was decreased to 0.0016 µg Karate/cm2. Subsequently, the number of escaped females decreased, whereas the number of living females increased with a further reduction of the application rate. The dose-response curves of Karate for the dead and escaped females together as well as for the dead females only, which were calculated from five replications at every tested application rate (while considering the estimated natural mortality rates) are shown in Figure 1. The LR50 value of Karate for the dead and escaped females together lies at 0.00046 µg/cm2 leaf area. Calculation of the LR50 on the basis of the dead females only led to an increase of the LR50 value for Karate to 0.03122 µg/cm2 leaf area in comparison to the LR50 value of escaped and dead females together. The difference between the two values is large (by a factor of 68). The question is, which LR50 value should be used for the assessment of the pesticide. Is it right to count the females, which were trapped in the glue barrier as dead? Or should this phenomenon indicate that there is a repellent effect? Is it possible, that the mites which had escaped and been trapped in the glue barrier, would have lived, if they had had the possibility to escape to an untreated leaf part and had been able to escape to this leaf area? This question cannot be answered by the leaf disk test method. Tests with complete leaves The results of the water-treated control samples are shown in Table 2. The mean number of living females, which were found on the leaves was 17.6 in control 4, and higher in controls 1, 2 and 3 on day 7. About half of the females were located on the marked middle leaf part, i.e. the treated leaf part, and the other half were seen on the remaining leaf part which was not treated. The portion of dead females was ≤ 4 %. Dead females were observed both on treated and on untreated leaf part. A maximum of 4 % of the test animals were found in the water or not at all. With that the validity criterion “mortality-escaping rate ≤ 20 %” was fulfilled (Blümel et al., 2000). The results of the four application rates of Karate, applied on the marked middle leaf part only, are shown in Table 3. With an application rate of 2.0 µg Karate/cm2 leaf area, no living females were seen. In the mean, 98 % of the females were dead. Most of these were found on the treated leaf part and only a very small number of females on the untreated leaf part. The portion of escaped females, that means here the number of females which had escaped and been trapped in the water barrier, was very small too.
31
Figure 1. Dose-response curves with direct application of Karate to females of Typhlodromus pyri on bean leaf disks ( dead and escaped females, dead females only) Table 2: Mortality of Typhlodromus pyri females on complete bean leaves – water treated control -
Treatment
Number of females (mean ± std) 7 days after treatment
alive on
dead on
in
not
treated
untreated
treated
untreated
water
found
leaf part
leaf part
leaf part
leaf part
Control 1
9.8 ± 3.5
9.4 ± 3.5
0
0.6 ± 0.5
0
0.2 ± 0.4
Control 2
9.8 ± 2.4
9.0 ± 2.3
0
0.8 ± 0.4
0.2 ± 0.4
0.2 ± 0.4
Control 3
9.8 ± 2.8
9.2 ± 2.8
0.2 ± 0.4
0.4 ± 0.5
0
0.4 ± 0.5
Control 4
10.2 ± 3.4
7.4 ± 3.5
0.8 ± 0.8
0
0.8 ± 1.1
0.8 ± 0.8
32
Tabl
e 3.
Eff
ect o
f Kar
ate
on fe
mal
es o
f Typ
hlod
rom
us p
yri o
n co
mpl
ete
bean
leav
es –
trea
ted
part
in th
e le
af c
entre
onl
y -
Tre
atm
ent
Num
ber o
f fem
ales
(mea
n ±
std)
7 d
ays a
fter t
reat
men
t
aliv
e on
dead
on
in
not
treat
ed
untre
ated
treat
ed
untre
ated
wat
er
foun
d
App
licat
ion
rate
in
µg/
cm2
leaf
par
t
leaf
par
t
leaf
par
t
leaf
par
t
Mor
talit
y ra
te
(Abb
ott)
in %
on
day
7
C
ontro
l
–
9.8 ±
3.5
9.4 ±
3.5
0
0.6 ±
0.5
0
0.2 ±
0.4
–
K
arat
e
2 0
0
17.6
± 1
.8
2.0 ±
1.9
0.4 ±
0.9
0
100
K
arat
e
0.2
0 0
17.2
± 1
.5
2.6 ±
1.7
0
0.2 ±
0.4
100
K
arat
e
0.02
0
0.4 ±
0.5
13.6
± 2
.1
3.2 ±
1.3
2.2 ±
1.3
0.6 ±
0.9
98
K
arat
e
0.00
2
4.2 ±
3.6
5.0 ±
1.9
2.6 ±
0.5
4.4 ±
2.3
3.6 ±
2.5
0.2 ±
0.4
52
32
33
Tabl
e 4.
Eff
ect o
f Kar
ate
on fe
mal
es o
f Typ
hlod
rom
us p
yri o
n co
mpl
ete
bean
leav
es –
unt
reat
ed p
art i
n th
e le
af c
entre
onl
y -
Tre
atm
ent
Num
ber o
f fem
ales
(mea
n ±
std)
7 d
ays a
fter t
reat
men
t
livin
g on
dead
on
in
not
treat
ed
untre
ated
treat
ed
untre
ated
wat
er
foun
d
App
licat
ion
rate
in
µg/
cm2
leaf
par
t
leaf
par
t
leaf
par
t
leaf
par
t
Mor
talit
y ra
te
(Abb
ott)
in %
on
day
7
C
ontro
l
-
9.2 ±
2.8
9.
8 ±
2.8
0.
4 ±
0.5
0.
2 ±
0.4
0
0.4 ±
0.5
-
Kar
ate
2
0 0
13.8
± 2
.4
5.2 ±
1.8
1.2 ±
1.0
0
100
K
arat
e
0.2
0 0
18.0
± 1
.4
1.4 ±
1.1
0.6 ±
0.5
0
100
K
arat
e
0.02
0.2 ±
0.4
0.2 ±
0.4
14.4
± 2
.1
0.6 ±
0.9
4.4 ±
1.5
0.2 ±
0.4
98
K
arat
e
0.00
2
2.0 ±
2.0
9.2 ±
2.6
3.8 ±
2.5
1.2 ±
2.2
3.6 ±
3.0
0.2 ±
0.4
41
33
34
A reduction of the application rate to 0.2 µg/cm2 led to nearly the same results as with 2.0 µg/cm2. The first surviving females were observed with an application rate of 0.02 µg Karate/cm2. They were seen on the untreated leaf part only. Most of the dead females were again found on the treated leaf part. An average of 2.2 females had escaped into the water barrier. The mortality rate was 98 %. A further reduction of the application rate to 0.002 µg Karate/cm2 led to a decrease of the effect to 52 %. Alive females were observed both on the treated and on the untreated leaf part. The mean number was nearly the same. With this application rate more dead females were found on the untreated leaf part than on the treated leaf part. The number of females which had escaped into the water was higher in this application rate than in the other three tested. Altogether, the present results have shown that this method works. A reduction of the application rate led to an increase of the living females first on the untreated leaf part and then on the treated leaf part. The number of dead females was reduced on the treated leaf part and increased on the untreated leaf part. The number of females found in the water increased with decreasing application rates. The number of animals not recovered fluctuated between 0.2 and 0.6 and can be accepted. Exchanging the treated and untreated part on the leaf, that is, placing the females on the untreated area in the leaf centre, led to the same results with the application rates 2.0, 0.2 and 0.02 µg Karate/cm2 (Table 4). The first surviving females were again observed with 0.02 µg Karate/cm2, but in comparison to the other test variant they were seen in equal numbers on both leaf parts. Most of the dead females were again found on the treated leaf part. The number of females trapped in the water was a little higher in this test variant than in the other. With the application rate 0.002 µg Karate/cm2 the mortality was 41 %. That was 11 % lower than in the test method with treated leaf center. Generally, these first results of the test method with the complete bean leaves showed that the number of escaped females can be reduced clearly in comparison to the leaf disk method. With the leaf disk method for instance, 88 % of the females were trapped in the glue barrier with an application rate of 0.002 µg Karate/cm2, while only 18 % escaped to the water in both complete leaf test methods. In particular the results of the test variant with the untreated part in the leaf centre shows, that Karate has no characteristic repellent effect. The females did not stay on the untreated leaf part in the centre of the leaf. The application rates 2.0 and 0.2 µg Karate/cm2 led to the same effect with all test designs used. With the application rate 0.02 µg Karate/cm2 the result of the complete leaf test method with treated leaf centre showed, that the females which had been found in the glue barrier with the leaf disk method can be counted as dead mainly. In the complete leaf method a portion of females escaped from the treated leaf part to the untreated leaf part and died here. The part of dead females found on the leaves increased from 65 % with the leaf disk method to 84 % with the complete leaf test method. With application rates < 0.02 µg Karate/cm2, the classification of glue-trapped females as dead probably leads to a general overestimation of the effect. This is shown by a comparison of the results of the leaf disk method (mortality rate = 94 %) and the complete leaf method with treated leaf centre (mortality rate = 52 %) with the application rate of 0.002 µg Karate/cm2. To confirm this statement, more investigations with the complete leaf test method and application rates < 0.002 µg Karate/cm2 are necessary. In particular the results of the test variant with the untreated part in the leaf centre shows, that Karate has no characteristic repellent effect. The females did not stay on the untreated leaf part in the centre of the leaf.
35
The application rates 2.0 and 0.2 µg Karate/cm2 led to the same effect with all test designs used. With the application rate 0.02 µg Karate/cm2 the result of the complete leaf test method with treated leaf centre showed, that the females which had been found in the glue barrier with the leaf disk method can be counted as dead mainly. In the complete leaf method a portion of females escaped from the treated leaf part to the untreated leaf part and died here. The part of dead females found on the leaves increased from 65 % with the leaf disk method to 84 % with the complete leaf test method. With application rates < 0.02 µg Karate/cm2, the classification of glue-trapped females as dead probably leads to a general overestimation of the effect. This is shown by a comparison of the results of the leaf disk method (mortality rate = 94 %) and the complete leaf method with treated leaf centre (mortality rate = 52 %) with the application rate of 0.002 µg Karate/cm2. To confirm this statement, more investigations with the complete leaf test method and application rates < 0.002 µg Karate/cm2 are necessary. References Abbott, W.S. 1925: A method of computing the effectiveness of an insecticide. J. Econ. Entomol.
18: 265-267. Bakker, F., Grove, A. , Blümel, S. , Calis, J. & Oomen, P. 1992: Side-effect tests for phytoseiids
and their rearing methods. IOBC/WPRS Bulletin 15 (3): 61-81. Blümel, S.; Bakker, F.M., Baier, B., Brown, K., Candolfi, M.P., Goßmann, A., Grimm, C.,
Jäckel, B., Nienstedt, K., Schirra, K.J., Ufer, A. & Waltersdorfer, A. 2000: Laboratory residual contact test with the predatory mite Typhlodromus pyri Scheuten (Acari: Phytoseiidae) for regulatory testing of plant protection products. In: Candolfi, M.P., Blümel, S., Forster, R., Bakker, F.M., Grimm, C., Hassan, S.A., Heimbach, U., Mead-Briggs, M.A., Reber, B., Schmuck, R. & Vogt, H.: Guidelines to evaluate side-effects of plant protection products to non-target arthropods. IOBC/WPRS Gent, 121-143.
Candolfi, M.P., Barrett, K.L., Campbell, P.J., Forster,R., Grandy, N., Huet, M-C., Lewis, G, Oomen, P.A., Schmuck, R. & Vogt, H. 2001: Guidance document on regulatory testing and risk assessment procedures for plant protection products with non-target arthropods. SETAC-Europe, 46 pp.
Hassan, S.A. 1992: Meeting of the Working Group ”Pesticides and Benefical Organisms”, University of Southampton, UK, September 1991. IOBC/WPRS 15 (3): 1-3.
Louis, F. & Ufer, A. 1995: Methodical improvements of standard laboratory tests for determinating the side-effects of agrochemicals on predatory mites (Acari: Phytoseiidae). Anz. Schädlingskunde, Pflanzenschutz, Umweltschutz 68: 153-154.
36
Pesticides and Beneficial Organisms IOBC/wprs Bulletin Vol. 25 (11) 2002
pp. 37 - 42
37
Data variability in carabid field studies (Coleoptera: Carabidae) and how to deal with small-scaled inhomogeneities of environmental conditions Thomas Schmidt, Richard Schmuck and Christian Maus Bayer AG, Agricultural Centre Monheim, Institute for Environmental Biology, D-51368 Leverkusen, Germany Abstract: A within-season field study was conducted to evaluate the potential effects of the insecticidal seed dressing Gaucho® (35 g and 70 g imidacloprid a.i. per 100 kg seed) on the carabid community of winter wheat fields. Evaluation endpoints were the number of species and the number of individuals (adults and larvae) recorded by pitfall trappings. Data were analysed on a large spatial scale (i.e. number of individuals per plot) and on a small spatial scale (i.e. number of individuals per trap and plot). On both levels a high variability in the numbers between replicates/traps was found mainly caused by habitat parameters varying on a small spatial scale. Including the habitat parameters soil surface structure, soil water content and wheat plant height the ANOVA-model with treatment and field as independent factors explained a major part of the observed variation. Besides abiotic habitat conditions, colonisation processes from the surroundings played a major role in the abundance of Pterostichus madidus. Key words: Carabidae, field study, pitfall traps, habitat variability, Pterostichus madidus, colonisation, winter wheat, seed dressing, imidacloprid, pesticide Introduction In recent years many efforts were made to introduce study designs and statistical methods in order to cope with natural variability in field studies (Brown 1998). The specific biotic and abiotic conditions of each field experiment, however, need to be identified as precisely as possible in order to minimize biases in data interpretation.
The field study presented in this paper is challenging a risk prediction from lab testing of side effects of plant protection products on beneficial arthropod species. In extended laboratory tests with four test systems (Aleochara bilineata (Coleoptera: Staphylinidae), adult Poecilus cupreus (Coleoptera: Carabidae), larvae of P. cupreus, adult and subadult Pardosa spec. (Araneae: Lycosidae)) seeds treated with one of two rates of a Gaucho® seed dressing had been used (35 g a.i. and 70 g a.i. per 100 kg seeds). Mortality and sublethal effects (i.e. abnormal behaviour, reduced feeding rate) had been recorded as testing endpoints. Only larval P. cupreus revealed an increased mortality when exposed to Gaucho®-dressed seeds (ca. 50% mortality at 35 g a.i., ca. 80% mortality at 70 g a.i. imidacloprid). Therefore carabid larvae were presumed to be most likely at risk in the field.
The purpose of the field study was to address the question whether effects under laboratory conditions with forced exposure would likewise be observed in the field. A study design was chosen which allows to differentiate the influence of various biotic and abiotic factors (including treatment) on the abundance of carabid species within the study field. Our study results emphasize the importance of recording abiotic conditions prevailing in study fields to avoid misinterpretation of field study results.
38
Material and methods Study design and time schedule The field study was conducted in the vicinity of the Agricultural Centre of the Bayer AG in Monheim/Germany. Four study fields were used (ca. 5 ha each, field 2 and 3 were halves of a larger field; Figure 1). Each of these four fields was divided into three plots (100 x 100 m, Figure 1): one plot was drilled with untreated seeds of winter wheat, one plot received winter wheat treated with 35 g a.i. imidacloprid per 100 kg seeds and one plot winter wheat treated with 70 g a.i. imidacloprid per 100 kg seeds. In each plot ten pitfall traps were set up in a regular spatial array of three trap rows with a distance of 20 m between the traps and a distance of 20-30 m to the plot margins (Figure 1).
field 1
field 2
field 3
field 4control
35g a.i.
70g a.i.
100m20m
20m30m
N
building
building
railwaytrack
Figure 1: Layout of the four winter wheat fields: In each field three plots were established (control, 35 g a.i., 70 g a.i. per 100 kg seeds). Fields 2-4 were surrounded by other agricultural fields. Buildings and a railway track were located at the southern and western border of field 1. 10 traps were established in the centre of each plot.
Seeds were dressed on 19.10.2000 and drilled on 26./27.10.2000. On 15.03.2001 the
study was initiated with the installation of 120 pitfall traps (diameter: ca. 12cm, trap solution: saturated NaCl-solution). In addition, 17 traps were set up around the margins of field 1, inside the field. Pitfalls were examined every second week over a total period of 20 weeks and all trapped beetles removed for further identification in the laboratory. The final trap evaluation was done in early August 2001. During the study the fields were treated with fertilisers, herbicides and plant growth regulators according to good farming practice except the use of insecticides.
Endpoints and levels of analysis Endpoints of the field study were the number of adult individuals, the number of species and the number of larvae trapped in the pitfall traps. Adult carabid species were identified to the species level while larvae were not. Catch data of species occurring mainly in the field (determined by a comparison between in-crop and off-crop pitfalls) and having a larval
39
overwintering mode were further subjected to a separate statistical analysis. In the analyses the catches of all sampling intervals were pooled.
The data were analysed on two spatial scale levels: on a relatively large scale the pooled catches of each plot (“plot view“) and on a smaller scale the catches of single traps resulting in ten values for each plot (“trap view“). For the “trap view” the calculation of means of the individual trap catches for each plot allowed to use more complex methods of analysis of variance than just calculations of means of the 4 plots which received the same treatment. In a two way-ANOVA we determined the influence of treatment and the influence of biotic and abiotic field conditions including four habitat parameters as covariates: size and number of solid soil particles (>3mm) per 0.1m2 surface, water content of soil (depth 1-2cm) and the mean height of ten wheat plants. These habitat parameters were measured in the immediate vicinity of each trap (about 1m2) for each plot resulting in 120 values for each parameter.
The data were log-transformed to meet the requirements of normal distribution (Shapiro-Wilk test) and equality of variances (test-values of Cochran-test with a significance level p>0.01 were accepted if the exclusion of outliers did not produce different results). Statistical analysis was performed using SPSS (version 10.0). Results and discussion The “plot view” At the “plot view“-level we analysed the mean values of the four replicate plots for each treatment. The control plots and the treatment plots did not differ in the number of species per plot (Table 1). Regarding the average number of individuals there was no significant difference between control and treated plots either (Table 1). In contrast, when the four study fields with the three plots each (Fig. 1) were compared with each other, significant differences were found for the number of individuals, indicating that the observed carabid abundance is primarily related to biotic and abiotic field conditions.
The same relation was observed for the number of carabid larvae per plot (Table 1), i.e. larval abundance was mainly a function of the biotic and abiotic conditions of the study plots.
Table 1: Number of individuals, number of species and number of larvae of Carabidae per plot at the “plot view“-level (mean ± s.d., n=4 per treatment)
10 trap catches were pooled per plot. F-values of two way-ANOVA are shown with significance levels for treatment and field effect (*: p<0.05; **:p<0.01; ***; n.s.: p>0.05). R2-values indicate the goodness of fit of the ANOVA-model.
number of individuals number of species number of larvae control 2755.8 ± 1988.9 33.8 ± 3.5 86.5 ± 58.8 35g a.i. 2169.0 ± 1555.2 32.5 ± 1.9 52.0 ± 29.1 70g a.i. 2155.5 ± 1099.4 34.0 ± 4.0 73.3 ± 73.0 Ftreatment 0.726 n.s. 0.207 n.s. 1.802 n.s. Ffield 9.717 * 0.559 n.s. 12.316 ** R2 0.699 0.359 0.764
The habitat heterogeneity In order to quantify the abiotic conditions between and within plots we recorded four habitat parameters, which, we assume, are appropriately indicative for defining “habitat quality” for
40
carabid beetles (Table 2). For number and size of solid soil particles (>3 mm), water content of soil and height of wheat plants highly significant differences were found between the 12 plots although the study plots were not classified as visually different during the plot selection procedure.
Table 2: Size and number of solid soil particles (> 3 mm) per 0.1m2, soil water content (%) and wheat height (cm) (mean ± s.d., n=30 per field)
Each parameter was measured in the near vicinity of each of the 120 pitfall traps (radius ca. 1m). Kruskal-Wallis H-test was used to test for significant differences of each of the four parameters between the 12 plots (three plots in each field; **:p<0.01; ***: p<0.001).
size of soil particles
number of soil particles
soil water wheat height
field 1 11.4 ± 2.8 15.7 ± 4.9 10.0 ± 1.6 67.3 ± 4.0 field 2 8.6 ± 2.5 21.3 ± 9.3 9.2 ± 1.5 63.9 ± 4.5 field 3 9.9 ± 2.6 27.0 ± 10.3 9.6 ± 1.4 66.5 ± 4.6 field 4 11.8 ± 6.1 8.1 ± 4.5 8.0 ± 2.0 86.2 ± 4.0 H (12,120) 25.257 ** 75.778 *** 40.563 *** 90.071 ***
Apart from between-field differences, a high variability of the recorded abiotic
conditions was recorded also within each plot. This with-in plot variability may have had an influence which was not accounted for by the statistical analyses of plot means. Because most carabid species respond very sensitive even to smallest differences in biotic and abiotic conditions (Thiele 1977) we recommend to record in each field study on carabid beetles at least some major habitat parameters on a small spatial scale to avoid mis-interpretation of study results.
The “trap view” At the “trap view”-level we calculated means of ten traps for each plot (Table 3). Highly significant differences in the number of adult individuals per trap and per plot were found between the fields.
There was also a significant difference in the number of individuals between treatments but the influence of biotic and abiotic field conditions was highly dominant over the possible influence of treatment as indicated by the differences in the corresponding F-values. The significance of the interaction between field and treatment effect reflects an inconsistent pattern: whereas in field 1 the highest numbers were found in the control plot (Table 3), field 3 had the highest numbers in the treatment plots. In addition, no dose-response relation was found between the two test rates which further supports the view that primarily abiotic field parameters determined the observed number of individuals per trap and plot. The herein chosen ANOVA-model which is mainly based on habitat conditions describes the observed variability quite well as indicated by the high R2-value. The number of species and the number of larvae per trap and plot did not differ between fields or treatments.
We further analysed the distribution of species that were predominantly encountered within fields and have developed a larval overwintering strategy (Turin 2000) since for these species a maximum exposure to seed dressing can be assumed. Four of the 5 analysed species (Nebria brevicollis, Pseudophonus rufipes, Trechus quadristriatus, Pterostichus melanarius) did not differ significantly in the number of individuals per trap and plot when control and
41
treatment plots were compared (ANOVA: Ffield-values with p<0.001; Ftreatment-values with p>0.05). In contrast, Pterostichus madidus was trapped in significantly lower numbers of individuals in plots treated with the lower seed dressing rate than in control plots (ANOVA: Ffield=11.1, p<0.001; Ftreatment=5.4, p<0.01; a posteriori LSD-test: p<0.05). However, no statistical differences were found between the control and plots treated with the higher seed dressing rate. When considering the number of P.madidus individuals on different fields it was evident that field 1 contained the majority of individuals (Figure 2). Field 1 differed from the other fields by the vicinity of buildings which bordered on two field sides. A colonisation handicap is evident from off-field pitfall catches around field 1: Whereas only 4.0 P. madidus individuals were trapped on average in the vicinity of the buildings the off-crop pitfalls next to open fields trapped an average of 11.0. Accordingly, it is reasonable to assume that the observed differences are related to this colonisation handicap rather than to treatment.
Table 3: Mean number of adult individuals, species and larvae of Carabidae per trap and per plot (± s.d., n=10 trap catches per plot) at the “trap view“.
F-values of two-way-ANOVA are shown with significance levels for treatment and field effect (*: p<0.05; ***: p<0.001; n.s.: p>0.05). Four habitat parameters were included as covariates. R2-values indicate the goodness of fit of the ANOVA-model.
number of individuals number of species number of larvae control 35g a.i. 70g a.i. control 35g a.i. 70g a.i. control 35g a.i. 70g a.i.field 1 414 ± 54 159 ± 49 288 ± 72 20 ± 3 17 ± 2 19 ± 5 8 ± 4.3 4 ± 1.9 6 ± 2.9field 2 101 ± 31 83 ± 29 93 ± 60 19 ± 3 15. ± 3 16 ± 2 6 ± 3.8 2 ± 1.4 3 ± 2.3field 3 109 ± 35 186 ± 83 156 ± 46 17 ± 3 18 ± 2 17 ± 3 4 ± 2.5 5 ± 2.9 3 ± 2.1field 4 479 ±176 441 ± 86 327 ± 92 17 ± 3 18 ± 2 19 ± 3 17 ± 8 9.2 ± 6 18 ± 12Ftreatment 3.280 * 1.896 n.s. 2.883 n.s. Ffield 40.032 *** 2.217 n.s. 1.627 n.s. Ftreatm. x Ffield 8.150 *** 1.647 n.s. 2.429 n.s. R2 0.779 0.067 0.440
Finally we analysed the number of individuals of species with peak abundance in spring and the species composition of the early sample intervals: no significant differences were detected between control and treatments. Conclusions A statistical analysis of our pitfall trap data on carabids strongly indicates that the observed differences in numbers of species and individuals between study plots are primarily related to small-scaled habitat variabilities and colonisation potentials rather than to an exposure to Gaucho®-treated cereal seeds. A major influence of habitat quality may not be a specific feature of the present field study but a more general feature for pitfall studies. It must therefore be requested in the interest of a scientifically reliable data interpretation of pitfall studies that habitat quality parameters are recorded in parallel to pitfall catches. The single trap analysis was found to be most indicative for highlighting the influence of habitat parameters and it is strongly recommended to use it in the analysis of pitfall catches.
42
The findings of this field study support the assumption of Candolfi et al. (2001) that mortality rates of less than 50% in (extended) laboratory tests are indicative for no detectable effect on populations of arthropod groups (here: carabids) under field exposure conditions. This assumption depends on the methods used and many other factors like habitat variability and study field size. For reliably concluding the absence of effects of a treatment, however, it is necessary to analyse not only pooled endpoints like number of individuals but separately those species with the strongest exposure to the examined treatment since carabids respond in a very species-specific manner to biotic and abiotic conditions of a habitat and corresponding surroundings (Thiele 1977, Turin 2000).
field 3field 2field 1
num
ber o
f Pte
rost
ichu
s m
adid
us
25
20
15
10
5
0
treatment
control
35 g a.i.
70g a.i.
Figure 2: Boxer Whisker plots of number of Pterostichus madidus individuals per plot Boxer Whisker-plots describe 50% of the data in the box, the data range between whiskers and median values as line. Data of three fields are shown (field 4 only with two individuals). Different letters on the top of the boxes within a field indicate significant differences between control and treatments ((ANOVA: p<0.05; LSD-test: p<0.05). Acknowledgements Thanks to R. Michel, B. Gines and A. Litwin for field and lab assistance, M. Daniels for field work, S. Umstätter for amending the language and J. Lückmann for continuous encouragement. References Brown, K. C. 1998: The value of field studies with pesticides and non-target arthropods. The
1998 Brighton Conference-Pests & Diseases: 575-582. Candolfi, M. P., K. L. Barrett, P. J. Campbell, R. Forster, N. Grandy, M.-C. Huet, G. Lewis,
P. A. Oomen, R. Schmuck, H. Vogt 2001: Guidance Document on Regulatory Testing and Risk Assessment Procedures for Plant Protection Products with Non-Target Arthropods. Proceedings of ESCORT II workshop, SETAC, Pensacola.
Thiele, H.-U. 1977: Carabid Beetles and their Environments. Berlin, Heidelberg, New York, Springer Verlag.
Turin, H. 2000: De Nederlandse Loopkevers, Nationaal Natuurhistorisch Museum Naturalis, KNNV Uitgeverij, European Invertebrate Survey-Nederland.
a
a
aa
a
a
abb
Pesticides and Beneficial Organisms IOBC/wprs Bulletin Vol. 25 (11) 2002
pp. 43 - 51
43
Results of the 8th and 9th IOBC Joint Pesticides Testing Programme: Persistence test with Phytoseiulus persimilis Athias Henriot (Acari: Phytoseiidae) S. Blümel & H. Hausdorf AGES, Institute for Phytomedicine, Spargelfeldstr. 191, A-1226 Wien [email protected] Abstract: Eleven insecticides/acaricides and 7 fungicides from the 8th and 9th Joint Pesticide Testing Programmes of the IOBC-WG “Pesticides and Beneficial Organisms“, which had moderately harmful or harmful effects (> 50%) on P. persimilis protonymphs in the lab-a-test (initial toxicity), were tested for the persistence of their residual effects on the phytoseiid. According to the method of Oomen (1988) bean plants (Phaseolus vulgaris) were treated with up to 3 different rates of formulated plant protection products, and with water as control, respectively, by the help of a commercial hand sprayer and were kept in the greenhouse at approximately 25°C, 75 ± 10% relative humidity and 16hL:8hD photoperiod. Three and 10 days, respectively, after the treatment single leaves were removed from the test plants and prepared as detached leaf cultures. The testing procedure subsequently followed the lab-a-test by placing prey mites (T. urticae) and the predator protonymphs on the leaf arenas and by assessing the mortality and the reproduction of P. persimilis at regular intervals during the 7 day trial period. The mortality and escape rate accounted for the main part of the total effect of the test products, whereas reproduction was rarely and only slightly reduced by the treatments.
Dicarzol 200 SP (a.i. Formetanaate), Dithane M 45 (a.i. Mancozeb), Masai 20 WP (a.i. Tebufenpyrad), Polo 25 SC (a.i. Difenthiuron), Topsin M 70 WP (a.i. Thiophanatemethyl) and Vertimec 018 EC (a.i. Abamectin) caused total effects of 100% at all application rates, both as 3 day old and as 10 day old residues. Bavistin 50 DF (a.i. Carbendazim), Euparen M 50 WG (a.i. Tolylfluanid), Kumulus 80 DF (a.i. sulphur) and Naja 050 EC (a.i. Fenpyroximate) residues resulted for all application rates and both residue ages in slightly or moderately harmful total effects, ranging from 55% to 97%. Confidor 200 SL (a.i. Imidacloprid) and NeemAzal-T/S (a.i. Azadirachtine) were either harmless at both lower tested application rates or caused slight to moderate harmful effects after treatment of the 3fold recommended field rate (56.2% respectively 87.2%) towards the phytoseiid as 10 day old residues. The 3 day old residues resulted in moderately harmful or harmful effects at least at the highest application rate (83.2% respectively 100%). Compared to results of the lab-a-test the effects of the aged residues were strongly reduced for Admiral 10 EC (a.i. Pyriproxifen), Amistar 250 SC (a.i. Azoxystrobin), Aztec 140 EC (a.i. Triazamaate), Impulse 500 EC (a.i. Spiroxamine), Phosdrin 145 Mengolie (a.i. Mevinphos) and Telmion (a.i. rape seed oil). Total effects caused by 3 day old residues ranged from 1% to 44% (except rape seed oil with 74.4%) increasing with the application rate, whereas 10 day old residues peaked at 13% total effect. Key words: side-effect testing, plant protection products, extended laboratory test, persistence test,
Phytoseiulus persimilis, phytoseiids Introduction Within the Joint Pesticide Testing Programmes of the IOBC-WG “Pesticides and Beneficial Organisms“, the predatory mite Phytoseiulus persimilis, which is one of the most frequently released beneficials in greenhouse crops, represents one of the standard test organisms. Both, the knowledge about the initial toxicity of fresh product residues and the persistence of effects
44
caused by plant protection products are of utmost importance for integrated plant production strategies in greenhouses, where P. persimilis is used for biological control of the two spotted spider mite (Tetranychus urticae). The persistence test is part of a sequential testing and decision scheme and follows the initial laboratory toxicity test, if an effect size of 50% is exceeded (Bakker et al., 1992). An effect size of 50% was found to be more appropriate to trigger higher tiered testing in predatory mites with regard to the correspondence of effects achieved in the laboratory and in the field (Bakker, 1998), than the 30% effect size, which was proposed for all test organisms by the IOBC classification scheme (Hassan, 1994). The results of the persistence test allow to predict introduction intervals for this phytoseiid after pesticide treatment with regard to the minimisation of harmful effects on the population development and the biocontrol effect of P. persimilis. Material and methods Test principle Phytoseiulus persimilis protonymphs were exposed to aged residues of the test compounds on detached bean leaves. Commercial formulations of 11 insecticides/acaricides and 7 fungicides from the 8th and 9th IOBC- Joint Pesticide Testing Programmes (= JPTP), which had caused effect values > 50% on P. persimilis protonymphs in the lab-a-test (initial toxicity), were tested for the persistence of their residual effects. The test was carried out according to the test method described by Oomen (1988) and Oomen et al. (1991), respectively. Application of test compounds Phaseolus vulgaris plants were treated with a commercial hand sprayer until a uniform spray deposit on the lower and the upper side of the leaves was achieved. Test rates applied were the predicted initial environmental concentrations (= PIEC: [µg a.i./cm²] = rate [g a.i./ha] x fd/100) for arable and glasshouse crops (fd = 1) (8th JPTP and 9th JPTP) with the exception of the orchard pesticides Match, Telmion and Zolone Flow (8th JPTP), where fd=0,4 was applied. For all products of the 9th JPTP the 3fold recommended field rate was also tested (to simulate multiple application). Bean plants, which were treated with tap water served as control samples. Test organisms Both, the two spotted spider mite Tetranychus urticae Koch as prey source and the phytoseiid mite species were obtained from mass-rearings at the BFL, Institute for Phytomedicine. Tetranychus urticae was reared on potted bean plants (Phaseolus vulgaris) in climatized greenhouse compartments at 25°C, 75 ± 10% relative humidity, 16hL:8hD photoperiod and was either kept as pure prey culture or served as prey infested plants for the Phytoseiulus persimilis rearing.
To obtain a sufficient number of predator protonymphs of uniform age for the test, three times the number of eggs which was needed as protonymphs, was collected from the mass rearing and were placed on detached bean leaf units (see below) for hatching at 25°C, 75 ± 10% relative humidity and 16hL:8hD photoperiod. Persistence test on detached leaves Three days and ten days after the application single bean leaves were detached from the test plants and placed upside down on a water saturated cotton layer in a petri dish (diameter 9 cm) with a perforated base to form a test unit. The petri dish was placed in a water tray to provide a continuous water supply of the cotton layer, which should offer both drinking water
45
for the predatory mites and a water barrier to impede their escape. Shortly before predator protonymphs were placed on the leaf arena by the help of a fine brush, about 30 to 60 mixed stages of spider mites were added as food source. Per treatment either 10 replicates with 15 predator protonymphs each (8th JPTP) or 8 replicates with 15 predator protonymphs each (9th JPTP) were used. The number of dead juvenile predators were counted with a stereomicros-cope and individuals missing were recorded. Mortality and escape of the predators was assessed on day 1 and 3 after the application and the dead individuals were removed at each evaluation date. The cumulative escape rate on day 3 comprised all mites which could not be found. The reasons to include escapers into the evaluation have been discussed extensively (Blümel et al. 2000; Blümel et al., 1993). The escape rate was calculated as the percentage of the number of mites present at the start of trial. The number of dead mites was cumulated until day 3 after application and added to the numbers of the escaped mites. This combined mortality-escape rate was set into relation to the numbers of phytoseiids present on the test arena at the start of the trial and termed as mortality rate. From day 3 until day 7 the egg production per female was evaluated daily. Eggs were removed from the test units after each evaluation. Predatory mites were supplied with prey every 2 days. The test units were kept at 25°C, 75 ± 10% relative humidity and 16hL:8hD photoperiod.
Mortality rates were adjusted for the control mortality (Abbott, 1925).
x % = (1− tc
) * 100
x %: mortality rate t: number of dead individuals in treated samples c: number of dead individuals in control samples
Possible changes in the number of females present on the test units during the reproduction period and the hatching of larvae from eggs between the assessment dates were taken into account by using the following formula:
R ry = )nF
nE (d4
d4 + ⎥⎦
⎤⎢⎣
⎡)/2nF+ (nF
nE d5 d4
d5 + ⎥⎦
⎤⎢⎣
⎡)/2nF+ (nF
nE d6 d5
d6 + ⎥⎦
⎤⎢⎣
⎡)/2nF+ (nF
nE d7 d6
d7
d4 to d7: examples for evaluation days R ry : Reproduction in replicate number y nE dx: number of eggs (in replicate number y) on day x nF dx: number of females (in replicate number y) on day x
The total effect size was calculated according to Overmeer & van Zon (1982):
E = 100% - (100%-Mt/100%-Mc)*Rt/Rc
Mean values of the mortality rate and of the reproduction per female were analyzed for significant differences between treatments with an univariate analysis of variance (ANOVA, Bonferroni – Test, SPSS) after a test for normal distribution of the data. Variance homogeneity was checked with the Levene - Test. Null hypothesis assumed that no significant differences were present between the mean values obtained with the different treatments. Percent data were angular transformed before processing with ANOVA. Results and discussion Similar to the lab-a-test the mortality and the escape accounted for the main part of the total effect of the test products, whereas reproduction was rarely and slightly reduced by the
46
treatments (Tables 1 and 2). The mean mortality in the control samples of the 8th JPTP reached up to 29.7%, whereas in the 9th JPTP 10% were only exceeded once. Due to the high number of replicates also tests with control mortalities > 20% - 29.7% may be considered as valid, with regard to the detection of statistically significant treatment effects.
For some of the test products, the main source of effect was the escape, which reached for Confidor 200SL, NeemAzal-T/S and Telmion rates between 30% and 80% (3 day old residues) and between 0% and 54% (10 day old residues), respectively.
The mean egg production in the control samples ranged from14.9 to 19.4 eggs per female in the 8th JPTP and 14.9 to 20.7 eggs per female in the 9th JPTP, which lies within the usual range of reproduction per P. persimilis female.
The total effect size reduction of aged residues compared to fresh residues varied for the different test products dependent from the field rate applied and the age of the residue. For Dicarzol 200SP, Dithane M 45, Masai 20WP, Polo 25SC, Topsin M 70 WP and Vertimec 018EC no reduction of the total effect (100% effect) was found for all application rates and both residue ages, compared to the effect size obtained in the initial toxicity test (lab-a-test). Thus these products have to be categorised as harmful for P. persimilis protonymphs.
However for Bavistin 50DF, Confidor 200 SL, Euparen M 50WG, Kumulus 80DF, Naja 050EC, and NeemAzal-T/S a total effect size reduction between 3% to 45% dependent from the application rates and the residue ages was observed (except Confidor 200SL, 3 day old residues), which allows a classification as moderately harmful respectively harmful compounds as aged residues for P. persimilis protonymphs. The release of the phytoseiids into a crop treated with these compounds could be carried out in compliance with an appropriate post application interval (mainly 10 days).
The highest reduction in the total effect size, as low as to harmlessness for P. persimilis protonymphs was achieved for Admiral 10EC, Amistar 250 SC, Aztec 140EC, Impulse 500EC, Phosdrin145 Mengolie and Telmion. Already 3 days after an application of these compounds the predatory mites could be released without being harmed.
The effect sizes achieved in the present study cannot be directly compared with results of other studies (e.g. Stolz, 1994) as different test rates and formulations of the plant protection products were used.
Higher tiered tests should be carried out to investigate, whether the test results from extended laboratory tests (such as the persistence test) with the 3fold recommended field rate can be extrapolated for a range of plant protection products to predict multiple application effects under field conditions. Acknowledgements We thank B. Walestin, BFL, Institute of Phytomedicine for her technical assistance. References Abbott, W.S. 1925: A method of computing the effectiveness of an insecticide. Journal of
Economic Entomology 18: 265-267. Bakker, F. M. 1998: Accuracy and efficiency of sequential pesticide testing protocolls for
phytoseiid mites. In: Ecotoxicology: Pesticides and Beneficial Organisms, eds. P.T. Haskell and P. McEwen, Kluwer Academic Publishers: 148-163.
Bakker, F. M., A. Grove, S. Blümel, J. Calis & P. A. Oomen 1992: Side-effect tests for phytoseiids and their rearing methods. IOBC/WPRS Bulletin 15 (3): 61-81.
47
Blümel, S., F.M. Bakker, and A. Grove 1993: Evaluation of different methods to assess the side effects of pesticides on Phytoseiulus persimilis. Experimental and Applied Acarology 17 (3):,161-169.
Blümel, S., C. Pertl & F. Bakker 2000: Comparative trials on the effects of two fungicides on a predatory mite in the laboratory and in the field. Entomologia experimentalis et applicata 97: 321-330.
Hassan, S.A. 1994: Activities of the IOBC/WPRS Working Group „Pesticides and Beneficial Organisms“. IOBC/WPRS Bulletin 17 (10), 1-6.
Oomen, P.A. 1988: Guideline for the evaluation of side-effects of pesticides on Phytoseiulus persimilis. IOBC/WPRS-Bulletin 11 (4): 51-64.
Oomen, P.A.; Romeijn, G. and Wiegers, G.L. 1991: Side-effects of hundred pesticides to the predatory mite Phytoseiulus persimilis, collected and evaluated according to the EPPO Guideline. EPPO Bulletin 21 (4), 701-712.
Overmeer, W. P. J. & A. Q. Van Zon 1982: A standardized method for testing the side effects of pesticides on the predacious mite Amblyseius potentillae (Acari, Phytoseiidae). Entomophaga 27: 357-364.
Stolz, M. 1994: Efficacy of different concentrations of seven pesticides on Phytoseiulus persimiis A.H. (Acarina: Phytoseiidae) and on Tetranychus urticae K. (Acarina: Tetranychidae) in laboratory and semifield-test. IOBC/WPRS Bulletin 17 (10), 49-54.
Tabl
e 1:
Res
ults
of t
he p
ersi
sten
ce te
st w
ith P
hyto
seiu
lus p
ersi
mili
s fo
r the
8th
JTP-
IOB
C
*si
gnifi
cant
ly d
iffer
ent f
rom
con
trol
T
RA
DE
NA
ME
g
or m
l pro
duct
/ ha
activ
e in
gred
ient
re
sidu
e ag
e [d
ays]
% e
scap
e (A
BB
OT
T)
% m
orta
lity
cont
rol
% m
orta
lity
(AB
BO
TT
) E
gg/fe
mal
e
[% c
ontr
ol]
Tot
al
effe
ct [%
]IO
BC
cl
ass/
A
DM
IRA
L 1
0EC
Py
ripr
oxife
n 0
23.1
* 12
.2
59.2
* 80
.2
67.2
2
500
g/ha
3 0
18.0
8.
1 10
0.1
7.3
1
10
4.
6 13
.3
9.2
99.2
9.
9 1
TE
LM
ION
R
ape
seed
oil
0 68
.3*
20.4
85
.8*
90.8
87
.1
3 20
000
ml/h
a
3 50
.8*
15.3
67
.7*
79.3
74
.4
3
10
1.
52
13.3
9.
2 10
0.2
9.1
1 PO
LO
25S
C
dife
nthi
uron
0
27.2
* 0
100*
0
100
4 20
00g/
ha
3
0.7
18.0
99
.2*
0 10
0 4
10
10
13.3
10
0*
0 10
0 4
VE
RT
IME
C 0
18E
C
abam
ectin
e 0
3.4
0 10
0*
0 10
0 4
750g
/ha
3
0 29
.7
100*
0
100
4
10
0
19.3
10
0*
0 10
0 4
EU
PAR
EN
M 5
0WG
T
olyl
fluan
id
0 15
.6*
20.3
92
.5*
0 10
0 4
3125
g/ha
3 4.
1 29
.7
48.8
* 66
.7
65.5
2
10
0.8
13.3
46
.9*
34.5
81
.7
3 K
UM
UL
US
80
DF
Sulp
hur
0 23
.9*
22.6
85
.4*
6.2
99.1
4
1000
0g/h
a
3 0
11.3
55
.9*
92.5
59
.2
2
10
8.
3 12
.7
74.8
* 54
.1
86.4
3
BA
VIS
TIN
50D
F C
arbe
ndaz
im
0 5.
2 25
.9
64.0
* 0
100
4 20
00g/
ha
3
0 24
.0
2.6
8.5
91.8
3
10
2.2
10.0
12
.9*
27.5
75
.9
2 D
ITH
AN
E M
45
Man
coze
b 0
22.1
* 21
.9
100*
0
100
4 45
00g/
ha
80W
P 3
0 24
.0
100*
0
100
4
10
12
.6
10.0
10
0*
0 10
0 4
TO
PSIN
M 7
0WP
Thi
opha
nate
- 0
14.8
* 21
.1
79.6
* 0
100
4 57
0g/h
a m
ethy
l 3
0 11
.3
98.5
* 0
100
4
10
0
12.7
97
.7*
0 10
0 4
48
Tabl
e 2:
Res
ults
of t
he p
ersi
sten
ce te
st w
ith P
hyto
seiu
lus p
ersi
mili
s fo
r the
9th
JTP-
IOB
C
n.t.:
not
test
ed, p
hyto
toxi
c
*sig
nific
antly
diff
eren
t fro
m c
ontro
l
TR
AD
E N
AM
E/
g or
ml p
rodu
ct/ h
aac
tive
ingr
edie
nt
fd P
IEC
resi
due
age
[day
s]%
esc
ape
(AB
BO
TT
) %
mor
talit
yco
ntro
l %
mor
talit
y (A
BB
OT
T)
Egg
/fem
ale
[%
con
trol
] T
otal
ef
fect
[%]
IOB
C
clas
s A
ZT
EC
140
EC
T
riaz
amaa
t 0.
4 0
3.5
10.8
70
.3*
85.6
74
.6
3
1.
0
7.2
69
.2*
93.9
70
.9
3 50
0 g/
ha
3.
0
0
83.2
* 76
.3
87.2
3
0.4
3 3.
4 3.
3 5.
1 10
4 1.
3 1
1.0
1.
7
11.2
* 98
.2
12.8
1
3.0
5.
9
43.9
* 10
6 40
.5
2
0.
4 10
5.
3 5.
8 8.
9 99
.9
8.9
1
1.
0
3.5
8.
9 96
.6
11.9
1
3.0
2.
7
11.5
98
.3
12.9
1
PHO
SDR
IN
Mev
inph
os
0.4
0 0
0 10
0*
0 10
0 4
145
ME
NG
OL
IE
1.
0
0
100*
0
100
4
3.
0
0
100*
0
100
4 34
48.3
g/h
a
0.4
3 14
.9
5.8
17.5
92
24
.3
1
1.
0
15.0
26.6
76
44
.3
2
3.
0
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
0.4
10
9.7
6.7
9.8
98
11.6
1
1.0
n.
t. n.
t. n.
t. n.
t. n.
t. n.
t.
3.
0
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
NE
EM
AZ
AL
A
zadi
rach
tin
0.4
0 66
.7*
5.0
87.7
* 79
.5
90.3
3
T/S
1.0
40
.4*
10
0*
0 10
0 4
3.0
48
.3*
10
0*
0 10
0 4
3000
g/h
a
0.4
3 29
.1*
2.5
34.2
* 89
41
.5
2
1.
0
42.7
*
56.4
* 96
58
.2
2
3.
0
60.7
*
80.3
* 86
83
.2
3
0.
4 10
4.
4 5
5.3
101
4.4
1
1.
0
11.4
19.3
10
8 12
.7
1
3.
0
38.6
*
53.5
* 94
56
.2
2
49
Tabl
e 2:
Res
ults
of t
he p
ersi
sten
ce te
st w
ith P
hyto
seiu
lus p
ersi
mili
s fo
r the
9th
JTP-
IOB
C
cont
inue
d 1
*s
igni
fican
tly d
iffer
ent f
rom
con
trol
TR
AD
E N
AM
E
g
or m
l pro
duct
/ ha
activ
e in
gred
ient
fd
PIE
C
resi
due
age
[day
s]%
esc
ape
(AB
BO
TT
) %
mor
talit
yco
ntro
l %
mor
talit
y (A
BB
OT
T)
Egg
/fem
ale
[%
con
trol
] T
otal
ef
fect
[%]
IOB
C
clas
s/
CO
NFI
DO
R
Imid
aclo
prid
0.
4 0
65.3
* 1.
7 10
0*
0 10
0 4
200S
L
1.
0
74.6
*
100*
0
100
4
3.
0
63.6
*
100*
0
100
4 35
0 g/
ha
0.
4 3
30.0
* 9.
2 35
.7*
69
55.6
2
1.0
74
.6*
88
.9*
0 10
0 4
3.0
80
.2*
91
.8*
0 10
0 4
0.4
10
0 15
.8
3.9
91.5
12
.1
1
1.
0
1.9
2.
9 10
6 -2
.3
1
3.
0
53.9
*
69.3
* 42
87
.2
3 N
AJA
050
EC
Fe
npyr
o-
0.4
0 5.
2 5.
8 10
0*
0 10
0 4
xi
mat
e 1.
0
0
100*
0
100
4 10
0 g/
ha
3.
0
2.5
1.7
100*
0
100
4
0.
4 3
0 4.
2 47
.4*
87
54.4
2
1.0
0.
9
74.8
* 69
82
.6
3
3.
0
2.6
95
.7*
77
96.7
3
0.4
10
0 2.
5 75
.2*
66
83.7
3
1.0
3.
4
52.1
* 80
61
.7
2
3.
0
4.2
71
.8*
74
79.0
3
DIC
AR
ZO
L
Form
etan
aat
0.4
0 0
0.8
100*
0
100
4 20
0SP
1.
0
0
100*
0
100
4
3.
0
0
100*
0
100
4 25
00 g
/ha
0.
4 3
0 6.
7 10
0*
0 10
0 4
1.0
0
10
0*
0 10
0 4
3.0
0
10
0*
0 10
0 4
0.4
10
0 7.
5 10
0*
0 10
0 4
1.0
0
10
0*
0 10
0 4
3.0
0
100*
0
100
4
50
Tabl
e 2:
Res
ults
of t
he p
ersi
sten
ce te
st w
ith P
hyto
seiu
lus p
ersi
mili
s fo
r the
9th
JTP-
IOB
C
n.
t.: n
ot te
sted
, phy
toto
xic
con
tinue
d 2
*s
igni
fican
tly d
iffer
ent f
rom
con
trol
TR
AD
E N
AM
E
g
or m
l pro
duct
/ ha
activ
e in
gred
ient
fd
PIE
Cre
sidu
e ag
e [d
ays]
% e
scap
e (A
BB
OT
T)
% m
orta
lity
cont
rol
% m
orta
lity
(AB
BO
TT
) E
gg/fe
mal
e
[% c
ontr
ol]
Tot
al
effe
ct [%
]IO
BC
cl
ass/
M
ASA
I 20W
P T
ebuf
en-
0.4
0 0
0.8
100*
0
100
4
pyra
d 1.
0
0
100*
0
100
4
3.
0
2.5
10
0*
0 10
0 4
500
g/ha
0.4
3 0
5.0
100*
0
100
4
1.
0
0
100*
0
100
4
3.
0
0
100*
0
100
4
0.
4 10
0
3.3
100*
0
100
4
1.
0
0
100*
0
100
4
3.
0
0
100*
0
100
4 A
MIS
TA
R
Azo
xyst
robi
n 0.
4 0
26.5
* 2.
5 30
.8*
82.8
43
.1
2 25
0SC
1.0
37
.6*
41
.9*
68.9
59
.9
2
3.
0
42.7
*
44.4
* 65
.1
63.9
2
1600
g/h
a
0.4
3 11
.0
10
11.1
97
.6
13.3
1
1.0
0
3.
8 98
.2
5.6
1
3.
0
15.6
24.1
81
.9
37.8
2
0.4
10
6.7
4.2
9.2
91.4
17
.1
1
1.
0
6.1
6.
9 87
.9
18.1
1
3.0
9.
6
16.5
88
.4
26.2
1
IMPU
LSE
Sp
irox
amin
e 0.
4 0
40.2
* 3.
3 68
.9*
38.8
88
.1
3 50
0EC
1.0
21
.4*
96
.6*
35.3
98
.8
3
3.
0
6.8
10
0*
0 10
0 4
1500
g/h
a
0.4
3 20
.7
3.3
27.6
10
3.8
24.8
1
1.0
n.
t. n.
t. n.
t. n.
t. n.
t. n.
t.
3.
0
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
0.4
10
4.4
5.0
7.9
102.
5 18
.9
1
1.
0
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
3.0
n.
t. n.
t. n.
t. n.
t. n.
t. n.
t.
51
52
Pesticides and Beneficial Organisms IOBC/wprs Bulletin Vol. 25 (11) 2002
pp. 53 - 61
53
Side-effects of fifteen insecticides on predatory mites (Acari: Phytoseiidae) in apple orchards J. Raul Rodrigues*, Nelson R. C. Miranda*, João D. F. Rosas*, Carmelinda M. Maciel* & Laura M. Torres** * Instituto Politécnico de Viana do Castelo, Escola Superior Agrária de Ponte de Lima, Dep.
Ciências da Planta e do Ambiente, Refóios do Lima, 4990-706 Ponte de Lima, Portugal; **Universidade de Trás-os-Montes e Alto Douro, Dep. Protecção de Plantas, Quinta de
Prados, 5000-911 Vila Real, Portugal. E-mail: [email protected] Abstract: To evaluate the toxicity of fifteen insecticides to the predatory mites Phytoseiids (Acari: Phytoseiidae), three field tests were carried out in an apple orchard in the period of 23 July to 03 September of 2000 in the region of Ponte de Lima, Northern Portugal. The experimental design was fully randomized, with five replicates (trees) per treatment. Five insecticides were tested at each time, using commercial formulations for all insecticides at recommended field rates. The control plot was treated with water. Assessments of the phytoseiid and P. ulmi density were performed after 4, 7, 14 and 28 days of the treatments. The remaining mobile stages of both mites were counted on 25 leaves per replicate. The results of toxicity of insecticides to the phytoseiids was calculated with the Henderson-Tilton formula, and divided into four categories corresponding to the standard for field methods of the IOBC Working group “Pesticides and Beneficial Organisms”. The species of Phytoseiid mites identified were in terms of their abundance, Euseius stipulatus (Athias-Henriot) (54%), Typhlodromus pyri Scheuten (41%), and other species (5%). Phosalone, endosulfan, deltamethrin, dimethoate, metidathion and vamidothion were harmful to the phytoseiid mites; dichlorvos, carbaryl, imidacloprid and pirimicarb were moderately harmful. Bacillus thuringiensis and fenoxycarb were harmless and diflubenzuron, teflubenzuron and diazinon showed a medium selectivity (slightly harmful).
Key words: apple tree; integrated pest management; predatory mites; insecticides, side-effects
Introduction The red spider mite Panonychus ulmi (Koch) is one of the major pests of apple orchards in Portugal. The control of this pest is based on the use of specific acaricides. Summer sprays containing organophosphates and other chemical families used against other pests and diseases, have been suspected to eliminate the natural predators of the red spider mite, such as phytoseiid mites (Acari: Phytoseiidae). Integrated Pest Management is now more and more used in the standard apple productions system in most European countries. Depending on specific and local problems, the accent is put on the reduction of broad spectrum pesticides, the application of selective and/or biological compounds, the use of biotechniques, the introduction of antagonists, mainly predatory mites, frequent monitoring, the protection of a specific and typical fauna in the orchard to create a more “stable” system, or, in most cases, a combination of these measures (Sterk et al., 1994).
Mites of the Phytoseiidae family received considerable attention in the last four decades because of their potential as biological control agents of phytophagous mites and, more recently, of thrips on various crops (McMurtry & Croft, 1997). The effectiveness of
54
phytoseiid mites in the biological control of tetranychid mites and the implementation of Integrated Pest Management programs has therefore stimulated widespread and systematic investigations on the side-effects of pesticides on this antagonist (Costa-Comelles, 1986). Several trials to evaluate the influence of pesticides on Phytoseiid mites in field conditions in an apple orchard were carried out in the Peninsula Ibérica during the last years (Costa-Comelles et al., 1995, 1996, 1997, Silva & Gonçalves, 1997, Abela & Jousseaume, 1999, Espinha et al., 1999a, 1999b, Rodrigues, 2000). In Portugal, studies began in the last decade in the University of Trás-Os-Montes e Alto Douro (Torres, 1999; Rodrigues, 2000) and has been continued in the High School of Agricultural in Ponte de Lima. A project to evaluate side-effects of pesticides on all sensitive stages of the predatory mites (Acari: Phytoseiidae) started recently. The tests will be carried out in a vineyards and apple orchards during three years in several portuguese regions (Rodrigues, 2001).
The aim of this study was to evaluate field toxicity of fifteen insecticides to Phytoseiid mites under field conditions in an apple orchard in single applications. Material and methods Fifteen insecticides were tested in single application in three trials with five insecticides per trial (Table 1). The trials were carried out according to the IOBC guidelines to evaluate side-effects of plant protection products to non-target arthropods (Candolfi et al., 2000). The experimental apple orchard was placed in a field at the High School of Agricultural in Ponte de Lima, Nordeast Portugal. It has a size of 2.3 ha and is planted with six years old Royal Gala apple trees planted in 4 x 1.3 m and grafted on M9-EMLA. Trees were pruned to approximately 2.3 m height.
The trials where carried out during the period between the last week of July and the first week of September 2000. During trial period the meteorological data were monitored. In the 2nd and 3rd week after treatments the rainfall was about 10 mm. Before the trials started, a first assessment of presence and homogeneous distribution and variance equality within the test plots was carried out. The experimental design consisted of 3 trees with 5 replicates per treatment in a fully randomized block. The control plot was treated with water. The test compounds were applied at recommended rates by the manufacturer using a volume of 1000 l/ha. The trees were sprayed with the insecticides by a knapsack using a hand-lance until run-off.
The assessment of the mobile stages of phytoseiid and red spider mites per leaf, was performed in laboratory with a stereoscopic loupe. The leaves were detached in a central tree of each replicate at five times: one day 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. The results of mortality of predatory mites were calculated with the Henderson-Tilton formula and divided into four categories corresponding to the standard for field methods of the IOBC Working group “Pesticides and Beneficial Organisms” (Sterk et al., 1994, Blümel et al., 2000). According this formula the percentage of
mortality 1001(%)12
21 ×⎟⎟⎠
⎞⎜⎜⎝
⎛××
−=PKPK , 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%).
55
Experimental plots were maintained without pesticide sprays during the observations period.
After the treatment, the mean number of mobile stages of predatory mites/leaf counted at each time of assessment in all 5 replicates, was calculated and statistically evaluated with an univariate ANOVA (SPSS 10). If statistically significant differences were found for the mean number of predatory mites/leaf, the HSD Tukey Test mean comparison was performed at 5% level. The identification of Phytoseiidae species were made using adequate Keys (Chant & Yoshida-Shaul, 1987; Miedema, 1987; Kreiter & Bourdonnaye, 1993; García-Mari et al., 1994). Table 1: Insecticides used in the trials.
Active ingredient Trial nº
Trade name Commercial product/ha
B. thuringiensis var. Kurstaky (32% p/p)
1 Bactur 2X WP® 0,75 kg
fenoxycarb (25% p/p) 1 Insegar 25 WG® 0,60 kg
diflubenzuron (25% p/p) 1 Dimilin WP 25® 0,40 kg
phosalone (30% p/p) 1 Zolone® 2,00 kg
teflubenzuron (150 g/l) 1 Nomolt® 0,32 kg
endosulfan (380 g/l) 2 Thiodan® 5,00 l
dichlorvos (500 g/l) 2 Nogos 50® 2,00 l
diazinon (600 g/l) 2 Basudine 600 EW® 0,85 l
deltamethrin (25 g/l) 2 Decis® 0,30 l
carbaryl (50% p/p) 2 Visene® 2,00 kg
dimethoate (400 g/l) 3 Digor® 1,00 l
imidacloprid (200 g/l) 3 Confidor® 0,50 l
metidathion (40% p/p) 3 Ultracide 40M® 1,00 kg
pirimicarb (50% p/p) 3 Pirimor® 0,50 kg
vamidothion (400 g/l) 3 Kilval® 1,25 l
Results
In this study, four species of phytoseiid mites where identified (Table 2). The species present were in terms of their abundance, Euseius stipulatus (Athias-Henriot) (54%), Typhlodromus pyri Scheuten (41%), Euseius finlandicus (Oudemans) (4%) and Neoseiulus barkeri Hughes (1%).
The mean density of Panonychus ulmi, was lower during the trials period. The highest values density of red spider mite were observed in the assessment realized14 days after the treatments, in the trial 1 (plot treated with dimethoate - 0,11 mites/leaf) and in the trial 2 (plot
56
treated with deltamethrin - 0,12 mites/leaf). For this reason we considere that mean population density as negligible. Table 2: Phytoseiid species identified during the trials period.
Species Nº abundance
Euseius stipulatus (Athias-Henriot) 310 54%
Typhlodromus pyri Scheuten 236 41%
Euseius finlandicus (Oudemans) 22 4%
Neoseiulus barkeri Hughes 7 1%
Predatory mites were homogeneously distributed within the plots before and after treatments in all trials. The mean density of phytoseiid mites in the assessment performed before the treatments was more than 2,3 mobile stages per leaf in all plots. Before the treatments, no statistically variation in the mean numbers of predatory mites between the different treated plots and the control plot was detected in all trials, whereas in the assessements performed after the treatments, a decrease in the mean number of predatory mites per leaf, was observed in the treated plots. After the treatments significant differences between treatments were found compared with the control plot in all trials (p<0,05). Trial 1: After the treatments, a decrease in the mean number of predatory mites per leaf was observed in the treated plots. Significant reduction in comparison to the control plot was found in the plot treated with phosalone (Fig. 1). Trial 2: Significant differences were found between the control plot and all treated plots after treatments (Fig. 2). The lowest value of predatory mites per leaf was observed in the plot treated with deltamethrin (0,37), that was significantly lower than the plots treated with dichlorvos and diazinon. Trial 3: In this trial, the mean number of predatory mites per leaf differed significantly between the control and all treated plots (Fig. 3). The mean value of predatory mites per leaf in the plot treated with dimethoate (0,09) was significantly lower than in the plot treated with imidacloprid (0,55 predatory mites/leaf). Discussion The phytoseiid species present in this study are categorized by McMurtry & Croft (1997) as Generalist Species – Type III (T. pyri and N. barkeri) and as Generalist Predators/Pollen Feeders – Type IV (E. stipulatus and E. finlandicus). These generalist species can maintain tetranychid populations at low densities. These species are also considered as mediterranean endemic species (Ferragut & Escudero, 1997; Kreiter & Bourdonnaye, 1993). Rodrigues (2000) and Villas (2001) describe their presence in apple orchards in this region.
57
0,00 0,50 1,00 1,50 2,00 2,50 3,00 3,50
control
B. Thuringiensis
fenoxycarb
diflubenzuron
phosalone
teflubenzuron
mobile stages of phytoseiids/leaf
Mean (T4 - T28) T0
2,33 a
2,20 A
2,06 A
1,17 AB
1,23 AB
0,37 B
1,1 AB
3,08 a
3,10 a
2,54 a
2,45 a
2,51 a
Figure 1: Mean density of Phytoseiidae per leaf in the different plots, before the treatments (T0) and within four weeks afterwards (mean of four assessments, T4-T28) for Trial 1. Means in same columns followed by different letters are statistically significantly different (p<0,05, HSD-Tukey multiple comparison)
0,00 0,50 1,00 1,50 2,00 2,50 3,00 3,50
control
endosulfan
dichlorvos
diazinon
deltamethrin
carbaryl
mobile stages of phytoseiids/leaf
Mean (T4 - T28) T0
3,04 a
3,07 a
2,82 a
2,75 a
3,3 a
3,07 a
0,79 BC
0,29 C
1,18 B
0,92 B
0,62 BC
2,31 A
Figure 2: Mean density of Phytoseiidae per leaf in the different plots, before the treatments (T0) and within four weeks afterwards (mean of four assessments, T4-T28) in Trial 2. Means in same columns followed by different letters are statistically significantly different (p<0,05, HSD-Tukey multiple comparison)
58
0,00 0,50 1,00 1,50 2,00 2,50 3,00 3,50
control
metidathion
pirimicarbe
vamidothion
dimethoate
imidacloprid
mobile stages of phytoseiids/leaf
Mean (T4 - T28) T0
2,90 a
3,25a
3,18 a
3,15 a
3,15 a
2,98 a
0,55 B
0,09 C
0,30 BC
0,46 BC
0,24 BC
1,13 A
Figure 3: Mean density of Phytoseiidae per leaf in the different plots, before the treatments (T0) and within four weeks afterwards (mean of four assessments, T4-T28) for Trial 3. Means in same columns followed by different letters are statistically significantly different (p<0,05, HSD-Tukey multiple comparison)
The present study was carried out in an apple orchard with a high density of phytoseiid mites and with low densities of red spider mite in the assessment realized before the treatments for all plots. McMurtry (1992) considers that relatively high predator: prey ratios (1:1 or higher for some species) may be required for generalist phytoseiids to control a tetranychid mite population at low densities.
During this study, the mean density of predatory mites per leaf decreased for the control plots in all trials. This decrease may be explained by the rain that occurred in middle August and/or by the migration process of female phytoseiids towards hibernation sites.
In Table 3 we present the results of toxicity of each pesticide during the trial period. As expected diflubenzuron, teflubenzuron and fenoxycarb were slightly toxic for the phytoseiids, and dimethoate, deltamethrin, metidathion and vamidothion were harmful for the Phytoseiidae mites. Also phosalone was harmful. This product is used in Integrated Pest Management programs in apple orchards in Portugal, as a recommended product against codling moth and aphids (Gonçalves & Cavaco, 1997). The results obtained for phosalone correspond to those obtained by Espinha et al, (1999c) in Northern Portugal. Contradicting results were found in the literature about the effect of phosalone on phytoseiid mites, whereas this compound was classified as slightly harmful to T. pyri by Sterck et al. (1994) in Belgian orchards.
Pirimicarb and imidacloprid were harmful to moderately harmful up to 14 days after the treatment, but the predatory mite population soon recovered. Costa-Comelles et al., (1995), observed high toxicity of these insecticides in field conditions on the phytoseiid Amblyseius andersoni (Chant). In contrast, contradictory results were referred by Gendrier & Reboulet (1994) that considers these insecticides as harmless to slightly harmful to T. pyri. The poor selectivity of deltamethrin and dimethoate to the phytoseiids (Gendrier & Reboulet, 1994; Casas,
59
1995; Espinha et al., 1999c) is confirmed. Endosulfan that was harmful, is used in Portugal in Integrated Pest Management programs and considered harmless to Typhlodromus pyri Scheuten (Gendrier & Reboulet, 1994; Gonçalves & Cavaco, 1997). Table 3: Classification of insecticides toxicity, according to the IOBC Working Group “Pesticides and Beneficial Organisms” (% = percentage of reduction in phytoseiid population in comparison to the control plot; Cl = Class of toxicity).
T4
T7
T14
T28
Mean (T4-T28)
Active ingredient
Trial
Nº % Cl % Cl % Cl % Cl % Cl
B. thuringiensis 1 13,2 1 12,1 1 2,04 1 -6,6 - 6,9 1
fenoxycarb 1 60,9 3 53,3 3 20,7 1 -22,4 - 25,4 1-2
diflubenzuron 1 58,3 3 19,0 1 10,3 1 20,9 1 29,9 2
phosalone 1 81,2 4 88,6 4 90,6 4 45,1 2 79,4 4
teflubenzuron 1 51,3 3 31,5 2 24,0 1 19,0 1 34,0 2
endosulfan 2 83,6 4 72,2 4 78,0 4 60,5 3 75,0 3-4
dichlorvos 2 67,4 3 68,7 3 48,1 2 22,8 1 55,4 3
diazinon 2 56,8 3 42,8 2 32,6 2 41,2 2 44,2 2
deltamethrin 2 91,9 4 93,2 4 85,6 4 72,7 3 87,7 4
carbaryl 2 83,6 4 75,1 4 68,3 3 14,3 1 65,4 3
dimethoate 3 93,9 4 95,0 4 95,1 4 78,6 4 92,4 4
imidacloprid 3 76,5 4 63,7 3 52,1 3 -45,1 - 50,1 2-3
metidathion 3 78,8 4 88,8 4 86,4 4 55,9 3 79,9 4
pirimicarb 3 76,1 4 78,5 4 55,9 3 9,26 1 61,4 3
vamidothion 3 90,2 4 86,4 4 73,1 3 21,1 1 74,9 3-4
The results confirm a strong gradient in toxicity between organophosphates (Sterck et al., 1994), going from vamidothion and metidathion (both harmful) to diazinon which was slightly harmful. Among the carbamates, carbaryl and pirimicarb have the highest toxicity to the phytoseiid species present. Compared to the organophosphates, the gradient in toxicity amongst carbamates is also pronounced, but with lower toxicity levels, going from fenoxycarb (that was harmless to slightly harmful) to carbaryl and pirimicarb (both moderately harmful). Acknowledgements We gratefully acknowledge the comments and the revision on the manuscript by Isabel Mourão, Miguel Brito, José Ribeiro and Sofia Bacelar (High School of Agricultural Ponte de Lima).
60
References Abela V. & Jousseaume C. 1999: O mancozebe e os ácaros fitoseídeos: um estudo prático em
macieira em Portugal. In: Instituto Politécnico de Bragança, Escola Superior Agrária (Ed.) - V Encontro Nacional de Protecção Integrada. A Prática da Protecção Integrada no Limiar do Milénio. Bragança, 27 a 29 de Outubro de 1999, 179-184.
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, 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.
Candolfi M. P., Blümel S., Forster R., Bakker F. M., Grimm C., Hassan S. A., Heimbach U., Mead-Briggs M. A., Reber B., Schmuck R. & Vogt H. (eds) 2000: Guidelines to evaluate side-effects of plant protection products to non-target arthropods. IOBC, BART and EPPO, Joint Initiative, IOBC/WPRS Gent, 158 pp.
Casas J. I. 1995. Productos fitosanitarios y organismos útiles en frutales. Fruticultura profesional. Especial producción integrada, 70: 27-33.
Chant D. A. & Yoshida-Shaul, 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.
Costa-Comelles J., Bosh D., Botargues A., Cabiscol P., Moreno A., Portillo J. & Avilla J. 1996: Toxicidad de algunos insecticidas sobre el fitoseido Amblyseius andersoni (Chant) depredador de la araña roja Panonychus ulmi (Koch) en manzano. Agrícola Vergel, Agosto: 471-475.
Costa-Comelles J., Bosh D., Botargues A., Cabiscol P., Moreno A., Portillo J. & Avilla J. 1997: Acción de algunos acaricidas sobre los fitoseídos y la araña roja Panonychus ulmi (Koch) en manzano. Bol. San. Veg. Plagas, 23: 93-107.
Costa-Comelles J., Cabiscol P., Portillo J., Botargues A., Moreno A., Solé J., Bosch D. & Avilla J. 1995: Eficacia de algunos plaguicidas sobre el pulgon verde del manzano Aphis pomi Degeer y su accion en la acarofauna. Agrícola Vergel, Abril: 184-192.
Espinha I. G., Rodrigues J. R., Carlos C. & Nave A., 1999b: Efeitos secundários de insecticidas e acaricidas sobre ácaros fitoseídeos associados à macieira: Simpósio “Protecção integrada da macieira contra o aranhiço vermelho, Panonychus ulmi (Koch) em condições mediterrânicas”. Universidade de Trás-Os-Montes e Alto Douro, 7 de Junho de 1999.
Espinha I. G., Rodrigues J. R., Torres L. & Avilla, J. 1999a: Efectos secundários de algunos acaricidas sobre los ácaros fitoseiods (Phytoseiidae, Acari) asociados al manzano en el Norte de Portugal. Congreso Nacional de Entomología Aplicada – VII Jornadas Científicas de la S. E. E. A., Ed. Junta de Andalucia, Consejeria de Agricultura y Pesca. Colección Congresos y Jornadas 53/99: 169. Almeria, 08-12 de Noviembre 1999.
Espinha I. G., Torres L. M., Avilla J. & Carlos C. 1999c: Testing the side effects of pesticides on phytoseiid mites (Acari, Phytoseiidae) in field trials.XIVth International Plant Protection Congress (IPPC) – Plant protection towards the third Millenium-Where Chemestry Meets Ecology. Jerusalem, Israel, July 25-30, 113 pp.
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Ferragut F. & Escudero A. 1997: Taxonomía y distribución de los ácaros depredadores del género Euseius Wainstein 1962, en España (Acari: Phytoseiidae). Bol. San. Veg. Plagas, 23: 227-235.
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.
Gendrier J. P. & Reboulet J. N. 1994: Choix des produits phytossanitaires en vergers. Les références de l’ACTA pour 1994-95. Phytoma – La Défense des végétaux, 465: 39-42.
Gonçalves M. & Cavaco M. 1997: Protecção integrada de pomóideas. Lista dos produtos fitofarmacêuticos e níveis económicos de ataque. Direcção Geral de Protecção das Culturas (DGPC), Lisboa, 58 pp.
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. 1992: Dynamics and potential impact of ‘generalist’ phytoseiids in agroecossystems and possibilities for establishment of exotic species. Experimental & Applied Acarology, 14: 371-382.
McMurtry J. A. & Croft B. A. 1997: Life-Styles of phytoseiid mites and their roles in biological control. Annu. Rev. Entomol. 42: 291-321.
Miedema E. 1987: Survey of phytoseiid mites (Acari: Phytoseiidae) in orchard and surrroundig vegetation of northwestern Europe especially in the Netherlands. Keys, descriptions and figures. Neth. J. Pl. Path. 93 (2): 1-65.
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. 2001: Redução do uso de pesticidas na agricultura, uma realidade. Site: Escola Superior Agrária de Ponte de Lima (Last update: 21/11/01). URL: http://www.esapl.pt/web/raulrodrigues/page6.html, (accepted 29/11/01)
Silva C. & Gonçalves M. 1997: Avaliação biológica de quatro substâncias activas acaricidas (piridabena, fenepiroximato, fenazaquina e tebufenpirade) no combate ao aranhiço vermelho da macieira Panonychus ulmi (Koch) e toxidade sobre ácaros fitoseídeos Typhlodromus pyri Scheuten. IV Encontro Nacional de Protecção Integrada, Angra do Heroísmo, 3 e 4 de Outubro de 1997, 68-69.
Sterk G., Creemers P. & Merckx K. 1994: Testing the side effects of pesticides on the predatory mite Typhlodromus pyri (Acari, Phytoseiidae) in field trials. IOBC/WRPS Bull. 17 (10), 27-40.
Torres, L. 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 grupo de trabalho da UTAD. Simpósio “Protecção integrada da macieira contra o aranhiço vermelho, Panonychus ulmi (Koch) em condições mediterrânicas. Vila Real, Junho de 1999.
Villas, M. V. 2001: Contributo para o estudo dos ácaros fitoseídeos associados à macieira e vinha na região de Entre Douro e Minho. Relatório final de Curso. em Engenheria Técnica Agrícola em Hortofruticultura e Jardinagem. Escola Politécnica Superior de Lugo da Universidade de Santiago de Compostela/Escola Superior Agrária de Ponte de Lima, 82 pp.
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Pesticides and Beneficial Organisms IOBC/wprs Bulletin Vol. 25 (11) 2002
pp. 63 - 70
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Side effects of plant protection products on Trichogramma cacoeciae Marchal (Hym. Trichogrammatidae) Abdelgader, H. and S. A. Hassan Institute for Biological Control, Federal Biological Research Center for Agriculture and Forestry, Heinrichstr. 243, D-64287 Darmstadt, Germany Abstract: The side effects of 13 plant protection products (9 insecticides/acaricides, 2 fungicides and 2 herbicides) on adults and immature stages of the egg parasitoid Trichogramma cacoeciae Marchal (Hymenoptera, Trichogrammatidae) were studied in the laboratory. The results showed that one insecticide (Mimic) and one herbicide (Logran) were safe to the adults, whereas 6 insecticides and one fungicide were harmful. The other tested insecticides (Naja und Chess), fungicide (Amistar) and herbicide (Gesagard) were slightly to moderately harmful. Spraying the immature stages of T. cacoeciae (within the host eggs Sitotroga cerealla) showed that only one insecticide (Phosdrin) was harmful, two insecticides (Confidor and Masi) were slightly harmful and the remaining preparations (Neemazal, Impuls, Aztec, Dicarzol, and Chess) were harmless. Key words: Trichogramma, pesticides, side effects, beneficial insect, parasitoid Introduction Parasitoids of the genus Trichogramma occure naturaly worldwide and play an important role as natural enemies of lepidopterous pests on a wide range of agricultural crops. During the past two decades Trichogramma wasps have been used as biological control agents for pest suppression. Results of augmentative releases of Trichogramma can be affected by the use of broad-spectrum insecticides in or near release plots (Stinner et al. 1974, Ables et al. 1979, King et al. 1984). The use of Trichogramma in biological control has gained widespread interest in many countries.
At the moment, about 18 species of Trichogramma are being used in more than 23 countries. Approximately 10 different Trichogramma spp. are being mass-reared to control pests on corn, sugarcane, tomato, rice, cotton, sugar beet, apple, plum, vineyard, pasture, cabbage, chestnut, sweet pepper, pomegranate, paddy and forests in at least 23 countries. Therefore, studying the side effects of pesticides is of prime importance to save these beneficials and encourage their role as biological control agents. The procedures used for testing the side effects of pesticides on beneficial organisms in several countries. Since the Council Directive 91/414/EEC concerning registration procedures became operative, data on side effects of pesticides towards beneficial organisms are obligatory in Europe (Hassan 1994).
In the present study, the side effects of 13 pesticides of different groups as selected by the IOBC WG “Pesticides and Beneficial Organisms” for the 9th Joint Pesticide Testing Programme were tested on the egg parasitoid Trichogramma cacoeciae on both the most susceptible stage (adult) and less susceptible stages (pupae and larvae within host) in the laboratory.
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Materials and methods Pesticides The study included 9 insecticides/acaricides, 2 fungicides and 2 herbicides as shown in Table 1. Test rates were based on the recommended field rates. The rates applied for the present laboratory trials were the predicted initial environmental concentrations (PIEC) for foliage dwelling predators ( Barrett et al. 1994):
PIEC [µg a.i./cm2= field dose rate in g a.i./ha x fd/100 (fd = 0.4)]. Table 1. Pesticides tested in the study
a- Insecticides / Acaricides
Trade Name Active Ingredient
Recommended field rate
(g product/ha)
Test rate PIEC 0,4
(µg product/cm2) Mimic 240 Flow Tebufenozide 600 2.4 Naja 50 EC Fenpyroximate 1000 4.0 Chess 25 WP Pymethrozine 120 0.5 Aztec 140 EC Triazamaat 500 2.0 Dicarzol 200 SP Formetanaat 2500 10.0 Neemazal T/S 1% Azadirachtine 3000 12.0 Confidor 200 SL Imidacloprid 350 1.4 Masai 20 WP Tebufenprad 500 2.0 Phosdrin 145 Mengolie Mevinphos
3450 13.8
b- Fungicides
Amistar 250 SC Azoxystrobin 1600 6.4 Impuls 500 EC Spiroxamine 1500 6.0 c- Herbicides Logran 75 WG Triasulfuron 20 0.1 Gesagard 50 WP Promethryn 20 0.1
Initial toxicity test The initial toxicity was tested by exposing the adult parasitoids (the most susceptible life stage in terms of exposure) to fresh dry residues of the plant protection products applied on glass plates. The test followed the standard method described by Hassan et al. (2000). Accordingly the exposure cage consisted of an aluminium frame bearing ventilation holes. The upper and lower surfaces of which were covered by foam material to provide pads for two glass plates. These glass plates were treated with various pesticides using a hand sprayer to achieve an even spray deposit of 2 mg/cm2 on the glass plate. After drying, the glass plates were then fitted onto the square aluminium frame forming the floor and ceiling of the test unit. One side of the aluminium frame has two different holes. The smaller hole was used to introduce the
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starting population of the parasitoid and the second bigger hole was used to introduce the host eggs (Sitotroga cerealla) to be parasitized and the food (honey/gelatine) for the insects. In order to deter the parasitoids from remaining on the untreated sides of the frame, the exterior edges of the glass plates were covered with black card. The cage was held together with clamps. The cages were connected to a vacuum pump via a tube system to prevent possible accumulation of plant protection product vapours. The side effects of tested pesticides was determined through mortality assessment and the capacity of parasitism. Larvae and pupae toxicity tests The method described by Hassan and Abdelgader (2001) was followed to study the side effects on immature stages of T. cacoeciae within the host eggs. For these tests about 1 day old Sitotroga eggs were glued in discs (ca. 1 cm in diameter) on strips of paper and parasitized by T. cacoeciae for 24 hours. The parasitized eggs were left for 2/7 days (larvae/pupae tests) at: 26 ± 2°C, 75 ± 20% RH. These parasitized egg discs were then sprayed with the appropriate pesticide. Six replications (each one disc) were made for each pesticide. The percentage adult emergence and/or the development of black eggs were used to assess the effects of tested pesticides.
The study included also a dose-mortality study with the harmful insecticide Phosdrin on the immature stages (larval stage / 2 days after parasitism) within host. The sprayed parasitised host eggs were kept after spraying at different post-treatment temperatures (26°C, 15°C and 10°C). The objective was to investigate the toxic effects of this harmful insecticide at different doses and temperatures. Results and discussion Initial toxicity test The results of the initial laboratory tests on adult T. cacoeciae in term of the percentage mortality after 24 hours of exposure to the pesticides are presented in Figure 1. The results of 4 experiments revealed 100% mortality 24 hours after exposing adults T. cacoeciae to glass plates treated with Impuls, Aztec, Dicarzol, Confidor, Neemazal T/S and Masai. These six harmful pesticides, are classified in class 4 based on IOBC – classification (> 99% effect) (after Hassan et al. 2000). Phosdrin can also be classified as harmful (class 4) insecticide on T. cacoeciae, based on it’s high vapour pressure, which resulted in high mortality of adults of T. cacoeciae in the emergence tubes. In addition, Phosdrin caused 100% mortality of both larval and pupal within host eggs (Tables 4 and 5).
Table 2 shows the results of further tests with the 6 pesticides causing less than 100% mortality in the above experiments. The results reveal, that the insecticide Mimic and the herbicide Logran were harmless (< 30%) for adults of T. cacoeciae. Amistar, Naja and Gesagard were slightly harmful (30-79%), whereas Chess was moderately harmful (80 - 99%) based on the IOBC classification for initial laboratory experiments on the most susceptible life stage. The result with Logran on the parasitic wasps can be expected since this chemical being a herbicide is targeted mainly against weeds. Mimic being one of the moult inhibiting insect growth regulator affects insects by stimulating a premature moult in the larvae of lepidoptera. This chemical can be considered as highly selective acting only on larvae of Lepidoptera and thus safe to bees, predatory and parasitic insects (Brunner 1998). The other slightly to harmful pesticides would need further testing before a final conclusion on their side effects on T. cacociae can be drawn.
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Table 2. Rate of parasitism (eggs/female) of adults Trichogramma cacoeciae exposed to treated glass plates
First Experiment Second Experiment
Treatment Eggs/female ** RP Class Treatment Eggs/female ** RP Class
Control 43.17 a - - Control 44.13 a - -
Naja 20.63 b 52.20 2 Logran 33.97 ab 23.03 1
Amistar 20.90 b 51.58 2 Gesagard 19.07 bc 56.80 2
Mimic 45.60 a -5.64 1 Chess 1.70 c 96.15 3 ** Figures followed by the same letter were not significantly different at 0.05 Multiple Range Test RP = Percentage Reduction in parasitism rate compared to the control Class = IOBC Classification Pupae toxicity tests The pesticides, which were harmful or moderately harmful during the initial laboratory test, were tested further on the pupal stage of T. cacoeciae within host eggs and results are presented in Tables 3 and 4. The results revealed that Phosdrin was harmful, Confidor and Masai were slightly harmful and Neemazal, Impuls, Aztec, Dicarzol and Chess were harmless to the pupae. Table 3. Effects of treating 7-day old parasitized host eggs with pesticides on adult emergence (First experiment)
TreatmentTreated
eggs **Emerged
Adults **Emerged Adult% RA Class
Control 956 a 900 ab 94.5
Neemazal 1037 a 690 c 66.6 29.3 1
Impuls 1050 a 979 ab 93.2 1.0 1
Aztec 974 a 943 ab 96.8 -2.9 1
Confidor 1202 a 768 bc 63.9 32.1 2 ** Figures followed by the same letter were not significantly different at 0.05 Multiple Range Test RA = Percentage Reduction in Adult emergence rate compared to the control Class = IOBC Classification
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Table 4. Effects of treating 7-day old parasitized host eggs with pesticides on adults emergence (Second experiment)
TreatmentTreated
eggs **Emerged Adults **
Emerged Adult% RA Class
Control 351 a 257.8 a 73.5 -
Phosdrin 235 c 0.0 d 0.0 100.0 4
Masai 301 ab 147.2 c 48.9 33.4 2
Dicarzol 340 a 216.8 b 63.7 13.3 1
Chess 287 b 217.0 b 75.7 -3.0 1 ** Figures followed by the same letter were not significantly different at 0.05 Multiple Range Test RA = Percentage Reduction in Adult emergence rate compared to the control; Class = IOBC Classification Larvae toxicity tests
The effects of pesticides on the larvae of T. cacoeciae within host eggs are presented in Table 5. The results revealed harmful effects of Phosdrin, slight harmful effects of Masai and no harmful effects of Dicarzol and Chess with regards to the percentage emerging adults.
With regard to the number of developed black eggs (i.e. number of larvae reaching pupal stage) there was a significant difference between three of the tested pesticides (Chess, Phosdrin and Dicarzol) and the control treatment. This might be taken as an indication of a toxic action of these pesticides on the larval development of T. cacoeciae. In case of Chess and Dicarzol fewer larvae reached the pupal stage, but were able to emerge normally as adults. On the other hand, the insecticide Masai had no effects relative to the control on the development of treated larvae to pupae, but the number of developing adults was significantly lower. Table 5. Effects of treating 2 – days old parasitized eggs (larval stage) with pesticides onn adults emergence
Treatment Treated
eggs **Emerged
Adults **Emerged Adult% RA Class
Control 377.7 a 319 a 84.5
Chess 196 c 161 c 82.1 2.7 1
Dicarzol 267.8 b 213.3 b 79.7 5.7 1
Masai 393.3 a 220.7 b 56.1 33.7 2
Phosdrin 263.3 b 0.0 d 0.0 100.0 4 ** Figures followed by the same letter were not significantly different at 0.05 Multiple Range Test RA = Reduction in Adult emergence rate compared to the control; Class = IOBC Classification
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The results of the rate response test with Phosdrin on larvae of T. cacoeciae and holding treated larvae at different post treatment temperatures are presented in Table 6. The results at higher temperature (26°C) showed harmful effects up to 0.125 fold of the field recommended rate (FR). As the post-treatment temperature decreased to 15°C, the 0.125 FR was moderately harmful (Class 3). By further decrease in the temperature to 10°C the toxicity of two other harmful rates at higher temperatures (i.e. 0.5 and 0.25 FR) became moderately harmful (Class 3). In all these cases the toxicity decreased as the temperature decreases. The results at the highest temperature tested (26°C) showed that a dose of 0.063 FR was slightly harmful to the treated larvae in term of the percentage developing adults. Table 6. Effects of treating parasitized host eggs, 2 days after parsitisation, with various rates of the insecticide Phosdrin (PH) and holding them at different post-treatment temperatures
Treatment 26°C 15°C 10°C Adult% RA Cl Adult% RA Cl Adult% RA ClControl 92.0 - 96.2 93.2 PH1 (FR) 0.0 100.0 4 0.0 100.0 4 0.0 100.0 4 PH2 (0.5 FR) 0.0 100.0 4 0.2 99.8 4 4.6 95.1 3 PH3 (0.25 FR) 0.0 100.0 4 0.5 99.5 4 6.0 93.6 3 PH4 (0.125 FR) 0.4 99.6 4 1.1 98.9 3 3.5 96.3 3 PH5 (0.063 FR) 51.9 43.6 2 42.0 56.3 2 9.3 90.1 3
RA = Reduction in Adult emergence rate compared to the control; Cl = IOBC Classification; FR = Field Recommended Rate Acknowledgement We thank the Alexander von Humboldt Foundation in Germany for financing the first author. References Ables, J.R., Johnes, R.K., Morrison, V.S., House, D.L., Bull, L.F., Bouse and Carlton, J.B.
1979: New developments in the use of Trichogramma to control lepidopteran pests of cotton, pp. 125-127. In: Proceedings, Beltwide Cotton Production Research Conference. National Cotton Council, Memphis, TN.
Barrett, K.L., Grandy, N., Harrison, S, Hassan. S. and Oomen, P. 1994: Guidance document on regulatory testing procedures for pesticides with Non-Target Arthropods. SETAC Europe. British Library Cataloguing-in-Publication Data (ISBN 0952253526).
Brunner, J.F. 1998: Pest Management-Novel Chemicals and Biological Control. A paper presented at the 41st Annual IDFTA Conference, February 21-25, 1998, Pasco, Washington.
Hassan, S.A., Halsall, N., Gray, A.P., Kuehner, C., Moll, M., Bakker, F.M., Roembke, J., Yousef, A., Nasr, F. and Abdelgader, H. 2000: A laboratory method to evaluate the side effects of plant protection products on Trichogramma cacocciae Marchal (Hym., Trichogrammtidae), 107-119. In: M.P. Candolfi, S. Blümel, R. Forster, F.M. Bakker, C. Grimm, S.A. Hassan, U. Heimbach, M.A. Mead-Briggs, B. Reber, R. Schmuck and H.
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Vogt (eds.) 2000: Guidelines to evaluate side-effects of plant protection products to non-target arthropods. IOBC/ WRPS, Gent.
Hassan, S. and Abdelgader, H. 2001: A sequential testing program to assess the effects of pesticides on Trichogramma cacoeciae Marchal (Hym., Trichogrammatidae). Pesticides and beneficial Organisms. IOBC/WRPS Bulletin 24 (4): 71-81.
Hassan, S.A. 1994: Comparison of three different laboratory methods and one semi-field test method to assess the side effects of pesticides on Trichogramma cacoeciae. IOBC/WRPS Bulletin 17 (10): 133-141.
King, E.G., L.F. Bouse, D.L. Bull, W.A. Dickerson, W.J. Lewis, P. Liapis, J.D. Lopez, R.K. Morrison and Phillips, J.R. 1984: Potential of management of Heliothis spp. by augmentative releases of Trichogramma pretiosum, pp. 232-236. In: Proceedings, Beltwide Cotton Production Research Conference. National Cotton Council, Memphis, TN.
Stinner, R.E., R.L. Ridgway, J.R. Coppedge, R.K. Morrison and W.A. Dickerson, Jr. 1974: Parasitism of Heliothis eggs after field releases of Trichogramma pretiosum in cotton. Environ. Entmol. 3: 497-500.
Pesticides and Beneficial Organisms IOBC/wprs Bulletin Vol. 25 (11) 2002
pp. 71 - 80
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Toxicity of different pesticides to the predatory bug Macrolophus caliginosus (Heteroptera: Miridae) under laboratory conditions Rosemarie Tedeschi*, Luc Tirry, Marc Van de Veire & Patrick de Clercq Laboratory of Agrozoology, Faculty of Agricultural and Applied Biological Sciences, Ghent
University, Coupure Links, 653, B-9000 Ghent, Belgium * Di.Va.P.R.A., Entomologia e Zoologia applicate all'Ambiente "Carlo Vidano", Università
degli Studi di Torino, Via Leonardo da Vinci 44, 10095 Grugliasco (TO), Italy Abstract: Laboratory experiments were carried out to evaluate the toxicity of commonly used pesticides to the predatory bug Macrolophus caliginosus Wagner. Young M. caliginosus nymphs were exposed to fresh spray deposits of different concentrations of pesticides in a worst case laboratory test. Nymphal mortality and fecundity of surviving nymphs were recorded. The insecticide pymetrozine and the insecticide/acaricide abamectin did not affect nymphal development, whereas the fungicides sulphur and thiophanate-methyl were slightly and moderately harmful, respectively. The insecticides/acaricides diafenthiuron, dimethoate and chlorfenapyr proved to be very harmful to M. caliginosus; the latter also demonstrated a long persistence of toxicity. Except for abamectin, no effect of the pesticides on fecundity was observed for the adults surviving dose-response assays. Preliminary trials were carried out to test the recommended concentration of 8 new pesticides, in order to have an overview of their side-effects on M. caliginosus. Key words: Macrolophus caliginosus, Miridae, predator, integrated pest management, side-effects. Introduction Trialeurodes vaporariorum (Westwood) and Bemisia tabaci (Gennadius) are economically important pests in European greenhouses. The parasitoid wasp Encarsia formosa Gahan has been used for several years for the biological control of these whiteflies. However, this aphelinid proved to be less efficient at lower temperatures. In contrast, the polyphagous mirid bug, Macrolophus caliginosus Wagner, has demonstrated excellent activity, even at lower temperatures.
Nevertheless, this predator has not always been able to keep pest populations below acceptable levels. Given the fact that growers want reliable and cheap control, it is necessary to have selective chemicals available that ensure satisfactory pest control without harming the natural enemies of whiteflies and other pests. Thus, it is extremely important for growers who use M. caliginosus as a biocontrol agent of whiteflies to know the possible harmful effects of the more commonly used agrochemicals on this predator.
Testing the side-effects of pesticides on beneficial organisms has become obligatory for registration in several countries and the use of standard methods is very important for the validity of the results and their comparison between different countries. The current work reports on laboratory experiments investigating the susceptibility of M. caliginosus to fresh and aged residues of some insecticides, acaricides and fungicides. The trials were carried out according to IOBC/WPRS guidelines for assessing side-effects of pesticides on beneficial organisms (Hassan et al., 1994; Sterk et al., 1999) in the laboratory. The effects on nymphal survival and on the fecundity of the adults were assessed after exposure to treated glass
72
plates. The persistence of the compounds was checked by exposing the nymphs to treated sweet pepper plants. Materials and methods Insects First and 2nd instars of M. caliginosus used in the experiments were purchased from Biobest NV, Belgium. Chemicals Information on the pesticides used and the recommended concentrations is presented in Table 1. Data are expressed in terms of concentrations instead of rates per ha because the amount of fluid sprayed in vegetable greenhouses varies a lot depending on the crop and on the stage of the plants.
Table 1: Active ingredients, trade names, formulations and recommended concentrations of the pesticides (i: insecticide; a: acaricide, f: fungicide)
Active ingredient
Trade name
Formulation Recommended concentration
(mg a.i./l) Abamectin (i/a) Vertimec 18 g/l 5 Acetamiprid (i) Mospilan 20 % 100 Chlorfenapyr (i/a) Intrepid 240 SC 275 Diafenthiuron (i/a) Polo 500 EC 400 Dimethoate (i/a) Perfekthion 400 EC 160 Emamectin (i) - 5 % SG 15 Halofenozide (i) - 240 WG 150 Indoxacarb (i) Steward 30 WG 300 Methoxyfenozide (i) Runner 240 WG 150 Pirimicarb (i) Pirimor 50 DG 250 Pymetrozine (i) Chess 25 WP 200 Pyriproxyfen (i) Admiral 10 EC 100 Sulphur (f) Kumulus 80 DF 3200 Thiamethoxam (i) Actara 25 WG 100 Thiophanate-methyl (f) Topsin M 70 WP 500 Triazamate (i) Aztec 250 WP 70
Initial contact toxicity tests: toxicity to nymphs The initial toxicity assays were performed in drum cells (Van de Veire et al., 1996). The drum cell consists of a Plexiglas ring (diameter: 9 cm; height: 1.5 cm) and two round glass plates of the same diameter. The ring has 7 ventilation holes (diameter: 0.6 cm), covered with nylon gauze, and one hole connected to a plastic tube containing a cotton wick that serves as water source. This wick is placed in a Plexiglas support containing tap water.
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Each glass plate was treated on one side with pesticide solution at different concentrations using a Cornelis spray chamber, which is similar to the Potter tower (Van de Veire, 1992). The compounds were sprayed at a pressure of 1 bar for 2.5 s, which resulted in a very homogeneous spray deposit (1 mg fluid/ cm2). After leaving the plates to dry at room-temperature, they were assembled to the ring to form the drum cells. The ring itself was not treated; however, the mirids spent most of the time on the plates where the food was deposited.
For each replication, 10 nymphs (1st - 2nd instar) of M. caliginosus were placed in drum cells together with eggs of Ephestia kühniella (Zeller) (Nutrimac, Biobest NV, Belgium) as a food source; fresh eggs were supplied every two days. During the experiments, the drum cells were kept in an incubator at 25 ± 1°C, 65 ± 5% RH and a 16:8 h (L:D) photoperiod. The insects remained in the drum cells until adulthood; mortality was checked every two days.
For each compound, five concentrations plus a water treated control were tested; five replications were carried out for each concentration.
Effects on reproduction To assess the effects of sublethal doses of the compounds on reproduction, adults surviving toxicity tests were placed in Plexiglas cylindrical cages (diameter: 9 cm; height: 3.5 cm). Each cage was provided with a sharp pepper plant (Capsicum annuum cv. Cayenne) with its roots immersed in water. The plant served as a moisture source and an oviposition substrate. Eggs of E. kühniella were supplied every two days as food.
Three females and 2 males were introduced in each cage and kept there for 15 days. Since eggs were difficult to find, the plants were checked for first instars every two days for a period of 20 days.
Persistence The compounds causing more than 50% mortality to nymphs at the recommended dose in the residual contact test were used in a persistence test. For these tests, young sharp pepper plants (4-6 leaves-stage) were sprayed with 10 ml of pesticide solution at the maximum recommended concentration, using the “Cornelis” spray chamber, which is superior to the Potter tower for this application (Van de Veire, 1992), at a pressure of 1 bar for 10 s. In the spray tower, plants were sprayed with 2 spray nozzles (one at the top of the tower, and one at the bottom of the tower), in that way plants were treated to incipient run-off. After drying at room temperature, the plants were transferred to cylindrical cages (diameter: 9 cm; height: 3.5 cm) and after different time intervals, 15 young M. caliginosus nymphs (1st-2nd instar) were placed in each container and provided with E. kühniella eggs as food. Mortality was monitored and fresh food was given every two days. The insects remained in the cells until they became adults. Each treatment was repeated 5 times. In controls, untreated plants were used. Preliminary initial contact toxicity tests with some new IPM relevant chemicals. Preliminary trials with new IPM relevant compounds (acetamiprid, emamectin, halofenozide, indoxacarb, methoxyfenozide, spinosad, thiamethoxam, triazamate) were carried out as described above; however, glass plates were treated only with the recommended concentration for use in the field (Table 1).
Ten 1st-2nd instar M. caliginosus nymphs were used for each replication; 5 replications were used for each compound and a water treated control. The mortality of the insects was checked 24 hours and 6 days after treatment.
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Statistical analysis LC50 values and 95% fiducial limits were calculated from probit regressions using POLO PC (LeOra Software 1987) based on the procedure of Finney (1971). To correct for control mortality, Abbott's formula (Abbott, 1925) was used. Fecundity and persistence data were compared between treatments by 1-way analysis of variance (ANOVA); means were separated using Tukey's test (SPSS Inc., 1989-1993), after arcsin square root transformation in the case of mortality percentages. Results Toxicity to nymphs Toxicity of the tested pesticides to the nymphal stages of M. caliginosus is shown in Table 2. Table 2: Toxicity of different pesticides to Macrolophus caliginosus Wagner nymphs, residual effect of deposits on glass applying 1 mg pesticide solution per cm2
Active ingredient
na SLOPE±SE LC50 mg a.i./l ( 95% FL)
χ2 df LR50 µg a.i./cm2
glass Abamectin 250 1.25±0.31 56.2
(27.8-386.1) 36.780 23 0.0562
Chlorfenapyr 260 1.17±0.39 1.6 (0.1-3.6)
23.509 24 0.0016
Diafenthiuron 120 1.46±0.36 23.3 (6.4-47.1)
15.566 10 0.0233
Dimethoate 250 4.05±0.89 11.1 (8.3-13.9)
22.193 23 0.0111
Pirimicarb 220 0.80±0.24 56.0 (23.1-1,329)
22.909 20 0.056
Pymetrozine 320 1.67±2.20 1122.5 (-)
19.922 23 1.1225
Pyriproxyfen 170 0.85±0.31 42.0 (14.4-163.5)
11.492 15 0.042
Sulphur 140 1.15±0.49 3943.3 (1,958-35,877) b
9.023 12 3.9433
Thiophanate-methyl
250 0.94±0.52 312.3 (-)
36.427 12 0.312
a n = number of insects tested
b fiducial limits at 90% probability level (-) fiducial limits not calculated by POLO-PC
Comparing LC50-values with the recommended doses, the pyrrole chlorfenapyr was the
most toxic compound followed by the organophosphate dimethoate, the thiourea diafenthiuron, the juvenile hormone mimic pyriproxyfen and the carbamate pirimicarb. The
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Table 3. Fecundity of Macrolophus caliginosus Wagner adults surviving exposure to different deposits of pesticides on glass during the nymphal stage
Active ingredient
Dose (mg a.i./l)
deposit µg a.i./cm2
glass
na
no. of nymphs Mean (±SE)b
Abamectin 0 0 4 53.0 (4.18) ab 2.5 0.0025 5 35.8 (12.34) ab 5 0.005 5 39.4 (7.86) ab 10 0.01 5 19.6 (0.87) b 20 0.02 3 63.33 (7.22) a 50 0.05 3 22.0 (11.37) b Diafenthiuron 0 0 4 26.25 (14.04) a 10 0.01 4 17.25 (6.69) a 50 0.05 2 0 a Pirimicarb 0 0 5 39.2 (14.2) a 2.5 0.0025 5 19.0 (7.76) a 5 0.005 5 20.40 (9.39) a 10 0.01 4 1.50 (1.50) a 25 0.025 4 17.75 (7.64) a 50 0.05 5 28.80 (10.70) a Pymetrozine 0 0 3 35.7 (2.91) a 50 0.05 5 24.0 (3.59) a 100 0.1 5 25.4 (2.29) a 150 0.15 5 29.0 (8.25) a 200 0.2 5 41.20 (7.37) a 250 0.25 5 43.80 (5.78) a Pyriproxyfen 0 0 5 5.0 (1.79) a 0.6 0.0006 3 18.08 (7.07) a 1.25 0.00125 5 10.80 (4.21) a 2.5 0.0025 2 5.50 (5.50) a 5 0.005 4 19 (4.06) a 10 0.01 3 9.0 (1) a Sulphur 0 0 4 41.25 (15.80) a 250 0.25 3 33.33 (15.41) a 500 0.5 4 55.0 (8.22) a 1000 1.0 4 54.75 (3.33) a 2000 2.0 4 37.25 (17.76) a 5000 5.0 4 25.0 (10.42) a Thiophanate 0 0 4 31.5 (6.91) a Methyl 25 0.025 4 36.5 (9.21) a 50 0.05 3 40.0 (13.80) a 125 0. 0125 4 47.75 (4.13) a 500 0.5 2 16.0 (15) a
a n = number of replications b Means (± SE) within an active ingredient followed by different letters are significantly different (P<0.05).
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fungicide thiophanate-methyl was moderately harmful while sulphur was slightly harmful with LC50-values of 312.3 and 3943.3 mg a.i./l respectively. The bacterial fermentation product abamectin and the pyridine azomethine pymetrozine were harmless to nymphs of M. caliginosus with LC50-values of 56.2 and 1122.5 mg a.i./l, respectively.
Effects on reproduction Fecundity of adults surviving dose-response tests was not significantly affected for most of the compounds tested (Table 3).
However, the results of abamectin should be considered with care: the fecundity of predators which had been in contact with 10 mg a.i./l abamectin during nymphal development was significantly lower than that of those treated with 20 mg a.i./l, but there were no differences with the control. Table 4: Duration of harmful activity of chlorfenapyr, diafenthiuron and dimethoate to young nymphs of Macrolophus caliginosus Wagner, persistence test on sharp pepper plants sprayed to incipient run-off
Active ingredient na Concentration mg a.i./l
Days after treatment
% Mortality Mean (±SE)b
Chlorfenapyr 5 0 27 20.0 (3.65) a 5 200 5 97.3 (2.66) b 5 200 12 100.0 (0) b 5 200 17 100.0 (0) b 5 200 27 100.0 (0) b
Diafenthiuron 5 0 23 22.7 (10.46) a 5 400 5 49.3 (6.53) a 5 400 13 33.3 (4.71) a 5 400 23 22.7 (4.52) a
Dimethoate 5 0 23 8.0 (5.33) a 4 160 5 84.0 (5.42) b 5 160 13 42.7 (5.81) c 5 160 23 24.0 (8.84) ac
a n = number of replications b Means within a column followed by different letters are significantly different Persistence The persistence of the toxicity was investigated only for chlorfenapyr, diafenthiuron, and dimethoate; these compounds proved to be harmful to M. caliginosus in initial contact toxicity tests. The mortality percentages obtained for each product were first transformed in arcsin square root and then compared with the water treated control (Table 4).
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Chlorfenapyr showed a very long persistence: 27 days after treatment, the mortality was still 100%. Dimethoate was very toxic 5 days after treatment (84% mortality); toxicity decreased after 13 days (42.7% mortality), until, after 23 days, survival became similar to that in the control. No significant differences were found between treatment with diafenthiuron and the control. Preliminary tests with some new IPM relevant chemicals The toxicity of the tested chemicals, used at the recommended concentration in an initial toxicity test, is shown in table 5. Spinosad, emamectin, triazamate, halofenozide and methoxyfenozide proved to be harmless to M. caliginosus nymphs after an exposure period of 6 days. Indoxacarb was moderately harmful, and the neonicotinoids acetamiprid and thiamethoxam were harmful. Table 5. Initial contact toxicity of new formulations to M. caliginosus nymphs; residual effect of deposits on glass applying 1 mg pesticide solution per cm2
Active Dose tested µg a.i./cm2 Corrected mortality a (%)
ingredient mg a.i./l glass 1 day after treatment
6 daysafter treatment
Acetamiprid 100 0.1 100 - Emamectin 15 0.015 19.1 7.7
Halofenozide 150 0.15 0 0 Indoxacarb 300 0.3 2.4 84.6
Methoxyfenozide 150 0.15 2.4 0 Spinosad 100 0.1 0 11.5
Thiamethoxam 100 0.1 100 - Triazamate 70 0.07 0 0
a Mortality corrected with Abbott's formula (Abbott, 1925)
Discussion In greenhouse vegetables, pests and diseases often occur simultaneously. Despite the widespread use of biological control, the management of diseases and some pests still often relies on the use of chemical pesticides. The success of integrated pest management in protected vegetable cultivation will depend upon the right choice of pesticides. These must be active against the pests, but relatively harmless to beneficial organisms. The IOBC/WPRS Working Group ‘Pesticides and beneficial organisms’ has designed a sequence of toxicity tests to evaluate side-effects of pesticides on a number of beneficials (e.g., Hassan et al., 1994). Usually, this sequence starts with a worst case laboratory trial. Compounds which cause little mortality and do not affect reproduction in these trials are considered harmless and further testing under semi-field or field conditions is not required.
According to the laboratory trials, pymetrozine and abamectin can be considered safe for the predatory bug M. caliginosus even when taking into consideration that abamectin has some negative influence on the fecundity of the insects.
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Abamectin was harmful to Orius laevigatus (Fieber) at 4.5 mg a.i./l (Van de Veire, 1992) and moderately harmful to E. formosa at 8 mg a.i./l (Oomen et al., 1994).
The results obtained with sulphur and in particular thiophanate-methyl suggest that one has to be prudent before using these compounds in combination with M. caliginosus.
Sulphur spray deposits did not have negative effects on the predatory bug O. laevigatus (Van de Veire et al., 1996), or on the entomopathogenic fungi Verticillium lecanii (Zimm.), Beauveria bassiana (Bals.-Criv.), and Metarhizium anisopliae (Metsch.) at 3200 mg a.i./l. This compound was moderately harmful to Aphidius matricariae Haliday, Amblyseius andersoni (Chant), Chrysoperla carnea (Stephens) and slightly harmful to Phytoseiulus persimilis Athias-Henriot at the same concentration. It has been found to be harmful to E. formosa adults, but harmless to larval and pupal stages (Hassan et al., 1994).
Thiophanate-methyl is a systemic fungicide with a protective and curative action and has a relatively low toxicity towards other natural enemies, such as O. laevigatus, even at very high concentrations (15,000 mg a.i./l) (Van de Veire et al., 1996), and E. formosa at 350 mg a.i./l (Oomen et al., 1994).
Considering the results obtained in this study, the other products should not be applied in combination with M. caliginosus.
Chlorfenapyr not only showed a high acute toxicity towards nymphs of M. caliginosus, but was also characterized by a very long persistence.
Dimethoate was classified as harmful to Trichogramma cacoeciae Marchal, E. formosa, Aphidius matricariae Haliday and Harmonia axyridis (Pallas) at a concentration of 840 mg a.i./l (Hassan et al., 1988), but it was not toxic at 600 mg a.i./l to A. aphidimyza (Warner & Croft, 1982). The results of the persistence assays showed that it is better to wait at least two weeks after treatment before releasing M. caliginosus.
Pirimicarb was toxic to Aphidoletes aphidimyza (Rondani) at a concentration of 70 mg a.i./l (Warner & Croft, 1982). This aphicide proved also to be harmful to first instar nymphs of O. laevigatus at 250 mg a.i./l (Van de Veire et al., 1996). Pirimicarb played an important role in glasshouse vegetable crops before new biological systems were implemented against aphids. Even nowadays this carbamate is still frequently applied, especially against Myzus persicae Sulzer; however, the results of this study show that it must be used with care.
Pyriproxyfen was harmless to Dicyphus tamaninii at a concentration of 75 mg a.i./l (Castañe et al., 1996) and to O. laevigatus at the recommended rate (Van de Veire et al., 1996; Delbeke et al., 1997). In contrast, this juvenile hormone mimic was highly detrimental to the predatory pentatomid Podisus maculiventris (Say) (De Clercq et al., 1995).
Diafenthiuron was very toxic to the mirid bug but had a short persistence. It was also very toxic at the recommended rate to nymphs and adults of O. laevigatus (Van de Veire et al., 1996; Delbeke et al., 1997). Toxicity was also observed for nymphs and adults of P. maculiventris (De Cock et al., 1996). Thus, the use of this insecticide/acaricide in protected crops must be carefully evaluated.
Concerning the new formulations, of which only the recommended concentration was tested in preliminary laboratory trials, emamectin, halofenozide, methoxyfenozide, spinosad and triazamate proved to be harmless to the nymphs. It can be expected these compounds will be compatible with M. caliginosus, however, a study on the effects on reproduction is necessary to be sure. The other compounds need to be further tested, especially for persistence. In fact, some compounds, which seem to be harmful to nymphs in initial toxicity laboratory tests, have a short persistence; so, they can be used in combination with the predatory bug on condition that a short delay between the treatment and the introduction of the beneficial is assured, as demonstrated for azadirachtin (Tedeschi et al., 2001).
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In conclusion, the data of this study indicate that some of the tested pesticides (e.g., pymetrozine, sulphur (spray application), thiophanate-methyl, spinosad, triazamate, methoxy-fenozide, halofenozide, emamectin) are truly compatible with releases of M. caliginosus against whiteflies. For other compounds (e.g., chlorfenapyr, dimethoate, and diafenthiuron) further testing is necessary to evaluate their toxicity under semi-field and field conditions; this may help in defining practical guidelines to growers who routinely apply M. caliginosus to control whiteflies. Acknowledgements R. Tedeschi was supported by a post-graduate grant from the University of Torino (Italy). M. Van de Veire was supported by the Ministry of Traders, Small Enterprises and Agriculture, DG6, Research and Development. The authors are grateful to G. Smagghe for his assistance with the statistical analyses. References Abbott, W.S., 1925: A method of computing the effectiveness of an insecticide. J. Econ.
Entomol. 18: 256-267. Castañe, C., Ariño, J. & Arnó, J. 1996: Toxicity of some insecticides and acaricides to the
predatory bug Dicyphus tamaninii (Het.: Miridae). Entomophaga 41 (2): 211-216. De Clercq, P., De Cock, A., Tirry, L., Vinuela, E. & Degheele, D. 1995: Toxicity of
diflubenzuron and pyriproxyfen to the predatory bug Podisus maculiventris. Entomologia exp. appl. 74: 17-22.
De Cock, A., De Clercq, P., Tirry, L. & Degheele, D. 1996: Toxicity of diafenthiuron and imidacloprid to the predatory bug Podisus maculiventris (Heteroptera: Pentatomidae). Environ. Entomol. 25: 476-480.
Delbeke, F., Vercruysse, P., Tirry, L., De Clercq, P. & Degheele, D. 1997: Toxicity of diflubenzuron, pyriproxyfen, imidacloprid and diafenthiuron to the predatory bug Orius laevigatus (Het.: Anthocoridae). Entomophaga 42: 349-358.
Finney, D.J.1971: Probit analysis, 3rd ed., Cambridge University Press, London and New York.
Hassan, S.A., Bigler, F., Bogenschütz, H., Boller, E., Brun, J., Chiverton, P., Edwards, P., Mansour, F., Naton, E., Oomen, P.A., Overmeer, W.P.J., Polgar, L., Rieckmann, W., Samsøe-Petersen, L., Stäubli, A., Sterk, G., Tavares, K., Tuset, J.J., Viggiani, G. & Vivas, A.G. 1988: Results of the fourth joint pesticides testing programme carried out by the IOBC/WPRS - Working Group "Pesticides and Beneficial Organisms". J. Appl. Entomol. 105: 321-329.
Hassan, S.A., Bigler, F., Bogenschütz, H., Boller, E., Brun, J., Calis, J. N. M., Coremans-Pelseneer, J., Duso, C., Grove, A., Heimbach, U., Helyer, N., Hokkanen, H., Lewis, G.B., Mansour, F., Moreth, L., Polgar, L., Samsøe-Petersen, L., Sauphanor, B., Stäubli, A., Sterk, G., Vainio, A., Van de Veire, M., Viggiani, G. & Vogt, H. 1994: Results of the sixth joint pesticide testing program of the IOBC/WPRS-Working Group "Pesticides and Beneficial Organisms". Entomophaga 39: 107-119.
LeOra Software - 1987. Polo-PC: a user's guide to probit or logit analysis. LeOra Software, Berkeley, CA.
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Oomen, P.A., Jobsen, J.A., Romeijn, G. & Wiegers, G. L. 1994: Side-effects of 107 pesticides on the whitefly parasitoid Encarsia formosa, studied and evaluated according to EPPO guideline no. 142. OEPP/EPPO Bulletin 24: 89-107.
Sterk, G., Hassan, S.A., Baillod, M., Bakker, F., Bigler, F., Blümel, S., Bogenschütz, H., Boller, E., Bromand, B., Brun, J., Calis, J.N.M., Coremans-Pelseneer, J., Duso, C., Garrido, A., Grove, A., Heimbach, U., Hokkanen, H., Jacas, J., Lewis, G., Moreth, L., Polgar, L., Rovesti, L., Samsoe-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.
Tedeschi, R., Alma, A. & Tavella L. 2001: Side-effects of three neem (Azadirachta indica A. Juss) products on the predator Macrolophus caliginosus Wagner (Het., Miridae). J. Appl. Ent. 125: 397-402.
Trottin-Caudal, Y., Trapateau, M., Chemaly, G. & Millot, P. 1993: Toxicité des produits agropharmaceutiques sur Macrolophus caliginosus. Infos-Ctifl 95: 41-46.
Van de Veire, M. 1992: Laboratory methods for testing side-effects of pesticides on the predatory bug Orius niger Wolff. IOBC/WPRS Bulletin 15 (3): 89-95.
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 (2): 235-243.
Warner, L.A. & Croft, B.A. 1982: Toxicities of azinphosmethyl and selected orchard pesticides to an aphid predator, Aphidoletes aphidimyza. J. Econ. Entomol. 5: 410-415.
Pesticides and Beneficial Organisms IOBC/wprs Bulletin Vol. 25 (11) 2002
pp. 81 - 88
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Effects of Oikos (a. i. Azadirachtin A) on the vedalia ladybeetle Rodolia cardinalis (Mulsant) (Coleoptera: Coccinellidae) Umberto Bernardo 1, Gennaro Viggiani 2 1 Centro di Studio CNR sulle Tecniche di Lotta Biologica, Via Università 133, 80055 Portici, Italia. E-mail: [email protected] 2 Dipartimento di Entomologia e Zoologia Agraria, Università di Napoli, Via Università, 100, 80055 Portici, Italia. E-mail: [email protected] Abstract: The effects of Oikos (a.i.: Azadirachtin A) on the vedalia ladybeetle Rodolia cardinalis (Mulsant) (Coleoptera: Coccinellidae) were studied by using laboratory and field methods. Laboratory trials were carried out on larvae and adults of the predator fed with treated and water treated egg masses of the cottony cushion scale Icerya purchasi Maskell (Homoptera: Margarodidae). Oikos was tested at the recommended rate per ha (1500 ml) x 0.4, which corresponds to the predicted environmental concentration. The ingestion effect was evaluated on the larvae taking into account mortality, presence or absence of deformations and duration of pre imaginal development. In addition, longevity, egg fertility and progeny were studied on the latter. The ingestion effect was evaluated also on adults of R. cardinalis not derived from treated young stages, considering longevity, egg deposition and egg hatchability. In a field trial, 5 plants of Pittosporum tobira, naturally infested with I. purchasi and with presence of R. cardinalis were treated with Oikos at dosage of 150 ml/hl in water at pH 5.5 until dripping off and other 5 plants in the same conditions were treated with water at pH 5.5. Before the treatment the larvae of R. cardinalis were counted and pupae and adults removed. In 3 day intervals the new formed pupae were collected and the number of derived adults and their state were recorded. Adults obtained from the larvae fed on Oikos treated plants were paired; in absence of functional males, individuals obtained from the check were used. Both laboratory and field trials have shown detrimental effects of Oikos on pupal mortality, adult deformation, egg deposition and egg hatchability.
Key words: Rodolia cardinalis, Azadirachtin, laboratory test, field test, progeny, egg deposition, egg hatchability, pupal mortality, adult deformation.
Introduction The present interest in using natural products in pest control has promoted intensive studies of neem oil derivates. Several studies have been carried out to explore the wide potentiality of the Azadirachtins, both on pests and beneficials (Viñuela et al., 2000; Bernardo and Viggiani, 2001; Capella et al., 2001; Viggiani and Bernardo, 2001).
In this study the effects of Oikos (a. i.: Azadirachtin A; 3.2 %) on the vedalia ladybeetle Rodolia cardinalis (Mulsant) (Coleoptera: Coccinellidae) have been evaluated by using laboratory and field methods.
Material and methods The biological material used in the laboratory trials of the present study was obtained from a permanent rearing of R. cardinalis fed with the natural prey I. purchasi kept at the Centro CNR sulle Tecniche di Lotta Biologica, Portici.
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Laboratory trials Ingestion effect Trials were carried out on larvae and adults of the predator fed in petri dishes, kept at 25° C in a climatic chamber, with insecticide and water treated (both with pH 5.5) egg masses of the cottony cushion scale Icerya purchasi Maskell (Homoptera: Margarodidae). Application rates for Oikos were based on the recommended per ha rate (1500 ml) and calculated using the PIEC formula developed by Barrett et al., 1994 considering a 0.4 factor (0.192 µg/a. i. cm2) in 2 mg water/cm2 (application volume). Each egg mass measures 0.8-1.0 in length and 0.4-0.5 in wide. Egg masses were sprayed directly using a computer controlled spraying apparatus.
Larvae Twenty five first larvae of R. cardinalis, 24 h age, arranged in 5 replicates, were fed with egg masses of I. purchasi treated or untreated. The ingestion effects were evaluated taking into account mortality, presence or absence of deformations and duration of development in adults. In addition, longevity, egg deposition, egg hatchability and progeny were studied on the derived adults (5 replicates).
Adults New emerged adults obtained from the permanent rearing were fed with treated and untreated egg masses of I. purchasi. The ingestion effects were evaluated taking into account the egg deposition during the first 15 days and the egg hatchability.
Field trial Ingestion and contact effects Five plotted plants of Pittosporum tobira (Thumb.) (Pittosporaceae), 70-90 cm in height naturally infested with I. purchasi and with presence of R. cardinalis were treated 3 times within 2 weeks with Oikos at dosage of 150 ml/hl and five other plants, under the same conditions, were treated with water at pH 5.5 to run off on July 3, 10 and 17, 2001. Before the treatment the larvae of R. cardinalis were counted; pupae and adults were removed. At 3 day interval the new formed pupae were collected and the number of derived adults and their state were recorded. Adults obtained from the larvae fed on Oikos treated plants were paired; in absence of functional males, individuals obtained from the check were used. On these adults the egg deposition during the first 15 day life and the hatchability were recorded.
Results and discussion Laboratory trials Ingestion effect on larvae The results obtained are reported in Table 1. They do not show statistically significant larval mortality. On the contrary, the percentage of the adult emergence was significantly reduced. The preimaginal development lasted 19.93 days on average for the treated and 18.22 days for the water treated insects (not statistically significant).
The longevity for the adults obtained from the treated larvae at 25°C and fed with honey was 6.8 days (SD = 1.92) for the males and 13.3 (± 4.04) for the females, whereas in the check it was 28.8 (± 18.58) for the males and 26.4 (± 7.67) for the females (statistically significant for p<0,05; test two ways ANOVA for sex and treatment).
The egg deposition during the first 15 day life was 1 egg/female on average for the treated vs. 150 eggs/female for the check.
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Table 1. Ingestion effects of Oikos in laboratory trials on larval mortality, pupal mortality and adult emergence of R. cardinalis.
Larval mortality (%) Pupal mortality (%) Adult emergence (%) Repl. no.
Oikos Check Oikos Check Oikos Check 1 40 20 0 0 60 80 2 0 0 20 0 80 100 3 40 0 0 0 60 100 4 20 20 40 0 40 80 5 40 0 0 0 60 100
Average 28 8 12 0 60* 92 *Statistically significant (p<0,05; test ANOVA).
Ingestion effects on adults The results (Tab. 2) show a significant reduction of egg deposition and egg hatchability for Oikos. These effects appeared even after a few days of feeding. No significant difference of adult longevity was detected. Table 2. Ingestion effects of Oikos on adults in laboratory trials on egg deposition and egg hatchability of R. cardinalis.
Repl. no. No.of eggs/female deposited during first 15
days of life
Egg hatchability
Oikos Check Oikos Check 1 35 94 12 91 2 25 50 0 48 3 2 132 0 122 4 47 117 8 112 5 27 106 6 106
Total 136 499 26 479 Average 27.2* 99.8 5.2* 95.8
*Statistically significant (p<0,05; test ANOVA).
Field trial During the experimental period 3.7 - 7.8.2001 the temperature ranged from 18°C to 36
°C. One mm of rain was recorded on 20.7.2001. The initial population of larvae in the experimental plots is reported in Table 3. The results on pupa and adult formation are reported in Table 4 and Fig. 1.
84
Table 3. Larval population of R. cardinalis in the experimental plots before treatment
Date Repl. Oikos Check 3.7 L1 L2+3 L1 L2+3
1 50 100 3 23 2 22 2 22 22 3 0 10 0 28 4 0 23 15 2 5 24 8 33 5
Total 96 143 73 80 Average 19.2 28.6 14.6 16
Table 4. Adults (A) obtained from pupae (P) collected in the experimental plots.
Sampling dates
6.7 10.7 13.7 17.7
20.7 24.7
27.7
Repl.
P A P A P A P A P A P A P A
Oikos 1 1 1 11 1 3 1 0 0 0 0 1 1 1 1
Oikos 2 0 0 14 4 13 8 20 9 2 0 0 0 0 0
Oikos 3 8 8 3 3 0 0 0 0 0 0 0 0 0 0
Oikos 4 17 17 10 10 0 0 0 0 3 2 3 2 2 2
Oikos 5 0 0 3 1 18 10 13 5 0 0 2 2 8 6
Total 26 26 41 19 34 19 33 14 5 2 6 5 11 9
Aver. 5.2 5.2 8.2 3.8 6.8 3.8 6.6 2.8 1 0.4 1.2 1 2.2 8Check1 4 1 32 31 38 34 7 7 1 0 1 1 2 2
Check2 0 0 62 59 3 3 8 8 2 2 0 0 0 0
Check3 22 17 5 5 3 3 38 33 7 7 0 0 1 1
Check4 0 0 8 8 12 12 6 6 1 1 0 0 0 0
Check5 0 0 14 12 40 40 9 9 2 2 0 0 0 0
Total 26 18 121
115
96 92 68 63 13 12 1 1 3 3
Aver. 5.2 3.6 24.2 2.3 19.2 18.4 13.6 12.6 2.6 2.4 0.2 0.2 0.6 0.6
85
Fig. 1. Reduction of pupa and adult populations on Oikos treated plants
Starting from 3 days after the first treatment an increasing reduction of the pupa and adult formation was recorded for Oikos. This effect was significantly reduced after 7 days from the last treatment (Fig. 1). The previous data were evaluated using the Henderson-Tilton formula (based on the pre-count from 3.7., L1, L2 and L3). A significant pupal mortality was detected for Oikos after 7 days from the first treatment (Fig. 2).
Fig. 2. Pupal mortality
In considering the total number of pupae and adults collected during the whole experimental period a significant reduction was recorded in the Oikos plot. Moreover, in the
010203040506070
3.7 6.7 10.7 13.7 17.7 20.7 24.7 27.7
%
OikosCheck
T
T T
T=treatment
0
20
40
60
80
100
3.7 6.7 10.7 13.7 17.7 20.7 24.7
%
PupaeAdults
T T T
T=treatment
86
same plot, a percentage (17.85) of abnormal adults, mostly with undeveloped elytra, was
recorded (Fig. 3).
Fig. 3. Total formation of pupae and adults (normal and abnormal) The egg deposition and egg hatchability (average per female) of the adults obtained from
pupae collected during the experiment are reported in the Fig. 4. Field data confirm the laboratory results of the detrimental effect of Oikos on R. cardinalis fertility. Egg hatchability was nearly completely stopped
During the experiment the complete suppression of I. purchasi was observed in the check plot. In contrast, the pest population persisted longer in the Oikos plot and some plants died. Consequently adults of R. cardinalis, coming from outside the experimental plots, colonized this plot and produced eggs. Thus, the derived adults were sterile.
Fig. 4. Egg deposition and egg hatchability (average per female) of the adults during first 15 day life
254179 147
458409413
0100200300400500
pupae adults normal adults
No.Oikos Check
0
10
20
30
40
50
60
70
80
90
3/7 6/7 10/7 13/7 17/7 20/7 24/7 27/7Pupal sampling dates
No.
Oikos (egg dep.)Check (egg dep.)Oikos (egg hat.)Check (egg hat.)
T
TT
87
Discussion and conclusion Neem oil or derived products have been considered safe for several beneficial arthropods (Capella et al., 2001). Some data show that they have no detrimental effects on predatory mites (Phytoseiidae) (Bernardo and Viggiani, 2001), on the predator Anthocoris nemoralis (Hemiptera: Anthocoridae) (Pasqualini et al., 1999) and on the parasitoid Ageniaspis citricola (Hymenoptera: Encyrtidae) (Villanueva-Jiménez et al., 2000). However, several predators: Hibana velox (Araneae: Anyphaenidae) (Amalin et al., 2000); Macrolophus caliginous (Hemiptera: Miridae) (Tedeschi et al., 2001); Chilocorus nigrita (Coleoptera: Coccinellidae) (Krishnamoorthy et al., 1998); Chrysoperla carnea (Neuroptera: Chrysopidae) (Vogt et al., 1998); and parasitoids: Encarsia formosa (Hymenoptera: Aphelinidae) (Del Bene et al., 2000), Aphytis melinus (Hymenoptera: Aphelinidae) (Krishnamoorthy et al., 1998) appear to be affected, mostly in a limited manner, by neem products. More commonly they caused mortality of several stages, adults shorter longevity, malformations, reduction of fecundity and fertility; in some cases a reduced number of pupae and adult emergence was obtained. Concerning C. carnea, it has been shown that NeemAzal-T/S was harmless under field conditions (Viñuela et al., 1996).
Most of the literature data are either not comparable, because results were obtained using different methodology and commercial products, or incomplete, because limited to laboratory or semi-field results. Field studies, in particular, on more beneficial arthropods, are needed, for understanding more in depth the ecotoxicological profile of neem products.
The remarkable effects of Azadirachtin (Oikos) on the predator R. cardinalis, in both laboratory and field trials, include this product in the category 4 of the IOBC classifications. Some cautions or limitations for use in citrus orchards should be taken. References Amalin, D.M., Pena, J.E., Yu, S.J. & McSorley, R. 2000: Selective toxicity of some
insecticides to Hibana velox (Araneae: Anyphaenidae), a predator of citrus leafminer. Florida Entomologist 83 (3): 254-262.
Barret, K.L., Grandy, N., Harrison, E.G., Hassan,S. & Oomen, P. (Eds.) 1994: Guidance document on regulatory testing procedures for pesticides with non target arthropods. Society of Environmental Toxicology and Chemistry-Europe. UK.
Bernardo, U. & Viggiani, G. 2001: Side effects of some pesticides on predatory mites (Phytoseiidae) in citrus orchards. IOBC/wprs Bulletin 24 (4): 97-101.
Capella, A., Guardone, A. & Domenichini, P. 2001: Azadiractina: caratteristiche, attività biologica e strategie di impiego su melo e ortive in coltura protetta. Notiziario sulla protezione delle piante 13 (N. S.): 59-63.
Del Bene, G., Gargani, E. & Landi, S. 2000: Evaluation of plant extracts for insect control Journal of agricolture and environment for international development 94 (1): 43-6.1
Krishnamoorthy, A. & Rajagopal, D. 1998: Effect of insecticides on the California red scale, Aonidiella aurantii (Maskell) and its natural enemies. Pest Management in Horticultural Ecosystems 4 (2): 83-88.
Pasqualini, E., Civolani, S., Vergnani, S., Cavaza, C. & Ardizzoni, M. 1999: Selettività di alcuni insetticidi su Anthocoris nemoralis. L’informatore agrario 46: 71-74.
Tedeschi, R., Alma, A. & Tavella, L. 2001: Side-effects of three neem (Azadirachta indica A. Juss) products on the predator Macrolophus caliginous Wagner (Het., Miridae). J. Appl. Ent. 125: 397-402.
88
Viggiani G.& Bernardo U. 2001: La lotta alla mosca delle olive, Bactrocera oleae (Gmelin), in agricoltura biologica. Notiziario sulla Protezione delle Piante 13 (N. S.): 199-203.
Villanueva-Jiménez, J.A., Hoy, M.A. & Davies, F. S. 2000: Field evaluation of integrated pest management-compatible pesticides for the citrus leafminer Phyllocnistis citrella (Lepidoptera: Gracillariidae) and its parasitoid Ageniaspis citricola (Hymenoptera: Encyrtidae). J. Econ. Entomol. 93 (2): 357-367.
Viñuela, E., Händel, U. & Vogt, H. 1996: Evaluacion en campo de los efectos secundarios de dos plaguacidas de origen botánico, una piretrina natural y un extracto de neem, sobre Chrysoperla carnea Steph. (Neuroptera: Chrysopidae). Bol. San. Veg. Plagas 22: 97-106.
Viñuela, E., Adam, A., Smagghe, G., Gonzàlez, M., Medina, M. P., Budia, F., Vogt, H.& 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 (2): 165-177
Vogt, H., González, M., Adán, A., Smagghe, G. & Viñuela, E. 1998: Side-effects of azadirachtin, via residual contact, on young larvae of the predator Chrysoperla carnea (Stephens) (Neuroptera, Chrysopidae). Boletín de Sanidad Vegetal, Plagas 24 (1): 67-78.
Pesticides and Beneficial Organisms IOBC/wprs Bulletin Vol. 25 (11) 2002
pp. 89 - 95
89
Side-effects of the microencapsulated Fenitrocap/IPM-400 (fenitrothion) and Pyrinex (chlorpyrifos ethyl) on Apis mellifera L. (Hymenoptera: Apidae) Gino Angeli, Michele Berti, Elena Gottardini, Fabiana Cristofolini, Diego Forti Agricultural Institute of San Michele all’Adige (Tn), Italy Abstract: Laboratory and field trials have been carried out to evaluate the side-effects of the microencapsulated insecticides Fenitrocap, IPM-400 (a.i. fenitrothion) and Pyrinex (a.i. chlorpyrifos ethyl) on Apis mellifera L.. In laboratory foraging honeybees have been exposed to Fenitrocap and Pyrinex by ingestion and topical treatments, and regression line and LD50 have been accordingly determined. Field investigations have been conducted applying one or two repeated microencapsulated treatments on Phacelia tanacetifolia before and during blooming, and in an apple orchard with and without mowing before treatment. In laboratory, exposure to the encapsulated formulations Fenitrocap and Pyrinex has resulted to be less toxic both topically (19.44 and 20.0 ml/l, respectively) and by ingestion (0.41 and 0.72 ml/l, respectively absorption) compared to the exposure to an emulsifiable fenitrothion formulation (0.45 by topical treatment and 0.02 ml/l by ingestion). Open field applications of insecticide on P. tanacetifolia have shown that adult honeybees mortality rate was lower with treatments before than during blooming. During blooming it was higher than usual only within 48 h after treatments. Further field investigations on apple orchards treated with IPM-400 and Pyrinex have shown a remarkable adult mortality and the reduction of some productive parameters only when lawn was blooming. The outcomes confirm that these formulations are moderately dangerous and that their use should be restricted when crops and other plants in the field are blooming. Key words: Apis mellifera L, microencapsulated insecticides, side-effects, laboratory test, field test, Fenitrocap, IPM-400, Pyrinex Introduction Microencapsulation increases insecticides’ activity and persistence (Cardarelli, 1976; Barker et al., 1979; Atkins & Kellum, 1984; Videau, 1990). Moreover, the recent encapsulated formulations are much less toxic to mammals through ingestion, inhalation, and topical contact than the traditional formulations are (Ivy, 1972; Lowell et al., 1977). And as opposed to traditional formulations, several investigations have shown that recent encapsulated insecticides are more selective to some beneficial organisms thanks to the lack of emulsifying agents and to a lower exposition to high doses of insecticide (Videau, 1990; Forti et al., 1992; Lester et al., 1999; Civolani & Pasqualini, 1999). The capsule walls of nylon-type polymer slow the release of the active ingredient and therefore microencapsulated and traditional formulations are under the same pesticide regulations for agricultural use. The mode of action is still the same; yet, encapsulation modifies the accumulation of lethal dosage. In order to apply the encapsulated insecticides by usual sprayers as well as to obtain the suitable dosages for noxious insects, the capsules should not exceed 60 µm in diameter. Nevertheless, diameters smaller than 3-5 µm, cause capsule walls to become too thin. Accordingly, the particles in the commercial formulations are about 5 to 60 µm in diameter, which is the same size as some entomophilous pollen. Laboratory and field trials have been carried out to determine the effectiveness of the microencapsulated insecticides
90
Fenitrocap, IPM-400 (a.i. fenitrothion) and Pyrinex (a.i. chlorpyrifos ethyl) on Apis mellifera L.. Purpose of the studies as well as the outcomes are herewith discussed. Material and methods Bees rearing and origin The A. mellifera of Carnica race used in the laboratory and field trials are reared in the apiary of Agronomy Institute of S. Michele a/Adige (Tn-Italy). During the tests, the colonies were of at least 8.000 to 15.000 bees according to the season and each colony covered minimum 7-8 frames including 2-3 brood frames. These colonies were disease-free and had not received any varroacide chemical treatments within the past 2 months before trials either during trials themselves. Pesticides The formulations used have been the microencapsulated insecticides Fenitrocap (fenitrothion CS, 23.15% a.i., 3.00 ml/l), IPM-400 (fenitrothion CS, 36% a.i., 1.90 ml/l), Pyrinex ME (chlorpyrifos ethyl CS, 23% a.i., 2.10 ml/l) and the reference products Perfekthion (dimethoate EC, 37.4% a.i., 1.50 ml/l), Folithion (fenitrothion EC, 48.5% a.i., 2.00 ml/l) and Steward (indoxacarb WG, 30% a.i., 0.167 ml//l). Formulated insecticides were diluted in distilled water to obtain concentrations equivalent to field applications rates of 12 hl ha-1. Laboratory tests In laboratory, using the EPPO guideline N°170 (EPPO/OEPP, 1992), foraging honeybees were exposed by ingestion and topical treatments to five different dosages of Fenitrocap and Pyrinex. Mortality rates have been assessed within 4, 24, 48, and 72 hours after the treatments in order to determine a regression line and LD50; the reference insecticides were Perfekthion and Folithion. Bees aged mostly 14 to 42 days were collected from frames and kept starving in laboratory up to 2h before exposure to the compound by ingestion or topical treatment. By feeding, each bee was supplied with 10 µl test solution diluted in 0.2 ml of fresh sucrose (500 gl-1); sucrose solution was used as control treatment. Anaesthetized bees were treated individually by topical contact with 1 µl of test substance onto the dorsal side of prothorax using PB600 Hamilton syringes; distilled water was used as control treatment. Four batches of 15 bees were utilized for each concentration of the insecticides tested. During the trials, the bees were kept starving in the ventilated incubator (25±1.5°C; 60±10 RH) with a daily supply of aqueous sucrose solution as food source. Field tests In field, three investigations were conducted with treatments of Fenitrocap, IPM-400, Pyrinex and the reference insecticides Reldan and Steward on a Phacelia tanacetifolia (Hydrophyllaceae) crop and on an apple orchard. Two, three bees colonies per product were placed in the test fields 2-3 days before the treatments in order to make sure that bees were foraging only in the adjacent area of the application. The tests and the reference products were applied in separate fields, each far from the others not less than 3 km in order to avoid that bees could be foraging in the same area. The effects of the treatments on bees were assessed shortly before and several times after treatments (t-1 day to t+14-18 days).
The results of the trials depend on the following parameters: assessment of foraging honeybees mortality, analysis and collection of contaminated pollen, transport to the hive and health condition of broods.
On a P. tanacetifolia crop (0.6 ha) one application of Fenitrocap was made before blooming of Phacelia, whereas a second treatment with the same product was made 28 days later when the crop was in full bloom.
91
Two investigations in apple orchards were carried out as well. The first trial meant to evaluate and compare the side-effects of Fenitrothion microencapsulated IPM-400 in two apple orchards (3 ha) treated in summer before and during lawn bloom. The second investigation was conducted in five apple orchards (0.8-0.9 ha) mowed prior to treatments in order to determine the side-effects of the two microencapsulated IPM-400 and Pyrinex compared with the emulsified formulations Dursban, Steward and an untreated control. Statistical analysis In order to assess the LD50-90 values, laboratory results were elaborated through control mortality according to Abbott and probit regression analysis, non-linear regression (CIRAD-CA/URBI Montpellier, version 4.6). For field results the significance of treatment effects was tested using t-test (Statistica, version 5). Results Laboratory tests In laboratory the LD50 (Table 1) and the dose-effect relationship (Figure 1 and 2) values concerning encapsulated formulations Fenitrocap and Pyrinex showed a lower toxicity by topical absorption, respectively 19.44 ml/l (95% confidence limits=15.054-25.112), and 20.0 ml/l (95% c.l.=14.877-25.328) and by ingestion, respectively 0.41 ml/l (95% c.l.= 0.294-0.564) and 0.72 ml/l (95% c.l=0.589-0.876), compared with an emulsifiable fenitrothion formulation, whose level was 0.45 ml/l (95% c.l.= 0.387-0.53.6) by topical absorption and 0.02 ml/l (95% c.l.= 0.016-0.028) by ingestion. Perfekthion proved to have the highest toxicity both by topical application and by ingestion, respectively 0.14 and 0.01 ml/l. Field tests Treatment of Fenitrocap on P. tanacetifolia: After insecticide field application the adult honeybees mortality (Table 2) was low before Phacelia blooming and not significantly different from what observed for the control (t=1.460; p=0.164); on the contrary, the treatment made during full blooming caused a higher mortality remarkably different from control data (t=2.229; p=0.041) and the previous treatment made in pre-blossom (t=-2,127; p=0.050). The analysis of collected pollen from the monitoring station in the treated area showed little presence of microcapsules (<0.1 capsule/100 pollen granules) only in samples where pollen of P. tanacetifolia was also present. Treatment of IPM-400 before and during lawn blooming The adult honeybees mortality after IPM-400 treatment was high in the not mowed apple orchard (Table 3), whereas it was reduced and significantly lower, when applications were made after mowing the lawn (t=-2,668; p=0.016). Moreover, with the mowed lawn the health conditions of the colonies and the average quantity of bees on comb and the quantity of collected pollen and honey, did not show significant differences between the hives in the treated area and the hives of the reference station. On the contrary, the treatments applied during lawn bloom resulted in a reduction of some productive parameters such as the foraging activity (40% lower within the first week after treatment) and the honey production (50% smaller within the first month after treatment). Further controls on the health conditions of the hives, made at the end of the season and in the following spring, showed a substantial increase in the population, which had been affected by the microencapsulated insecticide, however, it was still less numerous than the one in the reference station.
92
Table 1. LD50-90 by topical and ingestion treatment of the insecticides on A. mellifera. Values expressed in ml/l (p 0.05%).
Perfekthion Fenitrocap Folithion Pyrinex Treatment LD50 LD90 LD50 LD90 LD50 LD90 LD50 LD90
TOPICAL
0.136
0.379
19.443
41.492
0.455
0.939
20.0
44.100
INGESTION
0.011
0.032
0.407
3.161
0.021
0.060
0.719
1.967
Figure 1. Probit regression analysis by contact of Perfekthion (a), Folithion (b), Fenitrocap (c) and Pyrinex (d) on A. mellifera. Regression line: Fenitrocap, y = -7,80322 + (3,89302 * X); Pyrinex, y = -7,21560 + (4,11225* X); Folithion, y = -1,75659 + (4,07428 * X); Perfekthion, y = 1,73908 + (2,87662 * X)].
Figure 2. Probit regression analysis by ingestion of Perfekthion (a), Folithion (b), Fenitrocap (c) and Pyrinex (d) on A. mellifera. Regression line: Fenitrocap, y = 2,68039 + (1,44058 * X); Pyrinex, y = -0,44270 + (2,93142 * X); Folithion, y = 4,04144 + (2,86878 * X); Perfekthion, y = 4,84659 + (2,83173 * X)].
-0 ,8 -0 ,5 -0 ,2 0 ,1 0 ,4 0 ,7 1 1 ,3 1 ,6 1 ,9 2 ,2 2 ,5 2 ,8lo g (d os e)
1
2
3
4
5
6
7
p rob it a
b
c
d
0 0,3 0,6 0,9 1,2 1,5 1,8 2,1 2,4 2,7 3 3,3 3,6 3,9log (dose)
1
2
3
4
5
6
7
8
prob
it
a
b d
c
pr
obit
pr
obit
93
Table 2. Adult honeybees mortality (no. of dead bees) after Fenitrocap treatment on a P. tanacetifolia crop before and during blooming (T= day of treatment, d=day).
Pre-blossom Blossom Fenitrocap station Untreated station Fenitrocap station Untreated station
Check
Colonie 1
Colonie 2
Colonie 3
Colonie 4
Colonie 1
Colonie 2
Colonie 3
Colonie 4
T-0 T+1d T+2d T+3-5d T+6d T+7-8d T+10-12d T+13-14d
8 68 17 14
1 4
14 10
1 7 4 4 0 3 4 3
9 13
9 7 3 3 7 8
9 3 2 2 0 1 2 3
4 139
29 10 11
131 17 12
7 385
44 12
6 250
10 13
0 8 7 4 6 7 9 7
4 13
8 11
7 7
10 11
Sum 136 26 59 22 353 727 48 71 Table 3. Adult honeybees mortality after IPM-400 treatment in an apple orchard with a previously mown lawn and during lawn blooming.. (T= day of treatment, d=day, h=hour).
Mown in-bloom lawn In-bloom lawn IPM-400 station Untreated station IPM-400 station Untreated station
Check
Colonie 1
Colonie 2
Colonie 3
Colonie 4
Colonie 5
Colonie 6
Colonie 7
Colonie 8
T-1d T+6h T+1d T+2d T+3d T+4-6d T+7-9d T+10-12d T+13-14d
10 131 172
20 29 47 50 16 17
8 97
141 22 17 26 42 13 11
9 2 7 8 8 4
20 19 16
7 3 7 7
11 5
26 14 17
29 450 850 125
87 70 19 18 22
27 270 580 200 110
68 18 22 21
9 16
9 1
13 3 7
31 16
4 16
8 3 7 7
14 11 21
Sum 492 377 93 97 1670 1316 105 91 Side-effect of IPM-400, Pyrinex, Dursban and Steward By mowing in-bloom lawn, the adult bees mortality (Table 4) and the other parameters evaluated (such as foraging activity, honey production) resulted in slight side-effects coming from the use of the microencapsulated insecticides IPM-400 and Pyrinex. No differences were observed between the microencapsulated IPM-400 and Pyrinex (IPM-400/Pyrinex, t=0.370, p=0.720) and the other insecticide formulations (IPM-400/Dursban, t=1.610, p=0.145; IPM-400/Steward, t=-0.398, p=0.700). Remarkable differences were noticed between the insecticides applications and the untreated control (IPM-400/control, t=2,748, p=0.025; Pyrinex/control, t=3.426, p=0.034). In no case any toxicity was found in the hive brood.
94
Table 4. Adult honeybees mortality after treatment with IPM-400, Pyrinex, Dursban and Steward in apple orchards after lawn mowing.
Bloom mowing IPM-400 station
Pyrinex Station
Dursban station
Steward station
Untreated station
Check Colonies 1+2 Colonies 3+4 Colonies 5+6 Colonies 7+8 Colonies 9+10
T-1 d T+6 h T+1 d T+2 d T+(3/5) d T+6 d T+(7/8) d T+(9/14) d T+(15/19) d
27 13
127 9
37 49
114 64 30
60 0
28 23 83 68 51 65 40
26 35 85 27 18 31 49 45 22
35 75 61 41 7
40 39 42
229*
13 3
14 3
39 14 16 16 11
Sum 470 418 338 569 129 * The high mortality observed is ascribe at the interference of one other treatment made in the nearby orchard and confirmed with the analysis of the contaminated honey bees. Discussion Preliminary analysis (Angeli et al., 2000; 2001) of the characteristics of microencapsulated insecticides show the following aspects. The average size of capsules diameter was 12.9 µm for IPM-400/Fenitrocap and 9.8 µm for Pyrinex; with regard to <10 µm class, the highest number of capsules is obtainable. The drift of capsules (IPM-400) in the air was observed up to 15 m distance from the treated orchards; furthermore the spore-traps indicated that the capsules remained in the air for about 7 hours after the treatment. On microscope slide the persistence of IPM-400 and Pyrinex microcapsules was detected within 18 days after treatment whereas on treated vegetation it was observed up to 12 days after treatment. When there was no blooming in the field, the treated microcapsules were not attractive towards foraging honeybees. The pollen collected by foraging honeybees became contaminated by microcapsules only when the bees foraged on flowers previously sprayed with the microencapsulated formulations. When the bees foraged on treated flowers, capsules could be detected in their digestive apparatus (mid-gut) up to 14 days after the treatment. In the laboratory tests the microencapsulated formulations Fenitrocap and Pyrinex were less toxic than the emulsified ones, both by topical treatment and ingestion. By topical treatment the emulsified formulation Folithion were 43.2 to 44.4 times more toxic than Fenitrocap and Pyrinex; by ingestion Folithion proved to have a toxicity level 20.5 and 36 times higher than Fenitrocap and Pyrinex, respectively. The field investigations revealed a relevant difference of the selectivity level of the microencapsulated formulations towards foraging honeybees between treatments on P. tanacetifolia and on apple trees made before and during blooming. Open-field treatments with Fenitrocap on P. tanacetifolia, if applied in full blooming, caused moderate mortality in the days shortly after the applications. Moreover, microencapsulated Fenitrocap did not determine any crisis in the station throughout the following summer and spring.
P. tanacetifolia is a nectarean species but is poor in pollen, and as a consequence, the risk of large quantities of toxic pollen being eaten and carried back to the hives may be reduced. This aspect helps explaining the lower honey bees mortality in comparison to that observed on apple
95
tree and why neither significant bee losses nor any crisis in the hives were assessed after microencapsulated treatment applied in full blooming. Summer treatments with IPM-400 on apple trees produced contrasting results depending on whether the microencapsulated insecticide was applied before or after mowing of the lawn. On the one hand, the tests carried out after mowing proved a limited negative effect of the microencapsulated formulation, shortly after as well as long after the treatment, and this despite the monitoring stations being located inside a treated area 1-3 ha wide. On the other hand, a test performed in the apple orchard while several plants – such as Trifolium repens and Plantago major - were blooming showed quite different outcomes. The microencapsulated treatments caused a high mortality among foraging bees in the first week after applications. Chemical and visual analyses of residues in collected pollen and of foraging bees which were found dead in this period, showed the presence both of the active ingredient and of some insecticide capsules. In no case any toxicity in the hive brood was observed. These results show the moderate dangerousness of the microencapsulated formulations and the necessity to restrict their use when treated crops or other plants in the field are in full blooming. References Angeli G., Cristofolini F., Forti D., Gottardini E., 2000: Insetticidi microincapsulati e api.
Apitalia, 5: 16-23. Angeli G, Gottardini E., Cristofolini F., Forti D., 2001: Attività di fenitrothion microincapsulato
(Fenitrocap- IPM-400) su Apis mellifera Ligustica (Hymenoptera: Apidae). Inf. Fit., in press.
Atkins E.L., Kellum D., 1984: Microencapsulated pesticides: visual detection of capsules, quantification of residue in honey and pollen. Am. Bee J., 118: 800-804.
Barker R.J., Lehner Y., Kunzmann M.R., 1979: Pesticides and honey bees: the danger of microencapsulated formulations. Z. Naturforsch, 34 c: 153-156.
Cardarelli N., 1977: Controlled release pesticide formulations, CRC Press, Cleveland, pp. 210. Civolani S., Pasqualini E., 1999: Tossicità nel breve periodo di diverse formulazioni di insetticidi
su alcuni gruppi di entomofagi. Inf. Agr., 20: 87-90. EPPO/OEPP, 1992: Guideline on test methods for evaluating the side-effects of Plant Protection
Products on Honeybees. EPPO Bulletin, 22: 203-215. Forti D., Angeli G., Ioriatti C., Maines R., 1992: Valutazione dell’effetto collaterale di alcuni
insetticidi sull’acaro predatore Amblyseius andersoni Chant (Acarina: Phytoseiidae). Inf. Fit., 5: 57-59.
Ivy E. E., 1972: Penncap-M: an improved methyl parathion formulation. J. Econ. Entomol. 65(2): 473-474.
Lester P. J., Pree D. J., Thistlewood H. M. A., Trevisan L. M., Harmsen R., 1999: Pyrethroid encapsulation for conservation of acarine predators and reduced spider mite (Acari: Tetranychidae) outbreaks in apple orchards. Environ. Entomol., 28 (1): 72-80.
Lowell J.R., Dhal G.H., 1983: Effects of microencapsulated methyl parathion on beneficial insects. 10th Int. Cong. Plant Prot., Brighton.
Videau B., 1990: Microincapsulazione: nuove tecnologie. Informatore Agrario, 17: 51-54.
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Pesticides and Beneficial Organisms IOBC/wprs Bulletin Vol. 25 (11) 2002
pp. 97 - 106
97
Mimic-Confirm (a. i. Tebufenozide): a tool for a soft and ecologically sound pest control in pear orchards(1)
Edison Pasqualini, Stefano Civolani Dipartimento di Scienze e Tecnologie Agroambientali, Università degli Studi di Bologna, Via Filippo Re, 6 I-40126 Bologna (Italy). e-mail: [email protected] Abstract: An investigation regarding the side-effects of two insecticide application strategies has been carried out. Two chemical control programmes for codling moth (Cydia pomonella L.) and for the main leafroller species (Pandemis cerasana Hb.) have been compared. One programme (“soft”) based exclusively on tebufenozide applications and the other one (conventional) based on traditional insecticides use (the exclusively use of tebufenozide was chosen only for experimental requirement, without considering the resistance management).
The study has been carried out in the Ferrara District (Italy) on a pear orchard (cv. Abbè Fétel and Bartlett) in the 1998-99-00 years.
A randomised block design with four replicates was used to compare the two different chemical control programmes. For sampling visual and inventory methods (depending from the insect species and the developmental stage) were used. The main pest species sampled were C. pomonella L., P. cerasana Hb., Cacopsylla pyri L. and Quadraspidiotus perniciosus (Comst.), while Anthocoris nemoralis (F.), coccinellids and syrphids were the beneficial ones. The results showed the reliability of the control strategy based on tebufenozide (no difference with regard to damaged fruits in comparison with the traditional one). This strategy gave smaller side effects on the beneficials, mainly regarding A. nemoralis young larvae. It has been possible to measure the lower insecticide use: both in quantitative and qualitative terms (toxicological classes). Key words: tebufenozide, integrated control, pests, beneficials, pear Introduction The research about side effects of insecticides has evolved into a highly technical and precise science (Candolfi et al. 2000, Croft & Brown 1975, Croft 1990, Hassan et al. 1985, Hassan et al. 1994, Ishaaya & Degheele 1998, Sterk et al. 1999, Vogt 1994, Vogt & Heimbach 2000, Vogt et al. 2001, Pasqualini et al. 2001). Use of selective compounds, which do not harm beneficials (predators, parasitoids and pollinators), often permits natural biological control to succeed. As consequence it is possible to reduce the use of the pesticides and to limit resistance risks.
In Emilia-Romagna the use of IGRs (sensu lato) on main fruit crops and grapevines is recommended to control tortricid pests, leafminers, etc.. Mimic-Confirm (tebufenozide) and methoxyfenozide (RH-2485), the last one not yet registered in Italy, are two Moulting Accelerating Compounds (MAC group), which induce early lethal moulting on larval instar. Their activities mimic the endogenous ecdysone hormone (moulting hormone agonist). These compounds showed high selectivity for some important beneficials. Low toxicity and high effectiveness against the main tortricid moth species infesting fruit trees (Civolani et al., 1998; Civolani et al., 1999; Civolani et al., 2001a; Civolani et al., 2001b), and grapevines, give to this kind of products interesting chances to be recommended in IPM. The selectivity 1 Ecotoxicological study carried out by the CRPV (Centro Ricerche Produzioni Vegetali - Cesena) co-operation.
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of these recently developed active ingredients is very encouraging (Pasqualini et al., 1999: Pasqualini et al., 2001).
During three years a spray programme using the “soft” pesticide Mimic was compared to a conventional one in a commercial pear orchard in Emilia-Romagna Region (Italy).
The aim of the investigation was to assess the selectivity of two-control strategies for codling moth (Cydia pomonella L.) and leafrollers (mainly Pandemis cerasana (Hb.)) without interfering (or as less as possible) with the natural control of Cacopsylla pyri L. by Anthocoris nemoralis F.. Materials and methods Two insecticide programmes were compared: a “conventional” one, consisting in the use of insecticides commonly applied by fruit growers in Emilia-Romagna and a “soft experimental programme” using mainly Mimic-Confirm (Tab. 1) (the exclusively use of the last one insecticide was made only for experimental requirement, without considering the resistance management).
Both programmes were primarily timed for codling moth C. pomonella and leafroller P. cerasana, while the other pests (i.e. C. pyri, Quadraspidiotus perniciosus (Comst.)), etc., were controlled by the same compounds. A pear orchard (cv. Abbè Fètel and Bartlett, ten year old) was subdivided into eight plots of 350-400 trees (about 0,4 ha). A randomised block design with four replicates was used to compare the two different chemical control programmes. All insecticides were applied by a hand back sprayer calibrated to deliver 13 hectolitre/hectare. The central trees within each plot were sampled to check the pests and natural enemy populations (Tab. 2).
Sex pheromone traps and forecasting development models as well were used to decide on the timing of treatment. Monitoring and sampling Two sampling methods, visual control and inventory spray, were used to assess pest populations and beneficials insects. Visual sampling The visual sampling (not disruptive on leaves, shoots, fruits, etc.) was used directly in the field to estimate the pests and beneficials population (for example eggs in general, C. pyri larvae, etc.) not assessable by other methods (i. e. inventory spray). Sampling size is reported in each table explanation (see results). Inventory spray For inventory spray sampling two selected trees per plot had a cotton sheet (2 x 1.5 m) pegged beneath them to collect falling specimens. An application of deltametrin was made using 2.5-g a.i. hl-1 by back-pack-mounted mist blower. Each tree was treated until the point of insecticide run-off. Specimens were removed from the collecting sheets 6-h after spraying. Each treated tree and the eight surrounding ones were considered to be excluded from the next inventory sampling (Brown, 1989). Data processing The data collected were processed by Student “t” test (the data plot were transformed in log (x+1) before analysis. The data collected as percentage were transformed as arcsen √(x).
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Table 1. Treatments schedule for both control programmes
Date of treat-ment
Soft Common name and formulation
a.i. ha-1
(g/cc) Conventional Common
name and formulation
a.i. ha-1
(g/cc) Pest target
1998 17/03 mineral oil Oliocin 36000 mineral oil Oliocin 36000 Q. perniciosus25/03 o. methyl Metasystox SL 555 o. methyl Metasystox SL 555 H. brevis 19/04 tebufenozide Mimic SC 276 flufenoxuron Cascade DC 166,5 P. cerasana 25/04 dicofol+tetrad. Childion EC 480/180 dicofol+tetrad. Childion EC 480/180 E. pyri. 5/05 tebufenozide Mimic SC 207 diflubenzuron Dimilin WP 150 C. pomonella 18/05 amitraz Bumetan EC 742 amitraz Bumetan EC 742 C. pyri 26/05 tebufenozide Mimic SC 276 a. methyl Gusathion WP 750 P. cerasana 26/05 washing - washing - C. pyri 4/06 clofentezine Apollo SC 189 clofentezine Apollo SC 189 P. ulmi 9/06 amitraz Bumetran EC 742 amitraz Bumetan EC 742 C. pyri 4/07 tebufenozide Mimic SC 207 a. methyl Gusathion WP 937 C. pomonella 23/07 tebufenozide Mimic SC 276 a. methyl Gusathion WP 937 P. cerasana 28/07 ch. methyl Reldan EC 660 ch. methyl Reldan EC 660 Q. perniciosus15/08 triflumuron Alsystin SC 147 triflumuron Alsystin SC 147 C. molesta
1999 15/03 mineral oil Oliocin 36000 mineral oil Oliocin 36000 Q. perniciosus21/03 buprofezin Applaud WP 500 buprofezin Applaud WP 500 Q. perniciosus19/04 tebufenozide Mimic SC 276 flufenoxuron Cascade DC 148 P. cerasana 26/04r bromopropilate Neoron EC 750 bromopropilate Neoron EC 750 E. pyri. 8/05 tebufenozide Mimic SC 207 diflubenzuron Dimilin WP 150 C. pomonella 18/05 amitraz Bumetan EC 742 amitraz Bumetan EC 742 C. pyri 28/05 washing - washing - C. pyri 30/05 exitiazox Matacar WP 75 exitiazox Matacar WP 75 P. ulmi 21/06 washing - washing - C. pyri 26/06 tebufenozide Mimic SC 207 a. methyl Gusathion WP 750 C. pomonella 27/07 ch. methyl Reldan EC 660 ch. methyl Reldan EC 660 Q. perniciosus13/08 triflumuron Alsystin SC 147 triflumuron Alsystin SC 147 C. molesta
2000 19/03 mineral oil Oliocin 36000 mineral oil Oliocin 36000 Q. perniciosus30/03 buprofezin Applaud WP 500 buprofezin Applaud WP 500 Q. perniciosus 4/04 o. methyl Metasystox SL 555 o. methyl Metasystox SL 555 H. brevis 2/04 tebufenozide Mimic SC 276 flufenoxuron Cascade DC 166,5 P. cerasana 27/04 bromopropilate Neoron EC 750 bromopropilate Neoron EC 750 E. pyri 5/05 tebufenozide Mimic SC 207 diflubenzuron Dimilin WP 150 C. pomonella 15/05 amitraz Bumetan EC 742 amitraz Bumetan EC 742 C. pyri 26/05 tebufenozide Mimic SC 276 ch. methyl Reldan EC 660 P. cerasana 29/05 clofentezine Apollo SC 189 clofentezine Apollo SC 189 P. ulmi 12/07 tebufenozide Mimic SC 276 a. methyl Gusathion WP 937 C. pomonella 18/08 triflumuron Alsystin SC 147 triflumuron Alsystin SC 147 C. molesta
o. methyl = oxydemeton methyl; ch. methyl = chlorpyrifos methyl; tetrad. = tetradifon; a. methyl. = azinphos methyl Details about the sampling schedule are given in Table 2.
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Table 2. Sampling schedule for both control programmes.
Date sampling Sampling procedure Sampling target 1998
18 mar visual C. pyri eggs and Q. perniciosus 14 apr visual P. cerasana overwintering larvae 6 may inventory spray A. nemoralis and Coccinellids 7 may visual C. pyri eggs 18 may inventory spray A. nemoralis and Coccinellids 21 may visual C. pyri larvae 4 jun visual C. pyri honeydew 17 jun inventory spray A. nemoralis and Coccinellids 2 jul visual Coccinellids and Syrphids 9 jul visual Coccinellids and Syrphids 15 jul inventory spray A. nemoralis and Coccinellids 16 jul visual Coccinellids and Syrphids 17 jul visual Fruits (damage) 5 aug inventory spray A. nemoralis and Coccinellids 24 aug inventory spray A. nemoralis and Coccinellids 25 aug visual Fruits (damage)
1999 30 mar visual C. pyri eggs 8 apr visual P. cerasana overwintering larvae 14 apr visual C. pyri larvae 20 apr inventory spray A. nemoralis and Coccinellids 4 may inventory spray A. nemoralis and Coccinellids 13 may inventory spray A. nemoralis and Coccinellids 14 may visual C. pyri eggs 31 may visual C. pyri larvae 3 jun visual C. pyri honeydew 14 jun inventory spray A. nemoralis and Coccinellids 6 jul visual Coccinellids and Syrphids 14 jul visual C. pyri honeydew 29 jul inventory spray A. nemoralis and Coccinellids 20 aug visual Fruits (damage) 23 aug visual C. pyri honeydew 25 aug inventory spray A. nemoralis and Coccinellids
2000
13 mar visual C. pyri eggs 15 apr inventory spray A. nemoralis and Coccinellids 22 apr visual P. cerasana overwintering 22 apr visual C. pyri larvae 7 may inventory spray A. nemoralis and Coccinellids 25 may inventory spray A. nemoralis and Coccinellids 29 may visual C. pyri larvae 6 jun visual C. pyri honeydew 26 jun visual C. pyri honeydew 29 jun inventory spray A. nemoralis and Coccinellids 3 jul visual Coccinellids and Syrphids 28 jul inventory spray A. nemoralis and Coccinellids 2 aug visual C. pyri honeydew 17 aug visual Fruits (damage)
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Results The results obtained by the inventory samplings reveal differences in A. nemoralis young larvae population between both treatments (Fig. 1). Flufenoxuron, applied after blossom in the conventional programme to control P. cerasana, interferes more than tebufenozide with A. nemoralis young larvae population (for about 20 days after treatments). This is a very critical natural control period during which the young larvae of A. nemoralis play a decisive predatory role. The results regarding old larvae and adults appear in Table 3 and 4.
Figure 1. Young larvae of A. nemoralis sampled by inventory spray. (*) = significant differences at p=0,05 level on 6/05 and 15/07/1998; 13/05/1999.
As consequence the level of C. pyri population rose in the conventional plots (Table 7), as well as the number of fruits with honeydew (Table 10). The higher C. pyri population density assessed in the conventional plot is the reason of the higher level of A. nemoralis found in the same plots in the following period (June).
The results obtained by inventory spray sampling regarding coccinellids spp. are shown in Table 5.
The results regarding the C. pyri larvae population (first generation) obtained by visual sampling during the blossoming in the years 1999 and 2000 appear in Table 6. They reveal a slightly greater number of C. pyri larvae in the conventional programme. In Table 7 the C. pyri larvae population during the second generation is shown. In this sampling a higher number of larvae were observed in the conventional plot.
0
5
10
15
20
25
30
35
40
19/4
/98
6/5/
98
18/5
/98
17/6
/98
15/7
/98
5/8/
98
24/8
/98
20/4
/99
4/5/
99
13/5
/99
14/6
/99
29/7
/99
25/8
/99
15/4
/00
7/5/
00
25/5
/00
29/6
/00
28/7
/00
A. n
emor
alis
you
ng la
rvae
/she
et
Soft programme Conventional
*
*
*
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Table 3. Average number of A. nemoralis old larvae per sheet. (*) = significant differences at p=0,05 level
Programme and year Date 1998
6 may 18 may 17 may 15 jul (*) 5 aug 24 aug Soft 0 0 6,25 5,25 6,5 0
Conventional 0 0 7 16,75 2,25 0,25 1999
20 apr 4 may 13 may (*) 13 jun (*) 29 jul 25 aug Soft 0 0 1,5 15,5 8,5 5,5
Conventional 0 0 0 37 8,5 4,5 2000 15 apr 7 may 25 may 29 jun 28 jul -
Soft 0 0,75 5,5 29,5 8,75 Conventional 0 0,5 1 22,75 5,5
Table 4. Average number of A. nemoralis adults per sheet. (*) = significant differences at p=0,05 level
Programme and year Date 1998
6 may 18 may 17 may 15 jul (*) 5 aug 24 aug Soft 2,5 1,5 16 7,75 13,25 2
Conventional 2 0,75 10,5 21,5 14,25 1 1999
20 apr 4 may 13 may 13 jun (*) 29 jul 25 aug Soft 2,25 1 2,5 24,75 26 9
Conventional 2 0,5 1,25 38,75 25,5 10,25 2000 15 apr 7 may 25 may 29 jun (*) 28 jul -
Soft 1,25 3 12 38 16,5 Conventional 0,75 3 8,75 29,75 15,25
Table 5. Average number of coccinellid spp. adults per sheet. (*) = significant differences at p=0,05 level
Programme and year Date 1998
6 may 18 may 17 may 15 jul 5 aug 24 aug Soft 0 0 2 0,5 0,25 0,25
Conventional 0 0 0,75 0 0,25 0,25 1999
20 apr 4 may 13 may 3 jun 29 jul 25 aug Soft 0 0 0 0,25 1,5 0,75
Conventional 0 0 0 0 0,5 0,25 2000 15 apr 7 may 25 may 29 jun 28 jul -
Soft 0,25 0 0 0,5 0,25 Conventional 0 0,5 0 0 0
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Table 6. Average number of C. pyri larvae/flower (observed on 400 flowers/plot). (*) = significant differences at p=0,05 level
Programme 14 apr. 99 22 apr. 00 Soft 0,23 0,04
Conventional 0,30 0,06 Table 7. Average number of C. pyri larvae/shoot (observed on 50 shoots/plot). (*) = significant differences at p=0,05 level
Programme 21 may 98 31 may 99 (*) 29 may 00 Soft 5,10 3,48 1,43
Conventional 6,42 5,6 1,82
In summer some visual samplings in order to assess the predators of Aphis pomi (De Geer) colonies (100 per plot) have been carried out. The most common ones were syrphids and coccinellids. After the OP applications (see Table 1) syrphids disappeared (1998) or were clearly reduced in the conventional programme (Table 8). Although numbers of coccinellids were small and at several sampling dates no specimen at all were found in both treatments, altogether more individuals were found in the soft programme (Table 9). Table 8. Average number of syrphid larvae feeding on 100 A. pomi colonies. (*) = significant differences at p=0,05 level
Programme 2 jul. 98 9 jul. 98 (*) 16 jul. 98 6 jul. 99 (*) 3 jul. 00
Soft 3 4,5 5 8,8 1,9 Conventional 4 0 2 4,5 0,4
Table 9. Average number of coccinellid larvae feeding on 100 A. pomi colonies. (*) = significant differences at p=0,05 level
Programme 2 jul. 98 9 jul. 98 16 jul. 98 6 jul. 99 (*) 3 jul. 00
Soft 3,5 0 0 2,5 0 Conventional 0,5 0 0 0 0
During summer, until harvest, three visual samplings have been executed to estimate the damages caused by C. pyri honeydew on fruits (200 per plot). The results (Table 10) showed the higher level of damaged fruits regarding the conventional spray programme, mainly during the early summer.
The results obtained by fruit sampling (200 per plot) showed no losses caused by C. pomonella and leafrollers (P. cerasana, A. podana and A. pulchellana). A small difference regarding the fruits damaged by Q. perniciosus was found at harvest (Table 11) during the first two years.
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Table 10. Average-% of damaged fruits (by honeydew). (*) = significant differences at p=0,05 level
Programme and year Date 1998
4 jun. (*) 17 jul. (*) 24 aug. Soft 16,5 3,4 0,5
Conventional 33,2 6,9 0,5 1999
3 jun. (*) 14 jul. (*) 23 aug. Soft 23,5 2,67 0,33
Conventional 46,67 8,17 1,33 2000 6 jun. (*) 26 jun. (*) 2 aug. (*)
Soft 7,88 1,8 0,38 Conventional 15,81 3 0,75
Table 11. Average % of scale Quadraspidiotus perniciosus (*) = significant differences at p=0,05 level
Programme 24 aug. 98 (*) 20 aug. 99 2 aug. 00 Soft 0,38 0,38 0
Conventional 0 0 0
The saving regarding the kind and the amount of insecticides used in the soft programme is reported as environmental observation (Table 12).
Table 12. Comparison of insecticides (quantity and toxicity class) used in the conventional and replaced in the soft programme by tebufenozide(sum of three years).
Products Amount a. i./ha Toxicological classes Flufenoxuron 481cc Xi Diflubenzuron 450 g Not classified Chlorpyrifos methyl 660 cc Xi Azinphos methyl 4124 g T+ Tebufenozide 2967 g Not classified
Conclusions The three years study demonstrates that the “soft” insecticide programme based on the use of tebufenozide is really effective to control Codling moth C. pomonella and leafrollers (especially P. cerasana), and to limit other internal fruit feeders under the level of acceptance. In comparison to conventional programme currently in use in pear orchard, the soft strategy does not stimulate the resurgence of secondary pest (i.e. C. pyri) because of less interference with the activity of the predator A. nemoralis. A particular attention has to be paid to Q. perniciosus summer infestation. From an ecological point of view the soft programme could be of great interest in IPM project because of the saving in number and amount of a. i. used
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and less toxic products applied in comparison to the conventional one. With regard to the resistance management, the number of applications of tebufenozide needs careful consideration. In the long term, further selective insecticides will be needed to be used in combination with tebufenozide.
References Brown, K. C. 1989: The design of experiment to assess the effects of pesticides on beneficial
arthropods in orchards: replication versus plot size. In “Pesticides and non-target invertebrates”. Editor Jepson P. C., Intercept, pp. 71-80.
Candolfi, M. P., Blümel, S., Forster, R., Bakker, F.M., Grimm, C., Hassan, S.A., Heimbach, U., Mead-Briggs, M.A., Reber, B., Schmuck, R. & Vogt, H. (eds.) 2000: Guidelines to evaluate side-effects of plant protection products to non-target arthropods. IOBC/WPRS, Gent.
Civolani, S., Vergnani, S., Natale, E. & Pasqualini, E. 1998: Strategie di difesa da Cydia molesta su pomacee. Inf. Agr. 22: 71-75.
Civolani, S., Vergnani, S., Vallieri, E. & Pasqualini, E. 1999: Tebufenozide e methoxyfenozide (MAC): momento di applicazione su Pandemis cerasana. Inf. fitopat. 9: 55-62.
Civolani, S., Vergnani, S. & Pasqualini, E.,, 2001a: Tecnica di difesa per la generazione svernante di Pandemis cerasana (Hb.) (Lepidoptera Tortricidae) in Emilia-Romagna. Inf. fitopat., 10: 44-48.
Civolani, S., Vergnani, S. & Pasqualini, E., 2001b: Valutazione dell’efficacia di methoxyfenozide (MAC) su Cydia pomonella L. in Emilia-Romagna. Inf. Agr., 34: 63-64.
Croft, B. A. & Brown, W. A. 1975: Responses of arthropod natural enemies to insecticides. Ann. Rev. of Entomol. 20: 285-335.
Croft, B. A. (Ed.) 1990: Arthropod biological control agents and pesticides. John Wiley & Sons, New York. 723 pp.
Hassan, S. A., 1985: Standard methods to test the side-effects of pesticides on natural enemies of insects and mites. Bulletin OEPP-EPPO 15: 214-255.
Hassan, S.A., Bigler, F., Bogenschütz, H., Boller, E., Brun, J., Calis, M., Coremans-Pelseneer, J., Duso, C., Grove, A., Heimbach, U., Helyer, N., Hokkanen, H., Lewis, G.B., Mansour, F., Moreth, L., Polgar, L., Samsoe-Petersen, L., Sauphanor, B., Stäubli, A., Sterk, G., Vainio, A., van de Veire, M., Viggiani, G. & Vogt, H. 1994: Results of the sixth joint pesticide testing programme of the IOBC/WPRS-working group "pesticides and beneficial organisms". Entomophaga 39: 107-119.
Ishaaya, I. & Degheele, D. (Eds.) (1998): Insecticides with Novel Modes of Action. Mechanisms and application. 1st ed. Vol. 1. Springer Verlag, Berlin Heidelberg. 289 pp.
Pasqualini, E., Civolani, S., Vergnani, S., Cavazza, C. & Ardizzoni M. 1999: Selettività di alcuni insetticidi su Anthocoris nemoralis F. (Heteroptera Anthocoridae). Inf. Agr. 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.). Inf. fitopat. 6: 53-54.
Sterk, G., Hassan, SA., Baillod, M., Bakker, F., Bigler, F., Blümel, S., Bogenschütz, H., Boller, E., Bromand, B., Brun, J., Calis, J.N.M., Coremans-Pelseneer, J., Duso, C., Garido, A., Grove, A., Heimbach, U., Hokkanen, H., Jacas, J., Lewis, L., Moreth, L.,
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Polgar, L., Rovesti, L., Samsoe-Petersen, L., Sauphanor, B., Schaub, L., Stäubli, A., Tuset, J.J., Vainio, M., Van de Veire, M., Viggiani, G., Viñuela, E. & Vogt, H. 1999: Results of the seventh joint pesticide testing programme carried out by the IOBC/WPRS working group "Pesticides and Beneficial Organisms". BioControl 44: 99-117.
Vogt, H. (Ed.) 1994: Side-effects of pesticides on beneficial organisms: Comparison of laboratory, semi-field and field results. IOBC/WPRS Bulletin 17 (10): 178 pp.
Vogt, H. & Heimbach, U. (Eds.) 2000: IOBC/WPRS Working Group „Pesticides and Beneficial Organisms”. Proceedings of the meeting at Versailles, 27-29 October 1999.
Vogt, H. & Viñuela, E. and Jacas, J. (Eds.) (2001): Pesticides and Beneficial Organisms. Proceedings of the meeting at Castelló de la Plana, Spain, 18-20 October 2000. IOBC/WPRS Bulletin 24 (4).
Pesticides and Beneficial Organisms IOBC/wprs Bulletin Vol. 25 (11) 2002
pp. 107 - 113
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New restrictions for the use of pesticides to protect off-field non-target organisms Rolf Forster 1), Christine Kula 1), Volkmar Gutsche 2) and Siegfried Enzian 2) Federal Biological Research Centre for Agriculture and Forestry 1) Department for Plant Protection Products and Application Technique, Biology Division 2) Institute for Technology Assessment in Plant Protection Abstract: In accordance with national and EU policies (i.e. Directive 91/414/EEC) the placing of plant protection products on the market is granted, if it is established in the light of the current scientific and technical knowledge that the product has no unacceptable influence on the environment, having particular regard to its impacts on non-target species. As presented in earlier publications Member States may need to impose conditions or restrictions with the authorizations they grant due to the inherent toxicity of certain products, e.g. for the protection of non-target arthropods or non-target plants. New measures have currently been implemented into the national authorization of plant protection products, and have been selected on the basis of the risks likely to arise, based on a deterministic risk assessment (TER approach). The new restrictions mainly focus on the use of the best application technique available in order to reduce spray drift to a level which is safe for non-target arthropods or non-target plants. If drift levels achieved by using certain techniques are still unsafe, a 5 m buffer zone may be recommended if possible, depending on the plant health or plant protection situation. This new concept is deemed to support the technical progress while being economically acceptable and ecologically effective. Exceptions from this concept are possible, taking into account both agricultural and environmental conditions in the area of the envisaged use, e.g. for the use of hand held sprayers, small strips of natural or semi-natural habitates and landscape structure as suggested in earlier publications. Key words: side-effects of pesticides, protection of non-target arthropods, restrictions, landscape structure Introduction In 2000 based on a former proposal of Forster & Rothert (1998) the Biologische Bundesanstalt für Land- und Forstwirtschaft (BBA) and the Umweltbundesamt (UBA) implemented restrictions for the use of plant protection products (PPPs) in order to protect non-target arthropods (NTAs) and plants (NTPs). In November 2000 the BBA together with the UBA started to revise these restrictions, because the restrictions implemented in the year 2000 mainly relied on buffer or no-spray zones 5 m wide, which was considered not appropriate for certain uses, especially for herbicides, from an agricultural or plant health point of view. A new approach focusing on spray-drift reducing technique was therefore put on the platform. The approch is applicable for both, risk mitigation for NTAs as well as for NTPs. Legal Background, Directive 91/414/EEC According to Directive 91/414/EEC the protection of the environment should take priority over the objective of improving plant production. However, scientific evidence indicates that even when properly applied and for the purpose intended, the use of pesticides might severely
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affect non-target arthropods, in-crop and off-crop, at least on a short term temporal scale and short range spatial scale. In accordance with national and EU policies (i.e. Directive 91/414/EEC) the placing of plant protection products on the market is granted only, if it is established in the light of the current scientific and technical knowledge that the product has no unacceptable influence on the environment, having particular regard to its impacts on non-target species.
There is scientific evidence that spray drift reducing measures (e.g. spray-drift reducing technique, buffer zones) can significantly reduce the exposure of non-target arthropods inhabiting field edges and field margins and consequently the severity of effects. Directive 91/414/EEC therefore suggests, that Member States shall impose conditions or restrictions with the authorizations they grant. Measures must be selected on the basis of and be appropriate to the nature and extent of the expected advantages and the risks likely to arise. Member States shall ensure that, decisions taken take account of the agricultural, plant health or environmental conditions in the areas of envisaged use. The severity of effects on the meta-population level, however, cannot be evaluated without having regard to certain aspects, such as landscape structure and the intensity of chemicals used on a landscape level as both external factors are known to govern recolonization dynamics. Because these factors currently are not appropriately considered within risk assessment these have been integrated into risk management. TER Approach, Concept and Limitations At tier 1 laboratory toxicity studies with two indicator species are performed (Forster & Martin, 2001). LR50 data are to be generated for two sensitive species which will be used to calculate TERs (Toxicity-Exposure-Ratios) for in-field and off-field exposure scenarios. A TER in-field of 1 and an uncertainty (safety) factor of 10 for the off-field TER-calculation are considered appropriate values at tier 1 (test on glass-plates). Usually, the overall 90th percentile drift data published by BBA (2000) are used to estimate off-field drift deposition. Spray-drift values are corrected for 3-D by a factor of 2 for 2-D-tests (an additional Leaf Area Index-factor might be introduced as appropriate, if supported by appropriate data). Based on these data, the protection level needed (50, 75, 90 % or more drift reduction) is calculated.
Compared to real life the current TER approach does not seem to be very sophisticated, because neither the spatial nor the temporal distribution of NTAs and chemicals are taken into account. Therefore the TER approach is considered protective but not predictive. It is however used mainly at the first and second tier and higher tier data might be helpful to assess risk more realistically. New Restrictions, Wording and Scientific Background The restrictions are split into 3 main groups with 3 different classes each (i.e. 50, 75 or 90 % drift reduction). For better communication and technical purposes the restrictions are encoded, e.g. as NS 611-1, NS 612-1, NS 613-1. The following wording focusing on agricultural uses in field crops was agreed:
In a strip of at least 20 m to adjacent areas (except areas used for agricultural or horticultural purposes, roads, paths, and public places), the product must be applied using spray drift-reducing equipment
109
which is registered in the ”Index of drift-reducing equipment” (...), and is registered at least under drift-reducing class 50 % (75 %, 90 %).
A strip of 20 m, where drift reducing technique must be applied is essential, because off-
crop relevant spray-drift is produced within an area of 20 m in-crop for field crops, or 5 rows in orchards and 10 rows in vineyards. Therefore the technique available in combination with the recommended speed or pressure limits needs to be effective within a strip of 20 m, only (according BBA-Guideline VII 2-1.1, 1992).
The second group covers uses, where drift reducing equipment is not available and a buffer zone of 5 m does not interfere with the principles of good agricultural practice, as for example in fruit crops, where a choice might be appropriate (NS 611-10, NS 612-10, NS 613-10):
In a strip of at least 20 m to adjacent areas (except areas used for agricultural or horticultural purposes, roads, paths, and public places), the product must be applied using spray drift-reducing equipment which is registered in the ”Index of drift-reducing equipment” (...), and is registered at least under drift-reducing class 50 % (75 %, 90 %). Drift-reducing technique need not be used if a buffer zone of at least 5 m is kept to adjacent areas (except areas used for agricultural or horticultural purposes, roads, paths, and public places).
The third group is used, where drift reducing equipment or a buffer zone of 5 m alone do
not ensure the protection level needed. Both measures must be taken as long as these do not interfere with the principles of good agricultural practice (NS 621-10, NS 622-10, NS 623-10):
In a strip of at least 20 m to adjacent areas (except areas used for agricultural or horticultural purposes, roads, paths, and public places), the product must be applied using spray drift-reducing equipment which is registered in the ”Index of drift-reducing equipment” (...), and is registered at least under drift-reducing class 50 % (75 %, 90 %). In addition, a buffer zone of at least 5 m must be kept to adjacent areas (except areas used for agricultural or horticultural purposes, roads, paths, and public places).
However, in order not to hamper the protection of crops beyond acceptable limits special
considerations have been agreed for certain scenarios, where aspects such as landscape structure and the intensity of chemicals used on a landscape level can be considered. The restrictions need not be followed, if in a local risk assessment one of three conditions apply:
Neither drift-reducing technique nor a buffer zone is required 1. if the product is applied using portable sprayers, or 2. if adjacent areas (such as field margins, hedges) are less than 3 m
wide, or 3. if the product is applied in an area of a landscape which has been
listed by the BBA as a landscape with a sufficient proportion of natural and semi-natural structures (Currently not accepted for NS 621ff).
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The reasons behind these considerations are:
– Hand held sprayers are used on small areas or plots and produce less spray drift. – Strips less than 3 m usually demonstrate lower bio-diversity, but eradication of small
strips by mechanical means shall be avoided. – The proportion of natural and semi-natural structures on the landscape level is important
with respect to the recolonisation and recovery of populations and relative to farming intensity.
0
5
10
15
20
25
30
35
40
Anza
hl
2 4 6 8 10 12 14 16 18 20 Biotop Index
Histogramm
Figure 1: Landscape structure/proportion of buffered natural/semi-natural area for 475 regions in Germany (X-Axis: proportion (%) of natural/semi-natural areas in each region; Y-Axis: number of regions)
0
10
20
30
40
50
60
Anza
hl
0 1 2 3 4 5 6 7 8IcPS
Histogramm
Figure 2: Farming intensity/weighted means of PPPs for 475 regions in Germany (X-Axis: relative intensity of plant protection in each region; Y-Axis: number of regions)
111
Landscape structure We can easily understand, that the higher the intensity of the total pesticide use is, the higher is the need for natural or semi-natural areas which allow for a recovery or recolonization of affected populations. From literature an interval of 5 to 20 % is suggested for the conditions in Germany. Figure 1 gives the proportion of these areas (e.g. field margins, hedges) for some 475 regions in Germany. The proportion was estimated using the official data base ATKIS together with estimates based on a number of spot checks in different regions and a literature survey.
The graph shows a maximum of app. 20 % and a median of app. 7 % in these areas. Obviously, a considerable number of regions does not meet the suggested minimum of 5 %. Farming intensity The next figure (Fig. 2) gives the relative farming intensity for the same regions (475 regions in Germany), based on all PPPs used in 15 main crops.
Mindestausstattungnicht erfüllterfüllt
Figure 3: Areas of concern for 475 regions in Germany (shaded regions)
112
The calculation is based on the frequency of the pesticide use for 15 main crops (Rossberg, 1997, unpubl.), the proportion of each crop and the proportion of all cropped area within one region. The maximum is app. 7.5 and the median is app. 1. Regions of concern in Germany Finally, by merging both of these databases we can indentify regions of concern, where the proportion of natural and semi-natural areas is considered too low compared to the intensity of pesticide use to ensure longterm survival of NTAs (Fig. 3). For more details see Gutsche & Enzian (2002, in prep.). It is in these areas, which make up app. 30 %, where pesticide use has to be in compliance with the risk mitigation measures on the label in order to avoid unacceptable effects on NTAs. PPPs potentially involved As a conservative estimate we can pick up from figure 4 a proportion of about 30 % of the PPPs currently authorized in Germany which will be subject to risk mitigation or dismissal of authorizations in future
It is evident from this database, and the picture has not changed significantly over the last few years (Forster, 1998 and 1999), that primarily acaricides and insecticides (about 90 % in high risk for NTAs) make up the group that is subject to risk mitigation or dismissal of authorization. It is especially this group for which the Annex I listing and the granting of authorizations is currently questioned, especially if uses in fruit crops are considered.
Figure 4: PPPs most likely to be subject to risk mitigation or dismissal of authorizations in Germany (July 1998; 850 PPP) Conclusions Based on the conservative TER approach restrictions are deemed necessary in order to fulfil national and EU requirements. New restrictions are deemed in line with the recommendations of Directive 91/414/EEC as they theoretically consider the nature and extent of risks, take account of agricultural, plant health and environmental conditions. With respect to NTAs most insecticides will be subject to the restrictions and probably denial of authorizations (especially orchard uses due to high spray drift). While these restrictions are in compliance with the precautionary principle, as required by the European Commission, we must generally admit that further scientific evidence is needed, to clarify, whether the restrictions presented in this paper are essential for the protection of NTAs on the meta-population level or not.
13%
44%13%
20%
10% negligible risk for NTAs
low risk (species specific)
medium risk (species specific)
high risk (species specific)
high risk for NTAs
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References Forster, R. 1998: Effects of plant protection products on beneficial organisms: the current
authorization procedure in Germany. In: Ecotoxicology, pesticides and beneficial organisms, eds. Haskell, P. and McEwen, P., Kluwer Academic Publishers: 222-231.
Forster, R. 1999: Übersicht über die im Rahmen der Zulassung von Pflanzenschutzmitteln im Prüfbereich Nutzarthropoden ertelten Kennzeichungen – Zeitraum Juli 1993 bis Juli 1998. Nachrichtenblatt des Deutschen Pflanzenschutzdienstes, 51 (6): 152-154.
Forster, R. & Martin, S. 2001: Non-Target Arthropod Testing - The current German interim procedure based on proposals made at the ESCORT 2 workshop. Proceedings of the IOBC Meeting “Pesticides and Beneficial Organisms”, Castello 2000. IOBC/wprs Bulletin 24 (4): 1-6.
Forster, R. & Rothert, H. 1998: The use of field buffer zones as a regulatory measure to reduce the risk to terrestrial non-target arthropods from pesticide use. In: The 1998 Brighton Conference on Pests and Diseases, Conference Proceedings 3: 931-938
Gutsche, V. & Enzian, S. 2002: Quantifying the equipping of landscape by natural terrestrial habitats on base of the digital topographical data. Nachrichtenblatt des Deutschen Pflanzenschutzdienstes, (in prep.).
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Pesticides and Beneficial Organisms IOBC/wprs Bulletin Vol. 25 (11) 2002
pp. 115 - 120
115
Three-year study of the effects of Karate® applications in wheat on arthropod communities in a field margin Results and the problem of small numbers Bernd Freier, Stefan Kühne, Peter Kaul, Bärbel Baier, Eckardt Moll, Barbara Jüttersonke, Rolf Forster1
Federal Biological Research Centre, Kleinmachnow branch, Stahnsdorfer Damm 81, D-14532 Kleinmachnow, and 1 Messeweg 11/12, D-38104 Braunschweig, Germany Abstract: Effects of drift from Karate® (lambda-cyhalothrin, 7.5 g a.s. per ha) sprayed in a wheat field during BBCH69 were investigated in a 470-m field margin south of Berlin in the years 1998-2000. The field margin strip was divided into 8 similar patches which were either exposed to spray drift or left untreated as control patches. A tracer (BSF) was used to assess drift as specific three-dimensional deposition patterns in all margin patches neighbouring the treated wheat field. Serious drift occurred in 1998 and in 2000. From these drift patterns, isomortality lines for LD 50 were defined for important representative indicators to predict the theoretically expected effects in boundary areas. The actually measured effects in arthropod populations were lower.
The results of biocoenometer-suction samplings revealed significant differences (Wilcoxon-Test) between both groups in only a few arthropods. The mean density on the 1-m sampling line tended to be lower than that on the 5-m line. The pitfall trap data did not point out attributable to insecticide drift, either with respect to the total number of trappings (carabids, spiders) or to the diversity (carabids). Therefore, the effect of insecticide spray drift on the arthropod community in field margins seemed to be unimportant. No cumulative effects of insecticide spray drift in the same field margin patches were observed during the 3 years. The limitations of a species-specific evaluation of the sampling results due to small numbers of collected individuals is shown by examples of arthropod indicator groups. Key words: field study, lambda-cyhalothrin, field margin, spray drift, non-target arthropods Introduction Field crops border on other fields or field margins. Wide area structures such as woods, lakes, rivers and settlements are not located directly adjacent to crop fields, because strip biotops function as transitional zones separating these landscape structures. Kühne et al. (2000) recently calculated that in Germany the border lines between crop fields and field margins extend for a total length of nearly 1.6 million km. For this reason, it is particularly important to know whether and to what extend side-effects of insecticide drift on off-crop habitats adjacent to crop fields can occur. However, experimental data on side effects of insecticide treatments in crops on the non-target arthropod community in adjacent field margins are scanty (Davis et al., 1994). Details on drift sediment in field edges and immediate effects in non-target populations after a pesticide application as well as long-term effects considering recovery and re-colonisation processes during the cropping season and the following year or more are needed for a realistic risk assessment (Brown, 1998). Extensive field experiments with enormous methodological and man-power requirements are needed for proper study of such complex relationships in population dynamics of non-target arthropods. These experiments are always only case studies and generate data that can be used as reference values in theoretical risk assessments.
116
The aim of our 3-years field study was to obtain preliminary data by: 1. Measuring different insecticide drift in sediment as related to distance from the field and
vegetation height. 2. Assessing the immediate drift-related effects of insecticide applications in a wheat field on
arthropods in the adjacent field margin. 3. Testing for potential accumulation of insecticides in the field margin because the
insecticide applications took place on the same plots for 3 experimental seasons. 4. Gaining methodological experience on suitable indicator arthropods and representative
sampling methods. Material and method A detailed description of methodological approach is given in Freier et al. (2001). Therefore, this section contains only selected information on the side location, insecticide application and monitoring methods. Experimental site The experimental site was located in Osdorf, several km south of Berlin. A long field margin on the eastern edge of a 50-ha field was used for the study. This relatively uniform grass-dominated strip of vegetation was 6 m wide and 470 m long. For the duration of the experiment, the margin was divided into 8 similar plots, 4 plots were exposed to insecticide drift to yield a total of 4 replications (treatment group = Bi), and 4 plots were not exposed to insecticide drift (control group = Ki).
According to the vegetation analyses the field margin consisted of two plant communities: The middle strip was identified as a Lolio-Plantaginetum community and the strips to the left and right of the middle contained a Convolvulo-Agropyretum repentis community. A total of 34, 47 and 54 grass and herb species were identified in 1998, 1999 and 2000, respectively. Insecticide application The insecticide Karate® (lambda-cyholothrin, 7.5 g a.s. per ha) was sprayed as a conventional cereal aphid control measure against cereal aphids at the end of flowering (BBCH 69) using a sprayer with a working width of 18 m and boom height of 60 cm above the wheat. Only in 1999 was the insecticide applied according to the rules of good plant protection practise. The pressure, speed and nozzle type were changed to induce more spray drift in 1998 and 2000. Measurement of the contamination of field margin by spray drift The spray-related sediment levels on the treated wheat crop and in the margin were measured indirectly using the fluorescent marker BSF which was added to the spray. Pipe cleaners were used to catch the spray for laboratory measurements. Monitoring methods Biocoenometer surveys were performed to obtain the true arthropod densities. 1-m² cages were randomly placed along 1-m or 5-m lines (distances from field) in the margin and in the field. All plants inside the cages were cropped off and placed in bags. The remaining arthropods were caught using a suction sampler. The arthropod numbers were determined at the laboratory after cryopreservation. Sampling dates: 1 day before, 1 or 2 days after and 2 weeks after treatment. Additional sampling was performed 4 weeks after insecticide application in 1998.
Counting of grasshoppers was made under 1-m² cages randomly placed along the 1-m and 5-m lines in the field margin. Sampling dates: 1 day before and 1 day after insecticide application, than at weekly intervals.
117
Pitfall trap catches with 10-cm diameter cups were done on the 3-m line in margin and field. Sampling dates: 1 week before insecticide application and at weekly intervals thereafter. Results and discussion Drift was assessed in specific three-dimensional deposit patterns in all margin patches neighbouring on the treated wheat field. Figures on drift dispersion in all years and plots were published in Kühne et al. (2002). There were obvious differences between the years. Serious drift occurred in 1998 and in 2000 and, according to Anonymous (2000), basic drift values were exceeded by a factor of 5 to 10. In addition, the drift patterns also differed within the 4 replications within a given growing season. In 1999, the drift sediment distribution corresponded with the basic drift values reported by Anonymous (2000).
Based on the drift patterns and LD 50 values of the official registration procedure for Karate, isomortality lines for LD 50 were defined for important representative indicators to show the theoretically expected effects of 50 % mortality in the boundary area. Figure 1 shows these isomortality lines as borders of grey areas that represent mortality values of >50 % for Coccinella 7punctata on the vegetation surface (light grey) and ground (dark grey). The patterns suggest that a relatively high mortality by insecticide spray drift must be expected for this species under the spray drift conditions in the years 1998 and 2000. The calculations for other indicators demonstrate obvious differences of predicted mortality in the 6-m wide field margin: – Typhlodromus pypi – Strong effects in the whole boundary area, particularly in 1998 and
2000, – Lycosids – Some effects in margins within 2 m of the field in all years, – Aphidius rhopalosiphi – Effects within 1 m (1999) and 5 m (1998, 2000) of the field, – Chrysoperla carnea – No important effects in all years.
The question was whether these calculated mortality effects based on laboratory toxicity results and our sediment data reflected the true side-effects of spray-drift on arthropods in the field margin. Our sampling studies show that the actually measured effects on the arthropod populations were lower than expected. The results of biocoenometer-suction samplings demonstrate significantly (Wilcoxon-Test) lower densities in only a few arthropods in the treatment groups (table 1). The predicted sensitivity of mites was confirmed, although the statistical tests revealed significant differences in mean values in only one case. We found a slight tendency towards lower mean densities on the 1-m sampling line than on the 5-m line. This trend could be identified in an all over comparison of the findings in 60 taxa. On the 1-m line there were more cases with higher densities in control plots than in the plots exposed to insecticide drift.
The drift did not seem to affect the grasshopper community. Only one significant difference in the grasshopper counts of the two groups was found (table 1).
The pitfall trap data also did not point out any particular effects attributable to insecticide drift, either with respect to the total number of trappings (carabids, spiders) or to the diversity (carabids). Significantly lower numbers of individuals in the plots exposed to drift were observed in two tests (table 1).
The results indicate that the effect of insecticide spray drift on the arthropod community in field margins is rather unimportant. No cumulative effects of insecticide spray drift in the same field margin patches were observed during the 3 years of the study. However, these findings must be cautiously interpreted because for the relatively low effects of spray drift could be due to a number of factors, some of which are listed below.
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Figure 1. Isomortality lines (LR 50) (correspond to border lines of the grey areas) based on laboratory LR 50-data for adult Coccinella 7punctata and drift deposition data for Karate® measured in a field margin near Osdorf, in plots (B1-B4) of approximately 50 m lenght. 1. The effects might have been weaker in the field than in the laboratory due to - Overestimation of effects in laboratory tests, - The probability that habitats provide certain protection from drift (e.g. on leaf undersides)
and - Migration and other recovery processes.
2. Statistical problems preventing the identification of significant differences due to factors related to the experimental design and sampling methods. The main statistical problems were: 1. Small number of individuals found per sample, and 2. Too few replicates.
1 m
5 m
1 m
5 m
1 m
5 m
y
1 m
5 m
1 m
5 m
y
1 m
5 m
B1 B2 B3 B4
2000
1999
B1 B2 B3 B4
1998
B1 B2 B3 B45 m
1 m
Dis
tanc
e to
the
field
edg
e
5 m
1 m
(LR 50 = 0.2 g/ha) Vegetation surfaceMortality >LR 50 Ground
5 m
1 m Dis
tanc
e to
the
field
edg
eD
ista
nce
to th
e fie
ld e
dge
119
Table 1. Effects of Karate spray drift on arthropods in field margin near Osdorf. Significantly lower numbers of individuals were found in the 4 plots exposed to spray drift than in the control plots (n = 4, Wilcoxon-test, one tailed t-approximation). Values in bold print represent mean differences >50 %.
Time after application 5 m 1 m Biocoenometer samplings (238 statistical tests including 60 taxa) 1998 Mites + 2d p=0.0471 Aphids + 2d p=0.0336 Leafhoppers + 2d p=0.0336 Other carabids + 2d p=0.0258 Arthropods (sum) + 2d p=0.0336 p=0.0336 1999 2000 Grasshopper counts (10 statistical tests) 1998 1999 2000 Grasshoppers + 1d p=0.0331 Pitfall trap catches (36 statistical tests including 2 taxa) 1998 Carabids + 20d p=0.033 1999 Carabids + 27d p=0.078 2000
The following example illustrates the limitation of statistical analyses and data interpretations.
A biocoenometer sample as defined in the present study, covers an area of 2 x 1 m² = 2 m². The number of individuals found 2 days after insecticide application in 1999 was 1, 4, 7, 8 = mean 5.0 in the control group (K), and 3, 1, 6, 0 = mean 2.5 in the treatment group (B). These figures suggest that spray drift caused a reduction corresponding to the LR 50.
However, the number of individuals per sample was too low to determine whether this difference was significant. Assuming a normal distribution, α = 0.05 (type I error rate), β = 0.20 (type II error rate) and a variance s² = 10 for n = 4, a minimal difference of d > 6.4 individuals per m² would be necessary (calculation with CADEMO/MIWA), i. e., only a difference of more than 6.4 can be identified as significant. In reference to the example and assuming a normal distribution, the mean of B should be – 1.4, which is, of course, nonsense.
Furthermore, the number of replicates was too low to determine whether this difference was significant. If we also assume a normal distribution, α = 0.05 (type I error rate), β = 0.20 (type II error rate) and a variance s² = 10 for n = 4, a minimal difference of d = 2.5 would be necessary, which would just barely make a significant difference between the two means 5.0 (K) and 2.5 (B). However, this can be only achieved with n > 21 replications.
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An additional problem is that the number of individuals was not normally distributed, and when a binomial distribution is assumed, the number of replicates has to be even: n > 100.
In the present field study, no dramatic effects of spray drift were found. We assume that possible immediate insecticide effects on some pyrethroid-sensitive arthropods were compensated for by re-colonisation and recovery processes which must be considered in ecotoxicological evaluations (Duffield & Aebischer, 1994, Holland et al. 2000). However, our findings and statistical analyses indicate the necessity of giving more attention to experimental design and sampling methods in ecotoxicological field studies to achieve more sensitive identification of differences. Acknowledgements The authors would like to thank all colleagues who participated in this study during the activities in field as well as in laboratory. Also special thanks to Mrs. S. Wandrey, who kindly edited the English manuscript. References Anonymous 2000: Bekanntmachung über die Abtrifteckwerte, die bei der Prüfung und
Zulassung von Pflanzenschutzmitteln herangezogen werden. Bundesanzeiger 100, 26. Mai 2000: 9878-9890.
Brown, K.C. 1998: The value of field studies with pesticides and non-target arthropods. Proc Int. Conf. Brighton 1998, 2: 575-582.
Freier, B., Kühne, S., Baier, B., Schenke, D., Kaul, P. & Heimbach, U. 2001: Field study on effects of insecticide applications in wheat on the arthropod community of field boundaries. Mitt. BBA 383: 82-87.
Kühne, S., Freier, B., Baier, B., Schenke, D., Kaul, P., Jüttersonke, B., Heimbach, U., Moll, E. & Forster, R. 2002: Feldstudie zu Auswirkungen von Insektizidapplikationen in einem Saumbiotop infolge Abtrift. Agrarökologie, in press.
Kühne, S., Enzian, S., Jüttersonke, B., Freier, B., Forster, R. & Rothert, H. 2000: Beschaffenheit und Funktion von Saumstrukturen in der Bundesrepublik Deutschland und ihre Berücksichtigung im Zulassungsverfahren im Hinblick auf die Schonung von Nichtzielarthropoden. Mitt. BBA 378: 1-128.
Davis, B.N.K., Brown, M.J.; Frost, A.J.; Yates, T.J. & Plant, R.A. (1994): The effects of hedges on spray deposition and on the biological impact of pesticide spray drift. Ecotox. Environm. Safety 27: 281-293.
Duffield, S.J. & Aebischer, N.J. 1994: The effect of spatial scale of treatment with dimethoate on invertebrate population recovery in winter wheat. J. Appl. Ecol. 31: 263-281.
Holland, J.M., Winder, L. & Perry, J.N. 2000: The impact of dimethoate on the spatial distribution of beneficial arthropods in winter wheat. Ann. Appl. Biol. 136: 93-105.
Pesticides and Beneficial Organisms IOBC/wprs Bulletin Vol. 25 (11) 2002
pp. 121 - 123
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Off-crop pesticide drift deposition Heribert Koch State Agency for Agronomy and Plant Protection, Mainz, Germany e-mail: [email protected] Extended Abstract The deposition of drift on off-crop plant surfaces was investigated. A tractor mounted field sprayer (15m) was used to spray a fluorescent tracer as an indicator. Applications were done in a meadow and leaf samples were taken at 1, 3, 5 and 10 m distance from the edge of the boom. Measurements in the meadow allowed to set up driving direction and sampling lines almost perpendicular to the wind direction. Results are expressed as ng/cm² leaf surface and show a wide variability of deposits at the investigated distances due to the fact that wind is not constant rather than highly variable in time, speed and direction even on a small scale.
Deposits decrease fast with increasing distance. Drifting particles are smaller than 100 µm in diameter and are transported just by the air movement. We compared the standard nozzle (XR 110 03, 2 bar) and the nozzle AI 110 025, 3 bar which is registered as 50% drift reducing nozzle. Both configurations were operated at a water volume of 200 l/ha. The deposition pattern in the drift zone is characterised by single particles with a very low coverage on the plant surface indicating a very small surface portion accounting for the exposure. This exposure is not comparable to spray deposits under the nozzle boom within the sprayed area. Most of the off-crop plant surface is not contaminated by drift deposits. Measured low levels of drift exposure support other investigations showing almost minor effects of drift (Altes Land, BBA, Uni Gießen).
Table 1. Drift deposits on plant surfaces in a meadow: Deposits (ng/cm² per g/ha) off-crop after application with standard nozzles (XR 110 03, 2 bar) and 50%-drift reducing nozzles (AI 110 025, 3 bar). Coefficient of variation in brackets (CV %). 36 samples were investigated with XR 110 03 and 26 with AI 110 025 at 1, 3, 5 and 10 m adjacent to the sprayed area.
Distance to sprayed area (m) 1 3 5 10
XR 110 03 0.1347 (61.8)
0.0414 (77.6)
0.0179 (65.2)
0.0044 (81.0)
AI 110 025 0.0050 (131.8)
0.0010 (98.6)
0.0006 (71.8)
0.0003 (54.6)
XR 110 03 Samples < lq Samples < ld
– –
– –
– –
1 –
AI 110 025 Samples < lq Samples < ld
5 1
9 9
10 10
12 14
lq = limit of quantification, ld = limit of detection
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Table 2. Drift deposits on plant surfaces in hedgerows (ng/cm² per g/ha) after application with standard nozzles (XR 110 03, 2 bar). The front of the hedgerow was structured into 12 sampling sectors. Coefficient of variation in brackets (CV%). Mean deposits represent 28 samples per sector. 112 samples were taken per depth sector.
Height sector (cm) Depth sector (cm) 0 – 50 50 – 100 100 – 150
0.0238 (69.7)
0.0142 (144.2)
0.0048 (121.4)
0.0337 (74.3)
0.0162 (122.6)
0.0047 (93.0)
0.0717 (124.9)
0.0283 (106.7)
0.0136 (124.9)
150 – 200
100 – 150
50 – 100
0 – 50 0.1995 (140.0)
0.0534 (102.3)
0.0295 (172.0)
Samples < lq Samples < ld
1 2
6 8
14 2
Table 3. Drift desposits on plant surfaces in hedgerows (ng/cm² per g/ha) after application with 50%-driftreducing (AI 110 025, 3 bar). The front of the hedgerow was structured into 12 sampling sectors. Coefficient of variation in brackets (CV%). Mean deposits represent 20 samples per sector. 80 samples were taken per depth sector.
Height sector (cm) Depth sector (cm) 0 - 50 50 - 100 100 - 150
150 - 200 0.0047 (96.3)
0.0019 (99.4)
0.0006 (164.6)
100 - 150 0.0067 (72.8)
0.0013 (95.9)
0.0012 (87.7)
50 - 100 0.0125 (56.8)
0.0041 (82.1)
0.0022 (98.4)
0 - 50 0.0473 (178.0)
0.0073 (72.4)
0.0043 (172.1)
Samples < lq Samples < ld
2 3
22 7
30 20
50% drift reducing nozzles resulted in about 90% lower deposits compared to the standard nozzle XR 110 03. Trials were carried out at wind speeds between 2 - 5 m/sec. For the 50%-drift reducing nozzle we did not find any deposit above the limit of quantification at the 10 m point. This nozzle type produces only 0,3 % of the delivered volume in droplets smaller than 100 µm in diameter. Growers have been encouraged by the plant protection service to change their nozzle equipment. Drift will become less relevant in Germany as the change of nozzles proceeds.
123
Table 4. Drift deposit on plant surfaces (%) in relation to the nominal rate within the sprayed area (meadow), dose rate 50g/ha in 200 l/ha, field sprayer
Sprayed area Drift area 1 m 3 m 5 m 10 m
Standard nozzle 03 (36 samples) 1,347 0,414 0,179 0,044
50 % drift reducing nozzle (26 samples)
500 ng/cm²
0,05 0,01 0,006 0,002
References Koch, H., Weisser, P., Landfried, M. & Strub, O. 2002: Exposition durch Pflanzenschutz-
mittelabdrift an Blattoberflächen von Nichtzielpflanzen in terrestrischen Saumstrukturen. Zeitschrift für Pflanzenkrankheiten und Pflanzenschutz, Sonderheft XVIII: 1023-1030.