on Use and Production of Neem-lngradients and Pheromones

Transkript

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2 Practice Oriented Results on Use and Production of Neem-lngradients and Pheromones Organized by: H. Kleeberg and C.P.W. Zebitz (Eds.) 5th Workshop Wetzlar; Germany, Jan , 1996

3 Practice Oriented Results on Use and Production of Neem-lngredlents and Pheromones Proceedings of the 5 th Workshop; Wetzlar, Germany, Jan Printed by: Druck & Graphic, Giessen, Kiesweg Innenteil gedruckt auf 100% Recycling Papier Compiled and prepared by: H. Kleeberg and C.P.W. Zebitz Distribution: Trifolio-M GmbH, Sonnenstr. 22, D Lahnau 2 ISBN: Printed in Germany All rights of production, in whole or in part, e.g. in print or by film, radio, television, or any photo-copying are reserved.

4 CONTENTS Welcome address IX Development of NeemAzal 1 H. Kleeberg Neem and GLP: Good Laboratory Practice 7 Christine Kliche-Spory, H. Kleeberg, Beate Ruch, Ilona Schäfer, Annette Schlicht and R. Troß NeemAzal and analysis 9 R. Troß Summary of some environmental aspects of the Neem ingredient NeemAzal and NeemAzai-T/S 15 Beate Ruch, Christine Kliche-Spory, Annette Schlicht, Ilona Schäfer, Jenny Kleeberg, R. Troß and H. Kleeberg NeemAzal-T and NeemAzai-T/S against the rosy apple aphid - field trials in Switzerland M. Zuber Two years experience with the use of NeemAzal in organic fruit orchards 27 Jutta Kienzle, C. Schulz and C.P.W. Zebitz Wirkung der Neem-Extraktformulierung NeemAza!-T/S auf die Grüne Erbsenblattlaus Acyrthosiphon pisum im Labor (1995) 33 E. Hummel and H. Kleeberg Control of insects in Brussies" sprouts with NeemAzal-T/S 41 W. Manger and J. Koeckhoven Dreijährige Prüfung zur Wirksamkeit von Niemprodukten gegen tierische Schadorganismen im Feldbau 51 R. Gottwald Control of Colorado potato beetle (Leptinotarsa decemlineata Say) by an Azadirachtin-formulation (NeemAzal-T/S), by Bacillus thuringiensis tenebrionis (Novodor), and by combinations of both: short-term and long-term effects 59 T. Basedow and Andrea Peters

5 Control of the Colorado potato beetle (L ptinotarsa decbmlineata Say) in organic gardening systems with Bacillus thuringiensis var, san diego (m-one) and new products from the Neem tree (NeemAzaS-F and Align ) 67 M. Baumgart, Kirsten vom Brocke and M. He Crow Effects of NeemAzal" on vitality and fertility of Melolontha hippocastani 75 M. Rohde Effects of different NeemAzaS-formuIations on apple aphids and Aphis fafoae Scop. 81 C. Schulz, Jutta Kienzle, C.P.W. Zebitz The effect of NeemAzai-T/S on Dysaphis plantaginba, Rhopaiosiphum insertum and beneficial insects 93 G. Henkel, E. Mester and P. Heyne NeemAza!~T/S for the control of aphids on sour cherry and hop 101 Christine Hapke, C. Schulz, Jutta Kienzle and C.P.W. Zebitz Field investigations on the efficacy of NeemAzaS-T/S against Dysaphis plantaginea (Passerini) (Homoptera: Aphididae) and its effects on larvae of Chrysoperla carnea Stephens (Neuroptera: Chrysopädae) 105 Heidrun Vogt, U. Händel and Elise Vinuela Blattlausbekämpfung bei Schnittanemonen durch NeemAzai-T/S unter Berücksichtigung des Einflusses auf Nützilnge 115 Heike Döbeiin Die Bekämpfung der Larven des Seerosenblattkäfers Galerucella nymphaeae L mit dem Präparat NeemAzaS-T/S 123 E. Marklowsky Effect of NeemAzal-T/S on Tetranychus urticae Koch 129 M.K. Mironova and E.G. Khorkhordin NeemAzai-T/S gegen Blütenthrips (Frankliniella occidentalis) an Fuchsien unter Glas 139 R. Schmidt NeemAzai-T/S against Myzus persscae 141 F. Bollhalder and M. Zuber Comparison between NeemAzai-T/S and Syringa extracts: 147 Laboratory trials A.M. van den Berg and A. van der Schyf

6 Effects of NeemÄzai-T, NeemAzai-T/S and Delfin on the grape berry moth (Lobes/a botrana) and the predatory mite (Typhlodromus pyri) 151 J. Erbach and H. Hoist Mosquito control by NeemAzai - a pilot study in the Seychelles 155 P. Schröder and M. Kahnert NeemAzal-T/S against Musca domestica 161 M. Zuber and F. BoIIhaider Effects of NeemAzaS-containsng diet on the cockroach Periplaneta americana (L.) 165 G.-A. Böhm, K. Richter and K. Kleeberg Einsatz von Neem als Wachstumshemmer 175 A. Wudtke Effect of NeemAzal-T/S on the anthocorsd predator Orius majusculus Reuter 177 K. Drescher and G. Madel Effects of different NeemAzal-formuIations on larvae of the green lacewing Chrysoperla carn&a Steph. (Neuroptera, Chrysopidae) in laboratory and semi-field 183 Petra Hermann, C.P.W. Zebitz and Jutta Kienzie The effectiveness of plant oils as protectants of Mung bean Vigna radita against Callosohruchus chinensis infestation 189 M.H. Mansour Evaluation of certain Neem extracts against the tomato bug Cyrtopeltis tenuis (Reuter) 201 Nadia Z. Dimetry, A.A. Goma, A.A. Salem and A.S.H. Abd El-Moniem Effects of Neem on the 'banana-spotting bug" Amblyp&lta lutescens lutescens (Distant) (Hemiptera: Coreidae) in Australia 213 Ruth Huwer Effect of an aqueous Neem Seed Kernel powder extract (ANSKPE) and NeemAzal-F on the Liriomyza congesta (Becker) and Aphis craccivora (Koch) infesting broad bean at Zagazig region, Sharkia Governorate, Egypt 223 Shadia M. Omara, I.M. Kelany and H. Kleeberg

7 The effect of Neem-based products on bacterial and funga! growth 237 Emma Coventry and E. J. Allan Tissue culture of Aiadirachta indica (A. Juss) and its potential for the production of Azadirachtin 243 Antje Wewetzer Azadirachtin, saiannin and nlmbfn contents of seeds and callus of Neem (Azadirachta indica A. Juss) 251 Janakie P. Eeswara, A. Jennifer Mordue (Luntz), T. Stuchbury and E. J. Allan Inhibition of Vitellogenin and sex pheromone production in females of the Colorado potato beetle, sterilized by Neem ingredients (Azadirachtins) 263 D. Otto Chemical structure and application of sex pheromone of cotton bollworm in China 269 D. Wu } YYanandJ. Cui BMBF-project: Determination of attractants and pheromones of storage and material pests and their enzymatic production from vegetable and animal raw materials 5 " 273 Brigitte Trevin and Ch. Reichmuth

8 Welcome address Over the almost 30 years of Neem research in Germany activities may be assigned to three different phases. Except of the latest, all phases are linked with the person of Prof. Dr. H. Schmutterer at the Justus-liebig-University in Giessen. The beginning of Neem research in Germany was characterized by more or less solitary studies expressing the explorative character of this phase. However, the crude Neem extracts used in these studies exerted a remarkable efficiency against major pest insects recommending Neem insecticides, at least theoretically, as an appropriate pest control technology for small holding farmers in the tropics. Pushed by this scope, the first phase was soon taken over by a phase of highest research activity with the major effort to elaborate Neem pesticides and a Neem technology for the developing countries. Contemporaneously, growing interest in Neem reseach could also be seen in the basic sciences introducing more and more Neem-adepts" from different siences. The successful work during this phase made Neem also very popular with non-scientists, forced by newspapers, TV or radio, sometimes in a manner more spectacular rather than really informative. Thus, more and more questions arose to develop Neem produchts also for the German or European market, however, they left unheard for some time. Perhaps due to the retirement of some of the most active Neem scientists like Prof. Schmutterer and Prof. Rembold or due to a decreased interest in the public, a short quiet interphase with a strongly reduced Neem research led into the recent third phase which may be entiteld as the phase of Neem transfer to European farmers. This phase is strongly characterized by the efforts of private industries to step into the application for registration of a licenced Neem product and a tremendously growing interst of farmers in Neem products. These Neem products seem to be a welcome complementation of other nonpesticidal control methods in agriculture. And, thus, it is perhaps no accident that one of the driving forces of the third phase of Neem research, Dr. Hubertus Kleeberg, invented the Workshops on practice oriented results on use and production of Neem ingredients and pheromones" which celebrates its 5th anniversary this year. Thus, it is a great honour to me to welcome you to this meeting, dealing with, more than ever, the practical aspects of Neem applications in different crops, not neglecting also the aspechts of official restistration. I wish all of you a pleasant stay and a very fruitful workshop! Wetzlar, Jan. 22, 1996 Claus P. W. Zebitz

9 Hubertus Kleeberg Trifoiio-M GmbH Sonnenstr. 22, D Lahnau Development of NeemAzal During the past 8 years considerable progress was made in the extraction and fonnulation technology and with the analytical determination and standardisation of products based on different parts of the tropical Neem tree (Azadirachta indica A. Juss). We have concentrated our efforts on the development of Neem-products which may be used for the control of insects. For the different possible areas of application like plant protection, control of ectoparasites, protection of stored products, different formulations have to be developed. In table 1 the number of separate investigations for the development of different products are summarized. Improvements of the extraction process and the analytical tools are a never ending task in order to make the production process more efficient and increase our knowledge about the obtained extracts. For the standardisation of the active ingredient a.i. - which we have termed NeemAzal in order to stress its origin from the Neem tree as well as the major group of active substances, the Azadirachtins - an improvement of the existing analytical methods was necessary (1,2). In the course of the investigations, isolation and purification procedures of several compounds had to be developed in order to have standard substances of appropriate purity readily available. The different formulations had to be investigated mainly with respect to the content of a.i. and their storage stability, knowledge which is indispensable for a proper standardisation. Other physico-chemical studies include melting point, inflamability, spectroscopic properties, degradation in different compartments of the environment under different conditions. All the analytical or biological results concerned with the degradation of the a.i. show that it is very easily degraded. For those who want to use NeemAzal-fonnulations its efficacy surely is the most important factor. After intensive testing of the first formulation called NeemAzal- F we had to abandon the major fonnulation additive since it did not correspond to our toxicological and ecotoxicological demands. Practice Oriented Results on Use and Production of Neem-lngredients and Pheromones V H. Kleeberg & C.P.W. Zebitz (eds.) Copyright 1997 by Trifolio-M GmbH

10 Table 1: Number of investigations for our development of the active ingredient a.i. NeemAzal and different NeemAzal-formulations till Physico-chemical investigations: development of: extraction: analytics: characterization of the a.i. characterization of formulations others (like residue analysis, degradation in water, soil etc.) 5 years 8 years 25 investig 40 " 15 " a.i. Efficacy: Ecotoxicity: 6 Beneficials: laboratory test: field: Toxicity: 22 Phytotoxicity: NA-F NA-T NA-T/S 16^ number of investig total: 593 In this respect the fonnulation NeemAzal-T performed much better, however, soon first indications were found by Kienzle and Schulz (3) that the combination of this fonnulation with plant oil increased the biological activity considerably. Eventually plant oil was included in the fonnulation NeemAzal-T/S in addition to surfactants, which are produced from renewable resources. Investigations on the estimation of ecotoxicological risks concentrated on NeemAzal-T/S. Apart from effects on aquatic organisms (like algae, daphnia, fish) and earthworms (4) the possible influence of the product on beneficial insects was studied most intensively. -2-

11 Toxicological studies were carried out at least according to the requirements which are necessary for authorisation. They include many acute studies (see table 2) and several long term studies which make the estimation of possible effects on mutagenicity, carcinogenicity, reproduction possible. From all these studies it becomes clear, that practically no risk is involved in the contact with NeemAzal- T/S during preparation of the spraying solution, its application or the consumption of treated matter. In cases of skin irritation the undiluted formulation may be washed off with water. Surprisingly enough it turned out, that some plants show adverse effects after treatment with NeemAzal-T/S. Hence the influence of NeemAzal-T/S on the health of plants was studied. Especially some varieties of pears react very sensibly; even contact with small amounts of product may lead to phytotoxic effects (5). Other varieties of pears react indifferently. Fortunately this effect seems to be limited to pear-trees. In some cases flowers of ornamental plants may show brown spots to some degree (6). It can be concluded that NeemAzal-T/S should not be used in the flowering stage and precautions have to be taken that Neem-sensible pear trees are not treated (accidentally) with the product. Table 2: Summary of acute tokicological results of NeemAzal and NeemAzal-T/S (LD50-values in: mg/kg body weight). acute oral, rat acute oral, mouse acute dermal, rabbit acute inhalative, rat prim, skin irritation, rabbit primary eye irritation, rabbit skin sensitization NeemAzal (acitve ingredient) > >3365 >3000 > 0.72 mg/1 not irritant slight, reversible slight sensitization NeemAzal-T/S (formulation) > > > > 5.4 mg/1 slight, reversible not irritant no sensitization -3-

12 It is obvious that all these developments can not be borne and carried out by a small company alone. Without the financial support of the Gemian Ministry of Education and Research (BMBF) and the German Foundation for Environment (DBU) the project would not have developed in this manner. However, even this support would not have achieved these results if not a large number of excellent scientists had put tremendous energy into the research, improvement and application of the product (see acknowledgement). As a result of these efforts in different countries (like China, Egypt, France, Germany, India, Iran, Italy, The Netherlands, Russia, Sudan, Switzerland, Turkey, United Arab Emirates, United Kingdom) we have a considerable knowledge about the effects of NeeinAzal-formulations on pests like moths, aphids, beetles, white flies, bugs, mites and lice. This has led to several registrations of NeeinAzal-formulations for plant protection in orchards, vegetables, potatoes, ornamentals, forests, green houses and home & garden. However, still a lot of work has to be done like for example the optimisation of the application for the protection of stored products or the control of ectoparasites. It is generally agreed that the unique insectistatic effect of the Neem ingredients, which leads to feeding inhibition, inactivity and molting inhibition as well as fecundity reduction of the target insects, and its very rapid degradation under all conditions except in the fomuilated product are an optimum combination for pest control Furthermore the development of resistancies can not be expected and its safety to the environment has been proven. Thus we have well founded arguments for saying that NeemAzal-formillations fulfil even the most serious requirements! Acknowledgement: The author wants to thank the following persons for their very pleasant cooperation, continous efforts and personal encouragement: ML Andermatt, T. Basedow, S.A. Bilgrami, S. Brass, N. Dimetry, R. Gottwald, C. Hainmes, C. Hapke, M. Henrich, P.Hermann, H. Hoist, E. Hummel, S. Izhevski, I. Kelany, E.G. Khorkliordin, J. Kienzle, J. Kleeberg, C. Kliche-Spory, B.G. Kovalev, G. Madel, M.H. Mansour, L. Mayr, M. Mironova, V. Micheletti, C. Nicole, S. Oroumchi, D: Otto, S. Pillai, G. Rabindranath Rao, R. Rajan, S. Rajendirane, K. Raman, L.Rovesti, B. Ruch, I. Schäfer, A. Schlicht, C. Schulz, S. Schütz, R. Senrayan, D. Sreenivasa Rao, S. Subbiah, R. Troß, C.P.W. Zebitz, M. Zuber

13 1.) H. Kleeberg, S. Schlicht and P. Berg, Analytical Determination of Azadirachtin A and Azadirachtin B in Neem-Extracts in: Practic e oriented results on use and production of Neem-ingredients 4 n d pheromones (H. Kleeberg; ed), Proc. of 3. workshop, Druck & Graphik, Giessen (1994) i i 2.) R. Troß.NeemAzal and Analysis in: Prajctice oriented results on use and production of Neem-ingredients and pheromones (H. Kleeberg and C.P.W. Zebitz, eds.), Proc. of 5. workshop, Dmck & Cjiraphik, Giessen (1997)!I 1 3.) J. Kienzle, C. Schulz, personal information 1(1994) i i 4.) B. Ruch, et al., Summary of some environmental aspects of the Neem ingredient NeeinAzal and NeemAzal-T/S, in: Practice oriented results on use and production of Neem-ingredients and pheroinjones (H. Kleeberg and C.P.W. Zebitz, eds.), Proc. of 5. workshop, Druck & Graphik, Giessen (1997) 5.) M. Zuber, C. Schulz, J. Kienzle, personal information (1996) i I i 6.) E. Hummel, personal information (1995) '; -5-

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15 Neem and GLP: Good Laboratory Practice Christine Kliche-Spory, Hubertus Kleeberg, Beate Ruch, Ilona Schäfer, Anette Schlicht, Ralf Troß; Trifolio-M GmbH, Sonnenstr. 22, D-3!5633 Lahnau Abstract: Good Laboratory Practice" is a type of Quality Assurance System. In the European Community the principles of GLP have to be followed for experiments that are necessary for the registration of chemicals, drugs and pesticides. Since 1995 the analytical laboratory of Trifolio-M GmbH works according to GLP conditions Introduction Good Laboratory Practice contains instructions for an organisation on how to plan, carry out and document tests. The principles of GLP also fix t e extent of the control of the test facility by authorities. The aim of GLP is - to guarantee the high quality of all activities related to the respective tests. - to perform tests according to guidelines (like OECD, BBA: Biologische Bundesanstalt (Germany)). - to obtain test results that are rejproducible and can be verified by the authorities. - to produce test results that are In the European Community the use of the principli of GLP are laid down for experiments that are necessary for the registration of chemicals, < aigs and pesticides. The type of tests to be carried out under GLP-conditions are toxicologica and environmental (toxicol., ecotoxicol., phys.-chem., analytical,...) studies. Realisation of GLP at Trifolio-M accepted internationally We work on the registration of NeemAzal T/S as a plant protection agent. For that purpose GLP was realised in the analytical department of the company, because the prerequisites for the authorisation contain many tests that include anal) tical research. The most important elements of GLP can be divided ito 4 parts - the reliable quality of the test ft cility - the quality of the tests at the hi, hest possible level - the quality assurance unit (qau) - the archiving The management of the test facility is responsible for the quality of the test facility. This means for example, that the management has to assure, tha 1 an appropriate number of personal with up to date qualification is available. Necessary staif according to GLP is: the test facility manager, the study director, the quality assurance nanager and also the archivist. The last three positions need to be twice occupied. Practice Oriented Results on Use and Production of Neem-lngredlents and Pheromones V H. Kleeberg & C.P.W. Zebitz (eds.) Copyright 1997 by Trifo!io-M GmbH

16 If working steps in the GLP department can be standardised, the principles of GLP lay down to The study director is responsible to perform the tests. He has to guarantee to work according to the principles of GLP. The QAU is responsible for control measures, if the studies are carried out according to GLP. The original documents of tests and samples of test materials have to be archived. write standard operating procedures (SOP). They are required for the structure and organisation of the test facility, the equipment and its maintenance, for handling and calibration of test systems, waste utilisation, the quality assurance system and the documentation and archiving. Apart from the production of lots of paper these procedures help us to structurise our organisation and standardise the quality of our procedures and products. The first step in carrying out a study is to work out the study plan (by study director). The plan must fulfill formal requirements and minimum details about guidelines, testing materials, equipment and other. The accuracy of this plan is checked by the qau. If the accuracy of the study plan is confirmed, the study director, the qau and the testing facility manager authorise the plan by their signature. The original of the plan is archived. In the next step the study is carried out. During this time the qau controls, whether the study is performed according to the study plan, the sops and the principles of GLP. After finishing the study the study director produces the final report. The QAU checks the report, especially compares the raw data of the test with the results put down in the final report. The principles of GLP assure the comprehension of the tests for a long time. So all documents as the originals of SOPs, study plans, final reports, QA-reports and other documents, as for example handbooks, have to be archived for 30 years. Samples of testing materials are kept as long as they can be kept well. Practical Consequences of Quality Control The importance of quality control measures can be illustrated by the changes of the purity of neem ingredients. It has been described 4 years ago l that the quality of "pure" Azadirachtin A from four different companies varied between 39 and 96 % purity (specified purity of > 90 resp. 95 or 96 %). A recent study including Azadirachtin A from the same four companies shows a purity of > 95 % of all Azadirachtin A supplied. This example shows that the general introduction (not only of GLP) of quality control guidelines has direct consequences in practice. It becomes clear, that the routinely determined quality of standards used is a necessary prerequisite for the development and production of products with "standardised quality". This is especially true for natural products where rawmaterials may vary considerably. Conclusion Finally, the introduction of GLP changed a lot in the analytical facility of Trifolio-M We are convinced that we gained numerous advantages by GLP. GLP helps us to avoid mistakes. We now have more certainty about the reproducibility of the results and profit from an uninterrupted documentation. The areas of responsibility of every member of staff are now clearly defined and routine work is performed with greater efficiency. Reference: 1 H. Kleeberg, Lecture presented at the World Neem Conf, Bangalore (India): Feb. 1993; in press. -8-

17 Neem and Ana lytics Ralf Troß Trifolio-M GmbH, Sonnenstr. 22, D Lahnau Nowadays analytics plays an important role in the development of a plant protection agent, especially for those of botanical sources like the Neeir Tree. It is a non replaceable tool for the characterisation and the standardisation of such products. Another important function of analytics is the investigation of the persistence of i pesticide in the environment and the determination of residues. This is of great importance pr the risk estimation of a pesticide. Therefore the main tasks of analytic can be described Ipy the following three points: 1) Characterisation of the active ingredient(s) 2) Standardisation of the active ingredient(s) and the f srmulation 3) Residue analysis In the case of a botanical pesticide these responsibilities mean a great demand for the analytical methods. The requirements are much higher than they are for a chemical product, where the active ingredient usually is only one defined compound. A botanical pesticide on the other hand represents a mixture of different compounds, with possibly different activities. A complete analytical determination of all components is more ore less impossible, this is especially true for the investigation of their environmental persistence and their residual behaviour. These problems will be discussed relating to NeemAzal, an extract of seed kernels of the Neem Tree. NeemAzal it is a complex mixture of components, wich can not be defined exactly. For the characterisation and the standardisation of Neemi^zal it was desirable for us to determine the main components and some minor if possible. In he case of residue analysis it turned out to be impossible. Hence the analysis of one or a few eading compounds as representatives of the complete extract can be a feasible approach. For NeemAzal and its formulations we selected Azadirachtin A as the leading compound, which with an average content of 34 % represents the main component of the extract and furthermore is one of the most active ingredients. That means residue analysis regarding tc NeemAzal and its formulations can be carried out by the determination of Azadirachtin A. Practice Oriented Results on Use and Production of Noem-lngredients and Pheromones V H. Kleeberg & C.P.W. Zebitz (eds.) Copyright 1997 by Trifolio-M GmbH - 9 -

18 HPLC (High-Performance Liquid Chromatography) is the analytical method of choice as NeemAzal contains temperature labile compounds of high molecular weights,. Our target is to quantify Azadirachtin A in a quick, reliable and simple way. If possible the method should additionally allow the determination and quantification of analogues of Azadirachtin A, nimbin and salannin. However, this depends on the availability of the appropriate, well characterised standards. The main ingredients in Neem extracts can be divided into two groups compared to their relative polarity: - the more polar ones: - the more unpolar ones: members of the azadirachtin group and their analogues (azadirachtols and meliacarpins) members of the nimbin- and the salannin group To analyse all possible components by a one step method in an acceptable time, a gradient elution program is required. Additionally the resolution of the target compounds must be sufficient for a reliable quantification. We obtained the best results with RP-columns and a solution of acetonitrile and water as eluent. We developed a gradient program which allows us to determine the polar and unpolar components in one step. The fundamental requirements for the procedure are the following: HPLC-equipment with two pumps for high-pressure gradient elution UV-Detector, with detection at a wavelength of X = 214 nm reversed-phase column, packed with 5 \xn\ particles of octadecyl silica (RP-C18) and the dimension of 250 x 4 mm I.D. guard column with the same packing material, dimension : 5 x 4 mm I.D. Mobile Phase (eluent): eluent A: acetonitrile : water (38 : 62, v/v) eluent B: acetonitrile gradient setting: time [hh.min.ss] 00:00:00 00:02:30 00:06:00 00:09:30 00:13:30 flow [ml/rain] 1,20 % eluent A % eluent B

19 For the HPLC identification of the individual compounds in a sample one has to compare the chromatogram of the sample with those of standarcj solutions of each substance of interest under the same conditions. Therefore we isolated, uf to now, 7 components of NeemAzal by preparative HPLC up to a purity of about 95%. The substances are the following: Azadirachtin A Azadirachtin B Azadirachtin H Nimbin Salannin Desacetyl-Nimbin pesacetyl-salannin We can quantify these 7 compounds in NeemAzal. The others that are hidden behind the peaks in the HPLC-chromatogram (Fig. 1) of NeemAzal are partly unknown or we do not have enough standard of sufficient purity for a quantitative determination Azadirachtin A " CO X Lü i U! 8 3 V V 1 'S t i me m l n s? 1 a <s> CNJ Ö tx) cn Fig.l Chromatogram of NeemAzal»11 -

20 For the standardisation of NeemAzal we analysed several batches of it. The results of 9 batches are shown in the following table: Azadirachtin A Azadirachtin B Azadirachtin H Desacetyl-Nimbin Desacetyl-Salannin Nimbin Salannin Tab. 1: assay data of 9 batches of NeemAzal average- content 35,8 % 5,6 % 2,4 % 0,5 % 0,7 % 0,8 % 3,0 % 49,3 % With respect to the analogues of Azadirachtin A we can now assign some more peaks of the chromatogram (Fig. 2). Azadirachtin A 400" 300" region of Azadirachtin K J t i'me, min : 200- Fig. 2: Chromatogram section of NeemAzal. For comparison purposes we obtained pure Azadirachtin I and Azadirachtinin samples from T.R. Govindacharie and E.D. Morgan. The double peak at min was isolated by preparative HPLC and the NMR-data indicates Azadirachtin D as the main component. A patent specification of K Rembold (1) led us to assign Azadirachtin F to the second peak. Comparing the results of Govindacharie (2) with Fig. 2 it can be shown that Azadirachtin K is in the region between Azadirachtin B and Azadirachtinin. -12-

21 An interesting example for residue analysis is reported in the following: The analysis of honeydew from aphids fahae, treated with NeemAza.-T/S. Honeydew of about 1500 aphids was collected by C Hapke (University of Hohenheim) over a period of two days. Honeydew of aphids from untreated plants (control) and from aphids on plants treated with NeemAzal-T/S (50 ppm Azadirachtin A) was dissolved in methanol/water. Prior to HPLC analysis the solutions were concentrated to a volume of 1 ml by methanol evaporation. The results of the subsequent analysis ai]e: The chromatograms of both solutions show a small beak in the region of Azadirachtin A and Azadirachtin B. A comparison of both chromatograms with those of standard solutions of Azadirachtin A and Azadirachtin B give rise to the conclusion that this peak represents neither Azadirachtin A nor Azadirachtin B. This is confirmed by the analysis of a mixture of the treated honeydew sample and a standard solution of Azadirachtin A and Azadirachtin B. What is the implication of this result? Azadirachtin A could not be found analytically in the honeydew of aphids fabae on plants treated with NeemAzal-T/S. This doesn't rule ou the presnce of Azadirachtin A in the honeydew, but it shows that the concentration of Az dirachtin A is below the detection limit of the analytical method. The detection limit for Azadirachtin A of the method is 0? 3 ng/ml, wich corresponds to a content of Azadirachtin A in the honeydew of the tre; ted aphids of less than 0,3 \xg in total, Unfortunately it is difficult to obtain information on the amount of analysed honeydew. If we assume that an aphid produces 0,08 \i\ -1 \A honeydew per hour (3), 1500 aphids will then produce 120 pi \x\ honeydew per hour and after 2 days they will produce 5,8 ml - 72 ml honeydew. If the density of the honeydew is assumed to be 1 g/ml, one can estimate that the honeydew of the aphids from plants treated with NeemAzal-T/S contain less than 52 ppb Azadirachtin A (at the lowest assumed volume of 5,8 ml).

22 Conclusion The presented analytical method is very useful for the characterisation and standardisation of NeemAzal. For a more extensive characterisation further compounds of NeemAzal may be isolated and characterised. In the case of residue analysis, better sample preparation methods (i.e. solid phase extraction for example) have to be used in order to obtain lower detection limits, especially in view of the requirements for the registration of a pesticide by the authorities. References (1) H. Rembold, H. Forst er; Deutsches Patentamt, Offenlegungsschrift DE AI. (2) T.R. Govindachari, G. Sandhya, S.P. Ganesh Raj; Indian Journal of Chemistry, Vol. 3 IB, (1992). (3) Personal information of C. Hapke, Hohnheim.

23 Summary of Some Environmental Aspects of the Neem Ingredient NeemAzal and NeemAzaI~T/S Beate Ruch, Christine Kliche-Spory, Annette Schlicht, Ilona Schäfer, Jenny Kleeberg, Ralf Troß and Hubertus Kleeberg Trifolio-M GmbH, Sonnenstr. 22, D Lahnai), Germany Different toxicological and exotoxicological aspects have been studied for many different, usually ill-defined Neem-extracts 0 A reliable risk-estimation can only be possible for material which is well characterised and available in standardised quality. The risk estimation of a plant protective agent is part of the authorisation. For authorisation of a plant protective agent the applicant has to submit the results of extensive studies to the federal authorities. In Germany the responsible authority is the BBA" (Federal Biological Research Centre for Agriculture and Forestry). The experiments required for registration by the BBA include the following topics: Chemical and physical properties Residue analysis Efficacy Toxicology Environmental studies In the following a few aspects and results of the jenvironmental studies are shown. The environmental compartments of interest are water and soil. Air is negligible, because of a very low vapour pressure of NeemAzal. The describe^ tests were performed at BioChem (Karlsaihe), IF AH (Konstanz), IPSAB (Münster) and jtrifolio-m. Analytical investigations of the environmental aspects Concentrate on Azadirachtin A since this is the most abundant (ca. 34 % per kg weight) single chemical compound of NeemAzal and thus contributes most to the bioefficacy of this active ingredient (a.i.). Several other Azadirachtins (a group of tetranortriterpenoides typicafl for Neem) are present in NeemAzal at below 1 or a few % each. WATER In the direct phototransformation studies the quantum yield O which is the ratio of reacting molecules and absorbed photons are determined. From! the quantum yield the half life time ty a is calculated. The t/ a gives information with respect to the degradation by light. During this test (4 h) the ph was constant and therefore an influence o^ hydrolytically induced degradation can be taken into account appropriately. The estimated X Vl j of 6.5 mg/1 NeemAzal is 9.85 h and is below the critical value of the guideline which is 4\ days. This rapid degradation by light reduces a possible bioaccumulation of NeemAzal. The experiment - n-octanol/water distribution coefficient - is a model which may be used to describe the transfer of a substance from the aquatic environment into an organism and the potential bioaccumulation of the tested substance. This is of great interest for the estimation of (critical) concentrations of a.i. in the food chain. The result is given as the logarithm of the distribution coefficient and it describes the ratio of the Azadirachtin A concentration in n-octanol to the Azadirachtin A concentration in water. For NeemAzal the log p ow is determined to be According to the BBA guidelines a log p ow below 3 makes the authorisation more simple since a critical bioaccumulation can be excluded. Practice Oriented Results on Use and Production of Noem-lngredients and Pheromones V H. Kleeberg & C.P.W. Zebitz (eds.) Copyright 1997 by Trifolio-M GmbH -15-»

24 The next four tests consider aquatic organisms. Table 1 Water Test Result Guideline Direct photo-transformation study in purified water (NeemAzal) OECD-Draft Test Guideline (part A) n-octanol/water distribution coefficient (NeemAzal) OEDC Guideline 107 Side effect on aquatic organisms - Algae (NeemAzal-T/S) OECD Guideline Daphnia (Immobilisation test) (NeemAzal-T/S) OECD Guideline 202 Quantum yield O t vi = 9.85 h log p ow = 0.95 NOEC (No Observed Effect Concentration) NOEC = 22 mg /I 48 h EC 0 = mg/1 EC 50 = 1000 mg/1 t vz < 4 d log p ow < 3 NOEC > 1 mg/1 EC 50 >10mg/l - Rainbow Trout (Oncorhynchus mykiss) (Acute toxicity test; NeemAzal-T/S) OECD Guideline Brachydanio rerio (Acute toxicity test; NeemAzal) 96 h NOEC = 50 mg/1 LCo = 50 mg/1 LC 50 = 150 mg/1 LC 0 > 500 mg/1 NOEC > 1 mg/1 LC 50 > 10 mg/1 Algae were permanently illuminated for 72 h and none of the tested concentrations (up to 2500 mg/1) showed an inhibition or a stimulation of the growth rate. The NOEC (no observed effect concentration) is well above the critical value for authorisation. The Daphnia were also exposed to NeemAza!-T/S and afterwards the number of Daphnia which were unable to move were determined. The EC50 of the immobilisation test of the Daphnia is 100 times above the guidance value of the BBA. For the estimation of possible toxic effects of the formulation NeemAzal-T/S and the active ingredient NeemAzal towards Fish, acute toxicity tests were performed. In both cases the NOEC and the lethal concentration LC50 are far above the critical values. The comparison of test results between formulation and a.i. show that influences are mainly due to the formulation additives (like vegetable oils). From these results we can conclude that the large scale application of NeemAzal-T/S, even close to aquatic systems may be regarded as safe since no signs for an impact on water and fish are found.»16-

25 SOIL The registration of a plant protective agent also requires test reports about the behaviour in the soil. j Adsorption/Desorption data are necessary for the ^valuation of the migratory tendency of substances in different soils. The experiment gives information about the decrease in concentration when aqueous solutions of NeemAzal-it/S are in contact with different specified soils. In table 2 the amount of adsorped material is givfen in per cent for each soil. Knowledge of the leaching activity of a plant protective agent enables us to estimate the risks of a possible ground water contamination. In this lab experiment glas columns were filled with 3 different soils and aqueous NeemAzal-T/S solutions were applied on each surface. Subsequently the soils were watered with an amount of water equivalent to 200 mm rainfall. 200 mm rain correspond to V4 of the annual rain fall according to the degree of latitude in Middle Europe. The concentration of Azadirachtin A in the seepage water was analysed. The results (see Table 2) depend on the type of soil and $how that between 10% and 58% of the test substance remain in the soil in this test. j For the interpretation of the fate of NeemAzal-T/S in the soil we have to study a further experiment which is the degradation of NeemAzal-T/S in the soil. This test is based on a standard type soil which was treated with a solution of NeemAzal-T/S (a 20fold amount of the concentration used for pest control purposes) and t\\e concentration of Azadirachtin A was analysed. The BBA guideline requires the DT90-val ie which is the time when 90% of the concentration of the active ingredient is degraded in the soil. The BBA considers authorisation without restrictions or further experiments up t6 a DT90-value of 100 days.. The experimentally determined DT90-value for NeemAzal is 12.5 days. Table 2. - Soil - Test Results Remarks Adsorption/Desorption (NeemAzal-T/S) OECD Guideline 106 Leaching activity (Column method) (NeemAzal-T/S) BBA Guideline, part IV, 4-2 Degradation in soil (NeemAzal) BBA Guideline, part IV, * soil 1: sandy soil * soil 2: humic sand * soil 3: loamy sand A: adsorbed material soul*- A =j 7.5% soil 2*»A =(8.5% soil 3* -A== 7% concentration in seepage water soil 1* soil 2* soil 3* soil 2* 90% 55% 42% DT: disappearance time -17- no desorption test, because A < 25% DT90 < 100 d

26 But what may happen with the remaining NeemAzal-T/S which is seeping through the soil? To answer this question we have to look at the stability of the a.i. at different ph-values. Therefore tm of the leading component Azadirachtin A at 3 different phs at 30 C and 40 C were determined (see Table 3). Table 3 t. m of NeemAzal-T/S at different ph-values Temperature 30 C 30 C 30 C 40 C 40 C 40 C ph tl/2 236 h 105 h 21 h 60 h 27 h 5h These results show that degradation in water is comparatively rapid. The degradation increases with increasing ph. For ambient temperatures tm may be estimated to be of the order of some days. Side effects on earthworms (Eisenia fetida) Earthworms are important soil organisms. They contribute essentially to the areation and mixing of the soil and play a significant role in the decomposition of organic material. Therefore a study of the side effects of NeemAzal-T/S on earthworms has been performed (BBA draft guideline). Mortality: 10 earthworms were brought into contact with 5 different concentrations of NeemAzal-T/S in 500g of an artificial standard soil. Even at the highest concentration of 1000 mg NeemAzal- T/S per kg soil no mortality was observed and the earthworms showed no visible behavioural changes. As a reference substance for this test chloracetamide is used which shows a LD50 of 21.4 mg/kg soil. Weight development: Up to a concentration of 1000 mg NeemAzal-T/S per kg soil no negative effect on the weight development of adult earthworms has been recorded. Figure 1 Weight development of earthworms at different test concentrations concentration of NeemAzal-T/S (mg/kg soil)

27 As shown in the diagram (Figure 1), even an increase in weight is observed with increasing concentrations of NeemAzal-T/S. Tests with Neem leaves and Neem kernels incorporated in soil showed the same result (of a weight increase). Number of cocoons: No effects on the reproduction rate of adult earthworms Up to a concentration of 1000 mg NeemAzal-T/S per kg soil were observed.! The highest requested dose rate in the field is 3 1 NeemAzal-T/S per ha. With the assumption of a soil density of 1.5 and a penetration depth of 2.5 cm, ja theoretical concentration of 8 mg NeemAzal-T/S/kg soil may be expected. Since the the LD 5 j)-value was determined in the range of > 1000 mg NeemAzal-T/S/(kg soil) the formulation has to be classified as harmless to earthworms. Side effects on soil microflora Another test regarding the behaviour of the formulation NeemAzal-T/S in soil is the side effect on the soil microflora (BBA guideline IV 5 1-1). Two soils with different levels of microbial activity were treated with the highest requested concentration of NeemAzal-T/S which is applied in the field and the tenfold concentration. The effects of NeemAzal-T/S regarding the nitrogen-turnover and the dehydrogenase activity were determined in both soils after 3, 14 and 28 days after application. During this 28 day experiment NeemAzal-T/S had no significant influence on the nitrogen mineralisation in both soils. The dehydrogenase activity was also not affected. Thus NeemAzal-T/S represents no hazard to the activity of soil microflora. Degradation of Azadirachtin A The analytical method used to determine the degree of degradation of the formulation NeemAzal-T/S or the a.i. NeemAzal is HPLC. It is very difficult to quantify the degradation pathways of all ingredients of a NeemAzal. During our HPLC studies we found with the decreasing concentration of Azadirachtin A a new peak in the chromatogram, which is not clearly detectable in fresh Neem-seeds. I i Experimental data: column: RP-C-18 eluent: CH 3 CN/H 2 O detector: UV/214 nm gradient method Azadirachtin A cn c Qj Azadirachtinin Figure 2 0" 6 7 t i me, min Chromatogram of NeemAzal; region of Azadirachtin A and Azadirachtinin

28 The preparative isolation of this peak and identification through FAB-MS (Fast Atom Bombardment Mass Spectroscopy) and NMR (Nuclear Magnetic Resonance) shows that the first step for the degradation of Azadirachtin A is the isomerisation to Azadirachtinin. This evidence was supported by an Azadirachtinin standard supplied by Prof. Dr. D. Morgan (Keele University, England). COOMe OH MeOOC ""OH Azadirachtin A Azadirachtinin Figure 3 Rearrangement of Azadirachtin A to Azadirachtinin The epoxy group at Co, CM and the C7 hydroxyl group of Azadirachtin A undergo a rearrangement to Azadirachtinin. A point of interest is that Azadirachtinin also shows some biological activity 2). This gives us a clue to the observation of insectistatic effects of some Neem products with very low concentrations of Azadirachtins. Conclusions: NeemAzal can be regarded as an environmentally safe active ingredient for pest control. This view is supported by all available ecotoxicological tests summarized above. The fast degradation of the main component Azadirachtin A in water and soil shows that there is no hazard to aquatic organisms, no danger to groundwater and no influence on important microorganisms in the soil. References: 1) H. Schmutterer (ed.), The NeemTree, VCH Weinheim, Germany, ) S.V. Ley, E.D. Morgan et al, Tetrahedron 1989, 45 (16),

29 NeemAzal-T and WeemÄza!~T/ against the rosy apple aphid - field trials In Switzerland, Markus Zuber Andermatt BIOCONTROL AG, CH-6146 Grossdietwil Summary In 1995 Andermatt BIOCONTROL AG obtained a registration for the application of NeemAzal-T plus rape seed oil on a surface of 25 ha against the rosy apple aphid Dysaphis plantaginea. NeemAzal-T was compared to standard preparations for organic agriculture such as Pyrethrum HF and Parexan. The trials showed a much better effect of NeemAzai-T (plus rape s eed oil) against the rosy apple aphid than the standard products.there was also a very good effect of NeemAzal-T against Dysaphis devecta. Small scale field trials for precise field data with NeemAzal-T in full and half dosage and NeemAzal-T/S compared to Pyrethrum showed very good results with both NeemAzal-T in full concentration (0.1%, 50'000 ppm Azadirachtin/ha) and NeemAzal-T/5 (0.3%, 30'000 ppm Azadirachtin/ha). Further effects are discussed. 1. Introduction NeemAzal-T was introduced in Switzerland in The Swiss research institute for organic husbandry at Oberwil started with some very promising results with NeemAzal-T (0.1%, 50'000 ppm Azadirachtin/ha) plus rape seed oil (5 I/ha) against the rosy apple aphid Dysaphis plantaginea. These results confirmed the experiences made at Weinsberg (FRG) from In early 1995 NeemAzal-T was registered for large scale field trials on 25 ha of organic orchards in Switzerland. In adition to these trials small scale field trials were carried out by Andermatt BIOCONTROL AG in order to get more precise data on NeemAzal-T (0.1% or 0.05% plus rape seed oil) as well as on NeemAzal-L'S (0.3%; 30'000 ppm Azadirachtin/ha), the oily formulation, both products of Trifolio-M GrrjibH. 2. Large scale field trials 2.1 Material and methods Farmers who were taking part at these trials had to fulfill certain guidelines. Before treatment they had to check the relative attack by tie rosy apple aphid by counting 300 flower clusters (damage threshold: 3%). It was recommended to treat in early stage E. When treating with NeemAzal-T plus rape seed oil tley also had to treat with standard Practice Oriented Results on Use and Production of Neem-lngredients and Pheromones V H. Kleeberg & C.P.W. Zebitz (eds.) Copyright 1997 by Trifolio-M GmbH -21 -

30 preparations such as Parexan (Pyrethrine, Piperonylbutoxide + Rotenone) or Pyrethrum HF (Pyrethrine + Piperonylbutoxide). ii Ä A <m ^ Ä Ä A in A in m m m \x^> <&<$? t >y \+$p' \* $> \gp ^g*«pyrethrum HF or Parexan "buffer NeemAzal-T + rape seed oil Fig 1: Trial design for large scale field trials 2.2 Results NeemAzal-T (0.1% plus 0.5 % Agrirob) vs... - Parexan ( %): in 5 of 5 cases better results with NeemAzal-T against Dysaphis plantaginea - Pyrethrum (0.1 %): in 3 of 4 cases better results with NeemAzal-T against Dysaphis plantaginea in 2 of 3 cases better results with NeemAzal-T against Dysaphis devecta - Pirimor (0.1 %): (1 trial only) NeemAzal-T does not show the same efficacy as Pirimor against Dysaphis plantaginea but its effect is still quite satisfying. 3. Small scale field trials 3.1 Material and methods In order to get precise field data two small scale field trials were carried out in organic orchards with different concentrations and formulations of NeemAzai: NeemAzal-T (0.1 %) + rape seed oil (0.5%) NeemAzal-T (0.05%) + rape seed oil (0.5%) NeemAzal-T/S (0.3%) Pyrethrum HF (0.1%) untreated control -22»

31 pears O O O O O O O O O O O O O O O f Spartan O O O O O O O O O O! O * O O Q 0 0 Jdared , Boskoop O O O O O O O O O James Grieves O O O * O Ö O 0 O Jonagold O O J ^ O O O O O O O O O O O O O O O G Q Jdared Q Q/Q O O O O O O O O O O O O O O O O O Glocken O X > O O O O O O O O O O O O O O O D O O trial row (5 trees per replicate): 1: NeemAzai-T(0.1%) 2: NeemAza! T (0.05%) 3: NeemAza!-T/S (0.3%) 4: PyrethrumHF(0.1%) 5: control, untreated NeemAzai-T/5 Pig. 2: Trial design at Rosegg Treatments were carried out on aprii 24 at Herterenhof and on apri! 25 at Rosegg in the hollow ball stage (E2) just before blossoming. Before treating the number of the rosy apple aphids was assayed. At both places the relative attack had already passed the damage threshold. 3.2 Statistics: For assaying the results of the small scaie field trials two parameters were chosen: total number of colonies relative attack Grouping of colonies in order to the number of aph ds: A: 1-5 aphids B; 6-10 aphids C: aphids D: aphids E : > 75 aphids relative attack - (0,05m + O.lriB + O?ic /i/> + TIE) *100 number of infested twigs

32 3.3 Results The results were assayed 3 times after treatment. In the first two assays the relative attack was evaluated, in the last assay only the number of colonies and/or damages was counted. Herterenhof; rosy apple aphid Q _ 20 - NeemAzaH (0.1%) NeemAzal-T 1/2 (0.05%) NeemAzal-T/5 (0.3%) Pyrethrum HF control Fig. 3: Relative attack of the rosy apple aphid at Herterenhof (assayed 18 and 35 days respectively after treatment) KV o 150 o u ^ 125 H QJ " ioo H c , ^^ c o E <i.t <L E <y z: Fig 4: Number of colonies at Herterenhof (assayed 42 days after treatment)