INSECT REPELLING FOOD PACKAGI NG MATERIALS

The present iir ention concerns improved packaging mateπals that are insect repellent, non-tox.: to humans environmentally compatible and suitable for protecting food anc the like from insect mfestation

BACKGROUND OF THE INVENTION

Duπng packagm;, storage and distπbuuon. food packages are often exposed to a variety of conditions that bπng about insect infestation of the packaged food by penetration of insects through the packaging mateπal To prevent or repel such insect infestation, i: would be preferable to render the packaging mateπal impervious to insect penetranon This is preferable to treating the food itself, for a vaπety of reasons If the packaging mateπal itself is rendered impervious to insect penetration, less insect repellent material is required to create an effective barrier against insect penetration into the packaged food, when the insect repellents are concentrated in the packaging mateπal, relative to when the food itself is treated directly to achieve insect repellency Fi-rthermore, it is clearly a distinct advantage to avoid treating the food itself direct!) with insect repellents and to minimize the amount of insect repellents that is kxely to be absorbed by the food in order to achieve protection from infestation

insects

It should be pointed out however that most of the common insecticides in use todav, whether in agπculture or m domestic use, particularly the synthetic ones, are to a greater or lesser extent toxic to humans and animals, as well as hazardous or at least significantly cetπmental to the environment As a consequence, The search for naturalK occurππ: substances has become an impoπant aporoach m the development of ecciogically sound and compatible strategies for plant protection This stra_tegv seems :o be eminently suitable for adaptation in the food industry to facilitate the achie'. ment of effective pest and insect control Many known plants produce vaπous natural chemicals that protect them from insect attack. Extracts from such plants often are effective m controlling insects other than those that normally attack the said plant from which the extract was derived. The following is a list of vaπous publications dealing wit aspects of this subject. Anonymous (1992) Neem. A Tree for Solving Global Problems National Academy

Press, National Research Council, pp 141, Washington, DC Anonymous (1989) IMP User's Guide. First Pπnting SAS Institute Inc. pp.464,

Cary N C . USA Daniel, S H. and Smith, R.H (1990). The repellent effect of neem (Azadirachta indica A.Juss) oil and its residual efficacy against Callosobruchus maculatus (Coleoptera. Bruchidae) on cowpea. In Proceedings of the 5th International Working Conference on Stored-Prodacts Protection, eds F Fleurat-Lessard and P Ducom, Vol II, pp 1589-1596. Bordeaux, France, 1992 Highland, H A (1977) Chemical treatments and construction features used for insect resistance Package Development and Systems 13(3)251-256 Islam, B N (1986) Use of some extracts from Mehaceae and Annonaceae for control of Rice Hispa, Dicladispa armigera, and The Pulse Beetle, Callosobruchus chmen i In Proceedings of the 3th International Neem Conferance eds H. Schmutterer and K R S Ascher, pp 217-242 Nairobi, Kenya, 1986 Isman, M B , Koul, 0 , Luczynski, A and Kammski, J (1990) Insecticidal and Antifeedant Bioactivities of Neem Oils and Their Relationship to Azadirachtm Content Journal of Agricultural Food and Chemistry 38, 1406-1411 Jilani, G and Su. H C F (1983) Laboratory studies on several plant mateπals as insect repellents for protection of cereal grains Journal of Economic Entomology 76. 154-157 Jilani, G , Saxena, RC , and Reuda, B P (1988) Repellent and growth-inhibiting effects of turrnenc oil, sweetflag oil, neem oil, and Margosan-0 on red flour beetle (Coleoptera Tenebnonidae) Journal of Economic Entomology 81, 1226-1230 Jilani, G and Saxena, R.C (1990) Repellent and Feeding Deterrent Effects of Turmeπc Oil, Sweetflag Oil, Neem Oil, and a Neem-Based Insecticide Against Lesser Grain Borer (Coleoptera Bostrychidae) Journal of Economic Entomology 83, 629-634 Jotwani, M G and Smastava, K P (1984) A review of neem research m India in relation to insects In Proceedings of the 3th International Neem Conferance eds H Schmutterer and K R S Ascher, pp 43-56 Eschbom, Germany, 1983 Koul, O (1987) Antifeedant and growth inhibitory effects of calamus oil and neem oil on Spodoptera .itura under laboratory conditions Phytoparasitica, 15, 169- 180 Koul, O (1992) Neem allelochemicals and insect control In allelopathy, basic and Applied A pects ed

S J H Riavi and V Ria i, pp 389-412 Chapman & Hall,

London, 1992 Laudani, H., Davis. D.F. and Swank, G.R. (1955). A laboratory method of evaluating the repellency of treated paper to stored-product insects. Technical Associa_tion of the Pulp and Paper Industry 38, 336-341. Makanjuola, W. A. (1989). Evaluation of extracts of neem (Azadirachta indica A uss) for ±e control of some stored product pests. Journal of Stored Products Research 25, 231-237. Malik, M. M. and Naqvi, S. H. M. (1984). Screening of some indigenous plants as repellents or antifeedants for stored grain insects. Journal of Stored Products Research 20, 41-44. McDonald, L.L., Guy, R.H. and Spiers, R.D. (1970). Preliminary evaluation of new candidate materials as toxicants, repellents, and attractants against stored- product insects. USDA iVIktg Res. rep. 882, 8pp. Mordue, A.J., and Blackwell, A. (1993). Azadirachtin: an Update. Journal of Insect

Physiology 39_; 903-924. Navarro, S., Feriz . A.G., Dias, R., and Rinder, M. (1998). A device for testing resistance of packaging films to penetration by storage insects. (In press). Su, H.C.F., Horvay, R. and Jilani, G. (1982). Isolation, purification, and characterization of insect repellents from Curcuma longa L. Journal of Agricultural Food and Chemistr,' 30, 290-292.

Schmutterer, H. (1988). Potential of azadirachtin-containmg pesticides for integrated pest control ir. developing and industrialized countries. Journal of Insect

Physiology 34. "13-719. Subramanyam, B. and D W. Hagstrum. Resistance Measurement and Management. In Integrated Management of Insects in Stored Products ed.b_\ Subramanyam B. and Hagstrum D W.pp. 331-397. Marcel Dekker, Inc. New York

The relevant background information contained in these publications is incorporated herein, by reference

Over the last 25 years, intensive and pioneeπng research has been conducted on vaπous plant matenais including neem and its dεπvaπves, turmeπc and the like, etc As a consequence, the potential role of botanicals in the fields of antifeedants, repellents, toxicants and growth regulators has been established (Islam, 1986). Numerous plant substances have been isolated and tested on stored-product insects, and from among these, azadirachtin (extracted from neem) appears particularly promising as a potential stored-product protectant (Subramanyam and Hagstrum, 1996). Mixing πeεrr. extracts with other mateπals can boost their power. Among these so-called "promoters" are sesame oil, pyrethπns, and piperonyl butoxide

(Anon., 1992).

It has also been an age-old practice in rural India to mix dried neem leaves and turmeric powder wit stored gram or to place them among warm clothes to keep away insects (Jotwani and Srivastava, 1984, Koul, 1992). In some traditional storage practices, neem leaves are mixed with grain m storage for 3-6 months (Anon., 1992). Azadirachtin, alcohohc and aqueous extracts of neem seeds, and enriched formulations have revealed virtually no oral or dermal toxicity to mammals according to all tests earned out so far Neem flowers and leaves are even eaten as a vegetable in India. Burma and Thailand (Schmutterer, 1988) Ceπam neem products may even benefit human health The seeds and leaves contain compounds with demonstrated antiseptic, anti viral, and anti fungal activity There are also hints that neem has anti inflammatory, hypotεnistive, and anti-ulcer effects Also, tests in

Germany have proven that neem extracts prevent tooth decay, and neem is now used as the active ingredient in certain popular toothpastes in Germany and India (Anon ,

1992)

Therefore, the lack of acute toxicity in laboratory animals (oral LD₅₀ in rats >5000 mg'kg) and lack of evidence for chrome effects m animals, combined with a long histoπcal use of neem preparations in traditional medicine in India, should make any neem-based formulation highly acceptable as an alternative to the widely prevailing synthetic neurotoxin-type insecticides (Isman et al , 1990)

Turmeπc, Curcuma longa L , is a tropical herb of the Zingiberaceae family indigenous to southern Asia The aromatic yellow powder from its mature rhizomes was used in Asian countπes for many centuries as a yellow vegetable dye for silks and cottons It is still used in foods as a condiment, particularly as an essential ingredient of curry powder, m medicine as a stomachic, carmmativ e, anthelmmtic, laxative, and cure for liver ailment, and also as an ant repellent in India (Su et al , 1982) Jilani et al . (1988) have stated that turmeπc oil not only repels Trώυlium castaneum but also interfere with its normal reproduction and development

It is an object of certain aspects of the present invention to provide improved insect repelling food packaging mateπals It is a further object of certain aspects of the present invention to provide insect repelling food packaging mateπals that are non toxic to humans and animals

It is a further object of ceπain aspects of the present invention to provide improved insect repelling food packaging mateπals treated with insect repelling substances or combination of substances and optionally containing in addition appropπate non toxic synergists and insect repellent promoters

It is a further object of certain aspects of the present invention to provide improved insect repelling food packaging mateπals treated with natural non-toxic insect repelling substances or combination of substances and optionally containing in addition appropπate non toxic synergists and insect repellent promoters

It is yet a further object of certain aspects of the present invention to provide an improved method for protecting food from insect mfestanon by providing insect repelling food packaging mateπals treated with natural insect repelling substances or combination of such substances and optionally containing m addition appropπate non toxic synergists and insect repellent promoters

SUMMARY OF THE INVENTION

Thus there is provided m accordance with a preferred embodiment of the present invention an insect repelling food packaging mateπal compπsmg a food packaging mateπal treated with a non toxic to humans insect repelling substance or combination of substances and optionally containing in addition appropπate non toxic synergists and msec: repellent promoters

Neem seed extract can be mentioned as one example of such a non toxic to humans insect repelling substance Optimum range for treatment of packaging material with neem seed extracts has been found to be between 0.3% to 7.0%,, preferably between 5% to 6%

Turmeric extract can be mentioned as another example of such a non toxic to humans insect repelling substance. Optimum range for treatment of packaging mateπal with turmer.c extracts has been found to be between 5% to 40%,, preferably between 15% to 25%

Other suitable natural substances and combinations of substances can be considered as addraonal examples of such non toxic to humans insect repelling substances m accordance with additional preferred embodiments of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

Fuller details and aspects of the present invention will now be presented in the following experimental procedures and examples.

MATERIALS AND METHODS Repellents

The investigated Neem extracts were: NeemAzal T/S (1% azadirachtin Al) obtained from Eid Parry (India) Ltd., azadirachtin (30% puπty obtained from Trifoho-M GmbH as a NeemAzal powder)(Germany); cold pressed neem oil from seeds obtained from The House of Mistry Co. (England).

Extraction of turmeric: Powdered turmeπc rhizomes was extracted in a Soxhlet

extractor with petroleum ether (boiling point 40 to 60 °C) for 4 h Piperonyl butoxide (PBO)(90% technical grade) from laboratory stock was used to determine possible synergistic effect on repellency. In addition, natural pyrethrum extracts (50%) obtained from Yavnin Yave Industrial (Israel) Ltd. were tested for compaπson.

Insects

The test insects were adults of the lesser grain borer, Rhyzopertha domimca F. and the red flour beetle, Tribohum castaneum fHerbst.). Both species were reared on a mixture of broken wheat and 5% yeast (by weight). Cultures were kept at 27°C and 65% R.H. For the penetration test, only R. domimca was used. Adults that emerged at five day intervals were separated from rearing jars and were then placed in pre- treatment jars containing approximately 100 g of media until they were 10-15 d old. For repellency tests, emerging adults of both species were separated from rearing cultures at two-week intervals and were then held in pre-treatment jars as above until they were 7-21 d old.

Repellency test

The propensity of the tested extracts to repel insects (repellency test) was determined against T. castaneum and R. domimca adults using the method descπbed by Laudani et al. (1955) and McDonald et al. ( 1970). Filter papers (Filtrak 3 HW)( 10x20 cm) were treated with 4 ml of acetone solutions of the extracts at dosages of Turmeric at 800 μg/cm², NeemAzal T. S at 50 μg/cm² (containing 1% A.I. of azadirachtin), azadirachtin at 30% puπty, neem oil at 800 μg/cm², pyrethrum extract at 5 μg/cm² natural (50% A I ), and the mixtures of all the above with piperony 1 butoxide (PBO) at the dosage of 50 μg cm² Treated papers cut into 10x20 cm stπps were kept for 4 days m insect reaπng room Each treated stπp was attached lengthwise, edge to edge, to two untreated 5x20 cm stπps, to which acetone alone had been similarly applied, by Scotch tape on the reverse side Two glass πngs, 2 5 cm high and 6 4 cm i d , were then placed over the two matched papers so that the joined edges of the papers bisected the πngs Ten adults of each species, were then exposed separately on the test arenas side each glass πng, and their numbers on the treated and untreated halves were recorded after one hour and after eight hours exposure on the first day, and then at 9 a.m and 4 p m each day for 5 consecutive days. All tests were run at 27°C and 65% R H Each test was repeated four times The average of counts over each 5-day peπod was converted to percent repellency, as descπbed by Laudani et al. (1955) The mean repellency was then assigned a repellency class using the following scale

Percent Repellency class repellency

<0 1 0

0 1-20 I

20 1-40 II

40 1-60 in

60 1-80 IV

80 1-100 V Penetration test

Office paper (80 g m2)( 110 μm thick) was chosen as the test material after preliminary tests revealed its low resistance to penetration by R. dominica adults . The paper was cut into 28 mm diameter discs which were treated with 100 μl acetone solution of NeemAzal T/S, and azadirachtin, at dosages of 31.25, 62.5, 125,

250 and 500 μg/cm- ι A.I. azadirachtin), cold pressed neem oil from seeds at dosages

of 160, 320, 640, 1280, and 2,560 μg/cm², turmeric extract at dosages of 160, 320,

640, 1280, and 2,560 μg/cm², and natural pyrethrum extract (50% purity) at 2.5, 5,

10, 20, 40, 80, and 160 μg/cm² (A.I.). Paper discs treated only with 100 μl acetone served as control. After the solutions were applied to the paper discs, the acetone solvent was allowed to evaporate in the fumigation hood and the discs were then held at 27°C until used for bioassay. Penetration tests were carried out at delays of 1, 15, 30, 45, 60, and 75 days after treatment, or for as long as the treatment remained effective.

The device used for the penetration test consisted of two identical open-ended glass cylinders (24 mm i.d., 28 mm o.d., 26 mm height) each with four notches spaced at equal distances around the outer rim. The impregnated office paper, and a piece of wire-mesh (US standard No. 25) were cut into 28 mm diameter discs and were placed together on top of one cylinder. Then the second cylinder was placed over the wire-mesh. Tne two cylinders were then pressed together and held in place with two rubber bands, secured by the notches in the cylinder rims, (Navarro et al, 1998). Ten 10-15 d old R. domimca adults were placed inside the top cylinder and were kept in the test devices for 24 and 48 h. All tests were run at 27 °C and 65% R.H., and each test was repeated five times. At the end of each exposure period, the discs were examined on a black surface under a binocular microscope at 15x magnification. The number of perforations appearing as black circles were counted, and a comparative analysis was performed using the Student's t test for residual effect and the differences between control and dosages applied were determined using Dunπet^'s test (Anon., 1989).

RESULTS

Repellency

Table 1 shows the percent repellency and repellency class assigned to the substances tested. Against T. castaneum, the most effective substances were NeemAzal T/S (repellency class IV) and turmeric extract (repellency class IJJ and IV), while the least effective was azadirachtin (classes II and HI).

Table 1. Average repellency of several neem extracts and pyrethrum alone or in combination with pipcronyl buloxide (PBO) to Rhyzopertha dominica and Tribolntm castaneum adults during 5 days exposure (Roman numbers in brackets indicate repellency class values).

Tribolium castaneum Rhyzopertha dominica .

Substance Dosage (μg/cm²) without PBO with PBO without PBO with PBO

Turmeric 800 51.6 ( 111) 62.4 ( IV) 59.2 (111) 70.1 ( IV)

Neem Oil 800 50.1 (111) 57.8 (111) 55.2 (111) 51.4 (111)

NeemAzal T/S 50 63.8 (IV) 68.1 (IV) 57.2 (III) 60.5 (IV)

Azadirachtin 50 33.6 (11) 41.2 (111) 29.7 (11) 39.3 (II)

Pyrethrum 5 51.2 (111) 67.3 (IV) * *

Piperonyl butoxide 50 25.3 (11) 22.6 (II)

Control 16.2 10.4 (I) 5.8 (1)

* Insects were moribund

Against R. dominica, NeemAzal T S and turmeric extract provided the most effective repellency (class UI and IV), but exposure to the pyrethrum extract caused insects to die. It can be seen from Table 1 that for both insect species, there was no synergistic effect of PBO on the repellency values of the treatments.

Penetration

Tables 2 and 3 show a protective effect of up to 75 days provided by NeemAzal T/S at different dosages, against penetration by R. dominica over the two exposure periods of 24 and 48 h, respectively. Penetration through the paper discs was greatly reduced by all the dosages and for all the time delays after treatment. The residual effect of NeemAzal T/S extract for the confined exposure of 24 h period lasted until

the end of the 75 day experimental period except for the dosage of 31.25 μg/cm-.

The residual effect for the confined exposure of 48 h showed that the higher dosages of the extracts give a long lasting effect against penetration by insects (Table 3). Except for the 75 day time delay, all the dosages of NeemAzal T/S extract resulted in significantly lower penetrations than those of the control treatment for both exposure periods.

Table 2. Average number of perforations made by 10 Rhyzopertha dominica adults confined for 24 h on

Curcuma longa petrolium ether extract treated and untreated papers .

Time delay after exposure (days)

Dosage 1 15 30 45 60 - 75 (μg/cm²)

160 2.20 a a 0.80 a a 1.40 a a 2.00 a a 2.00 a a 1.60 a a

320 0.20 b b 0.40 b b 1.80 a a 2.20 a a 1.80 a a 1.60 a a

640 0.40 a b 1.00 a a 2.00 a a 1.40 a ab 1.20 a ab 2.40 a a b

1280 0.00 b a 0.00 b a 0.20 b a 0.40 a a 1.20 a a 1.40 a a

2560 0.00 b a 0.00 b a 0.00 b a 0.00 b a 0.20 a ab 0.80 a b

Control 1.30 a a 1.50 a a 2.10 a a 1.80 a a 1.90 a a 2.00 a a

* Values followed by the same letter within a column are not significantly different at the 5% level ** Values followed by the same bold letter within a row are not significantly different at the 5% I

Table 3. Average number of perforations made by 10 Rhyzopertha dominica adults confined for 48 h on Neem Azal T/S treated, and untreated papers .

Time delay after exposure (days)

Dosage (μg/cm²) 1 15 30 45 60 75

31 0 0..00 bb** bb** 0.0 b b 0.2 b b 0.4 b b 1.0 b b 3.6 a a

63 0.2 b b 0.0 b b 0.0 b b 0.4 b b 0.6 b a 1.2 b a

125 0.0 b b 0.2 b b 0.0 b b 0.2 b b 0.8 b a 1.4 b a

250 0.0 b a 0.0 b a 0.0 b a 0.4 b a 0.2 b a 0.6 b a

500 0.0 b a 0.0 b a 0.0 b a 0.0 b a 0.0 b a 0.2 b a

Control o.j a a 3.9 a a 3.7 a a 4.1 a a 3.5 a a 4.0 a a

' Values followed by the same letter within a column are not significantly different at the 5% level ** Values followed by the same bold letter within a row are not significantly different at the 5% level

Table 4 shows the protective effect of Neem oil at different dosages against penetration by R. dominica for time delays of up to 30 days for the two exposure periods.. The table shows that perforations through the paper discs were greatly reduced by the application of the neem oil at all dosage levels. The complete residual (no penetration by insects) effect of Neem oil for both the 24 h and 48 h exposure

periods was only obtained at the highest dosage of 2,560 μg/cm² after a one day time-delay. However, the tests showed that for both exposure periods the two higher dosages gave significant protection against penetration, until the maximum time- delay examined in this experiment of 30 days. At the 24 h exposure period all the dosages of neem oil resulted in significantly lower penetration than that of control at 30 davs after treatment.

Table 4. Average number of perforations made by 10 Rhyzopertha dominica adults confined for 24 and 48 h on Neem Oil treated and untreated papers .

Confined exposure for 24 h after time Confined exposure for 48 h after delay (days) time delay (days)

Dosage (μg/cm²) 1 15 30 1 15 30

160 1.0 a* a** 1.8 a a 1.0 b a 3.0 a b 6.0 a a 3.4 a b

320 0.6 a a 1.4 a a 0.8 b a 2.0 a a 2.4 a a 2.8 a a

640 0.8 a a 1.8 a a 1.2 b a 1.4 a a 3.2 a a 2.0 a a

1280 0.0 b a 0.6 a a 0.4 b a 0.8 b a 1.2 a a 1.0 b a

2560 0.0 b a 0.2 a a 0.2 b a 0.0 b a 0.4 b a 0.2 b a

1.5 a 2.1 a a 3.3 a a 3.9 a 3.7 a a

* At each exposure time, values followed by the same letter within a column are not significantly different the 5% level ** Values followed by the same bold letter within a row are not significantly different at the 5% level

Table 5 shows the effect of azadirachtin at different dosages against penetration by R. dominica over the two exposure times of 24 and 48 h. Although penetration was significantly reduced by azadirachtin especially at the confined exposure of 48 h, penetration was still apparent (Table 5).

Table 5. Average number of perforations made by 10 Rhyzopertha dominica adults confined on azadirachtin treated and untreated papers for 24 and 48 h, after 1 dav oftime delav.

Dosage 24 h 48 h

(μg/cm²)

31 0.8 a* 1.2 b

63 0.2 b 0.8 b

125 0.4 a 1.0 b

250 0.2 b 1.2 b

500 0.0 b 0.8 b

Control 1.3 a 3.3 a

*At each exposure time , val ues fol l owed by the same l etter wi thin a col umn are not significantly different at the 5% level

Table 6 shows the protective effect of pyrethrum extract at different dosages against penetration by R. domimca after a one and fifteen day time delay, at the two exposure times. Results showed that pyrethrum reduced penetration at all the

dosages except 2.5 and 5 μg/cm², though high dosages caused insect mortality

Table 6. Average number of perforations made by 10 Rhyzopertha domimca adults confined on pyrethrum extract treated and untreated papers for 24 and 48 h.

Confined exposure for 24 h Confined exposure for 48 after time delay (days) h after time delay (days)

Dosage (μg'cm²) 1 15 1 15

2.5 0.8 a* a** 0.6 b a 2.4 a a 1.2 b a

5.0 0.6 b a 1.2 a a 1.0 b b 2.8 a a

10.0 0.4 b a 0.6 b a 0.6 b a 0.4 b a

20.0 0.4 b a 0.2 b a 1.6 a a 1.8 b a

40.0 0.2 b a 0.2 b a 0.0^X b a 0.0^X b a

80.0 0.0 b a 0.0^X b a 0.0^X b a 0.0^X b a

160.0 0.0^X b a 0.0^X b a 0.0^X b a o.o b a

Control 1.3 a a 1.5 a a a a 3.9 a a

* At each exposure time, values followed by the same letter within a column are not significantly different at the 5% levei

** Values followed by the same bold letter within a row are not significantly differem at the 5% level

Insects were moribund

Table 7 shows a protective effect of up to 75 days provided by turmeric extract at different dosages, against penetration by R. domimca over the exposuperiod of 24 h. Penetration through the paper discs was reduced by higher dosages (1280 μg'cm² and 2560 μg'cm² ) for all the time delays after treatment. The residual effect of turmeric extract for the confined exposure of 24 h period was clear until the end of the 75 day experimental peπod at the highest dosage

Table 8 shows a protective effect of up to 75 days provided by Turmeric extract at different dosages, against penetration by R. domimca over the exposure period of 48 h. At higher dosages (1280 μg'cm² and 2560 μg/cm²), penetration through the paper discs was reduced for all the time delays after treatment The residual effect of turmeπc extract for the confined exposure of 48 h period was clear until the end of the 75 day experimental peπod at the highest dosage.

DISCUSSION

Repellency

Of the substances tested turmeric extract and NeemAzal T/S gave the highest levels of repellency, while azadirachtin had a low repellent effect on both insect species. Also, in no case did the addition of the synergist PBO have a significant influence on the repellency effect. McDonald et al., 1970 reported that pyrethrins 5 μg/cm2 gave 26.5 %, piperonyl butoxide (50 μg/cm2) gave 27.4 %, and the mixture of them (as a Standard) gave 57.3 repellency for the first week of repellency tests. Jilani and Su (1983) reported that most of the extracts that they investigated had a lower value than repellency class III (40.1-60%) which is considered as the standard for a promising repellent. They found that neem produced a maximum average repellency of only class El and turmeric extract (with petroleum ether) produced a 92.6% repellency (class V) at a dosage of 680 μg/cm². Neem seed petroleum-ether extract at a dosage of 680 μg'cm² gave a 81.5% repellency (class V), for the first week and at a dosage of 170 μg'cm² gave a 63% repellency (class IV) against T. castaneum. Jilani et al, (1988) reported that Turmeric oil extracted with n-hexane produced 93% repellency (class V) at a dosage of 800 μg/cm². Where neem leaves are mixed with grain in traditional practice, the grain is usually held in storage for 3- 6 months. Although the ingredients responsible for keeping out the stored-grain pests have not yet been identified, they seem to work well. In this context , repellency seems to be of primary importance (Anon, 1992). Jilani and Saxena (1990) reported that the repellency effect of neem oil (at 800 μg'cm²) and Margosan (Commercial derivatives of neem at 200 μg/cm² ) which were the minimum dosages that the authors applied against R. domimca, were 77% and 64% respectively (repellency class IV for both). However, in our expeπment, Neem oil at the same dosage gave class III repellency . This may be explained by the findings of Isman et al. ( 1990) who reported that azadirachtin content varied widely between different neem oil samples. This would lead us to conclude that the specific effectiveness of any particular neem oil sample should tested and confirmed before being applied to

practice in accordance with the present invention. In this respect it should be noted that two of the twelve oils that they investigated did not contain detectable levels of azadirachtin (detection limit=50ppm), while the remaining oils contained from 188 to 4,026 ppm.

Penetration

With respect to the neem extracts and derivatives, most promising results were obtained with NeemAzal T/S, for which the residual effect lasted for up to 75 days at the dosage of 500 μg'cm². The results with neem oil were less effective, while for azadirachtin, results showed that it had a protective effect, but to a limited extent. We found that azadirachtin at a dosage of 500 μg/cm² gave no penetration for the 24 h exposure, but at the same dosage at 48 h it gave 0.80 penetrations-' device. Malik and Naqvi (1984), using the device described by Highland et al. (1970), found that for azadirachtin, treated with 0.75 ml of 1% azadirachtin in acetone solution on 7 cm diameter Whatman No. 44 filter paper, (equal to 19.5 μg/cm²) gave no penetration for 24 h exposure, but for 48 h gave 0.25 penetrations/device. They found that penetration through control filter paper was 1.25 holes/disc for 24 h and 2.25 holes/disc for 48 h exposure. Using our penetration device, we found that penetration through the control was 1.30 holes/disc for 24 h and 3.30 holes/disc for 48 h exposure. Jilani and Su (1983) reported that the insects tended to make punctures which were not large enough for them to escape through duπng their 72-h observation period. During our tests, however, many times punctures made by insects were large enough to enable them to escape.

At the 2% concentration, the differences between the extracts were not significant, but all the treatments had significantly fewer punctures than the untreated, even though no treatment gave zero penetration (Jilani and Su, 1983). Same reported that at the 1 or 0.5% concentrations, the lowest number of punctures was observed in the neem extract treatment and these were significantly different from those treated with turmeric, fenugreek extracts and untreated control. Jilani and Saxena (1990) reported that R. domimca adults made significantly fewer punctures on filter paper treated with their test substances as compared with control. In their experiment, using filter paper treated with Margosan, they found fewer punctures than for papers treated with turmeric oil, sweetflag oil, and neem oil. At their highest concentration of 1000 μg/cm² neem oil gave no punctures for 24 h, but it caused 0.3 punctures for 48 h exposure. Margosan at the same concentration gave no punctures for both 24 h and 48 h. These results are in accordance with our results that relate to a one day time delay (Tables 2, 3 and 4). On the other hand, there are no records in the literature on the long term residual effectiveness using the penetration test. In this context our results showed that the most promising material tested was NeemAzal T/S which gave complete protection at the highest concentration for up to 75 days for 24 h and 60 days for 48 h exposure. The 30 days residual effect of neem oil was evident only at the highest dosage of 2560 μg cm² for the 24 and 48 h exposure tests where penetration was significantly lower than that of the control. Koul (1987) reported that neem oil as an antifeedant was effective against

Spodoptera litura larvae only at very high concentrations. Isman et al., (1990) reported that azadirachtin content varied widely between neem oil samples. They showed that there is a clear trend in which bioactivity of neem oils is related to azadirachtin content, and also that bioactivity of azadirachtin is enhanced by the presence of the oil as carried out by comparing bioactivity of pure azadirachtin to oil spiked with azadirachtin using the Peridroma chronic growth bioassay. They concluded that the presence of other constituents in these oils synergise or activate azadirachtin.

Isman et al., (1990) reported that advantages of neem preparations over pure azadirachtin include the presence of other potentially active constituents and the possibility that a botanical preparation may enhance the stability of azadirachtin and other active ingredients. Mordue and Blackwell (1993) reported that limonoid mixtures may be more effective than azadirachtin alone; also that neem oil itself has insecticidal properties unrelated to its azadirachtin content and that crude formulations may contain volatile repellent components.

The above data support our conclusion that NeemAzal T S ( which contains azadirachtin, other related Iimonoids and neem oil) gives better results than both azadirachtin and neem oil in preventing penetration by insects. Our results showed that natural pyrethrum caused a reduction in penetration. At high dosages (40, 80 and

160 μg'cm²) insects died, but at the lower dosages, penetration could be reduced for a short peπod. We can therefore conclude that the residual effect of some neem extract can last for a long time at the high dosages in storage conditions where the degradation problem caused by sunlight is less of a concern. This is supported by the observations of other investigators. (Anon, 1992; Daniel and Smith, 1990; Makonjuola, 1989; Mordue and Blackwell, 1993). Although Malik and Naqvi (1984) regarded the activity of plant substances in preventing insect penetration as an antifeedant effect, the insects fail to penetrate because they are repelled by the substances, and not because of an antifeedant effect.

With turmeric extract, in the repellency tests, most promising results were obtained for which the repellency class were IJJ and IV at the dosage of 800 μg/cm² for both insect species tested. The penetration results with turmeric extract showed that it had a protective effect in a dose dependent manner (Table 7 and 8). We found that turmeric extract at a dosage of 2560 μg/cm² gave no penetration for the 24 h and 48 h exposure up to 45 days. Jilani and Su (1983) reported that turmeric petroleum ether extract at all dosages applied reduced punctures significantly. Jilani and Saxena (1990) reported that R. dominica adults made significantly fewer punctures on filter paper treated with their test substances as compared with control. In their experiment, using filter paper treated with turmeric oil at a dosage of 1000 μg/cm² they found 0.0 punctures for 24 h, but it caused 0.7 punctures for 48 h. These results are in accordance with our results that relate to a one day time delay (Tables 7 and 8 ). On the other hand, there are no records in the literature on the long term residual effectiveness using the penetration test. In this context our results showed that turmeric was a promising material which gave complete protection at the high dosages (1280 and 2560 μg'c ²) for up to 45 days for 24 h and 48 h exposure. At the same dosages after 60 and 75 days delay, extract reduced punctures significantly.

On the basis of the repellency and penetration test results, it has been shown that turmeric and neem extracts affect insect behaviour in that they caused insects not to penetrate barriers treated with them. This effect of repelling storage insects, has been utilized in accordance with the present invention to treat packaging materials so as to impart to them resistance to the penetration of insects. This in turn protects the packaged food from insect infestation in an effective, user safe and environmentally friendly manner.

While certain embodiments of the invention have been hereinbefore particularly described, it will be apparent to anyone skilled in the art that many modifications and variations may be made, that do not deviate from the main features or spirit of the invention. The invention is accordingly not to be construed as restricted to such embodiments, but rather to its concept, spirit and general scope.