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Submission to the Genetic Engineering Approval Committee on Bt brinjal hybrids for open-air field trials in farmer’s fields.

Submitted by: Greenpeace No. 3360, 13th b Main, HAL IInd stage, Indiranagar, Bangalore 560032 Telephone: 080-41154861 Fax- 080 41154862 Contact person: Divya Raghunandan- +91 9845535406; [email protected]

Date: 14th July 2006

Trait Insect resistance- The Cry 1Ac gene of the soil bacterium Bacillus thuringenesis kurstaki (Bt). The genes have been introduced into the plant in order to give it protection from the Brinjal fruit and shoot borer, Leucinodes orbonalis and the fruit borer, Helicoverpa armigera. The plant becomes insect resistant because each cell of the plant produces the Cry proteins (pesticide) that kill the pests.

Variety It is extremely unclear as to how many hybrids of Mahyco are being considered for approval. M/s Mahyco has been requested to present details of the biosafety data for consideration of the GEAC as they have submitted their application to the GEAC for large scale trials and seed production of four Bt Brinjal hybrids namely MHB-4 Bt, MHB 9 Bt MHB 80 Bt and MHBJ-99 Bt containing cry 1 Ac gene during Kharif 2006 (Source: Geac 67th meeting [22/05/06]minutes)

The Committee considered the application for large scale trials and seed production of Bt Brinjal hybrids namely MHB-4 Bt, MHB 9 Bt, MHB 10 Bt, MHH 80 Bt, MHBJ-99 Bt, MHB-11 Bt and MHB -39 Bt containing cry 1 Ac gene submitted by M/s Mahyco. (Source: Geac 68th meeting[01/06/06])

Gene construct The Agrobacterium tumefaciens strain LBA4404 carrying the vector pMON 10518 (which carries cry1Ac, nptII and aad genes) was used in the transformation process. The cry1Ac gene is under the transcriptional control of the enhanced CaMV35S promoter (P-E35S). The aforesaid genes have been introduced by Agrobacterium mediated transformation, into young tissue of brinjal and transgenic plants have been regenerated by tissue culture, using kanamycin as the selection agent. Source: (http://www.envfor.nic.in/divisions/csurv/geac/brinjal_part-I.pdf)

Patent holder Monsanto 1. Potential allergenicity of Cry 1 Ac The document states “The cry1Ac gene, which encodes for an insecticidal protein, Cry1Ac, derived from the common soil bacterium Bacillus thuringiensis subsp. kurstaki (B.t.k) … The Cry1Ac protein produced in Bt brinjal is non-toxic to non-lepidopteran insects, birds, fish and mammals as these species lack receptors for the proteins on the surface of their gut cells. Also the acidic medium in gut of these organisms also makes Cry1Ac protein inactive.”

For Cry1Ac, there is concern over its potential allergenicity. Research considering the immunogenicity of the Cry1Ac toxin [1]indicates that

 Cry1Ac protoxin is a potent immunogen.  The protoxin is immunogenic by both the intraperitoneal (injected) and intragastric (ingested) route.  The immune response to the protoxin is both systemic and mucosal.  Cry1Ac protoxin binds to surface proteins in the mouse small intestine.

These research reports suggest extreme caution is required in the use of Cry1Ac in food crops.

The FAO/WHO Codex Alimentarius, who are developing international standards for GE food safety testing have adopted a “decision tree” approach[2]. This means that, should any evidence of possible allergy be found (as is the case with Cry1Ac), a very thorough and detailed assessment on the allergenic risks would need to be performed according to the FAO/WHO guidelines. Therefore, Cry1Ac must be examined thoroughly as a potential allergen. This has not yet been done. There are serious human, animal and health concerns associated with the Cry1Ac gene and no long term studies have been done to investigate the same.

“Animal studies cannot readily be applied to testing the risks associated with whole foods, which are complex mixtures of compounds, often characterised by a wide variation in composition and nutritional value. Due to their bulk and effect on satiety, they can usually only be fed to animals at low multiples of the amounts that might be present in the human diet. In addition, a key factor to consider in conducting animal studies on foods is the nutritional value and balance of the diets used, in order to avoid the induction of adverse effects which are not related directly to the material itself. Detecting any potential adverse effects and relating these conclusively to an individual characteristic of the food can therefore be extremely difficult. If the characterization of the food indicates that the available data are insufficient for a thorough safety assessment, properly designed animal studies could be requested on the whole foods. Another consideration in deciding the need for animal studies is whether it is appropriate to subject experimental animals to such a study if it is unlikely to give rise to meaningful information.”[3] Codex it self says since whole food experiments on animals cannot give conclusive evidence on the links between each and every nutrient component in the food and the potential adverse effect observed in an animal; toxicity and allergenecity tests with specific proteins need to be conducted. The safety assessment should take into account the chemical nature and function of the newly expressed substance and identify the concentration of the substance in the edible parts of the recombinant-DNA plant, including variations and mean values. Current dietary exposure and possible effects on population sub-groups should also be considered. [4].

According to our regulatory guidelines approval are sought for GM varieties and not genetic event. Each of the Genetically Modified hybrids of Brinjal needs to be studied as a separate plant and quantity of novel proteins in each pant part needs to be estimated separately .

2. Impacts on GE Bt eggplant on non-target organisms The document states “Mahyco R&D conducted multi location Field trials during the years 2004-05 & 2005-06. The protocol adopted to conduct these trials had specific mention of the assessment of the effect of Bt brinjal on non-target pests (sucking pest, secondary lepidopterans) and beneficial insects of brinjal crop. The vast data collected in all these years from various locations showed that non-target sucking pest counts (aphids, jassids, white fly, leafhoppers & thrips) did not vary significantly among Bt and non-Bt brinjal hybrids. The beneficial insects namely Chrysopa, lady beetle and spiders were also observed to be active in both Bt and non-Bt brinjal crops.”

These studies are simply is not enough to determine whether non target organisms are affected. “Observations” are subjective at best. Which Lepidoptera (butterflies and moths) were studied? And how? Importantly, long term experiments are necessary. For example, long-term exposure to Bt (Cry1Ab) pollen from two Bt maize types, MON810 and Bt11, has recently been found to cause adverse effects on larvae of the monarch butterfly, even though these strains of Bt maize contain less Bt in their pollen than Bt176. Although no short-term effects (4-5 days) were noted[5], longer-term studies (2 years) found over 20 % fewer monarch larvae reached the adult butterfly stage when exposed to naturally deposited Bt pollen[6]. Environmental risk assessments should include longer periods of exposure would improve the risk assessment[7]. The case of the monarch butterfly shows it is vital these studies are performed.

Bt proteins from natural Bt sprays degrade relatively quickly in the field as a result of ultraviolet light and lose most toxic activity within several days to two weeks after application[8]. In Bt crops, however, the Bt toxin is produced throughout the entire lifespan of the plants.

Direct Effects: GE Bt eggplant, like other Bt crops, could be harmful to non-target organisms if they either consume the toxin directly in pollen or plant debris, or indirectly by feeding on pests that have ingested the toxin. This could cause harm to ecosystems by reducing the numbers of important species, or reduce the numbers of beneficial organisms that would naturally help control the pest species.

The Bt toxins in GE eggplant are specifically toxic to Lepidoptera (butterflies and moths), but not all of these are pests. The potential for GE Bt crops to be directly toxic to non-target species was highlighted by research in the USA when it was demonstrated that pollen from one type of GE Bt maize (Bt176) was toxic to the much-loved Monarch butterfly[9]. More recently, it has been shown that long-term exposure even to relatively low levels of Bt in maize pollen causes adverse effects on larvae of the Monarch butterfly[10]. Importantly, these risks to non-target species were not identified until after commercialisation of Bt maize, and required several years of research for the long-term implications to be realized.

In addition, long-term exposure to Cry1Ac could quite possibly have harmful effects on silkworms similar to those observed to low level exposure of Bt to Monarch butterfly larvae. However, such long-term studies have not yet been performed.

Indirect effects: Data from Bt maize indicate that the beneficial insects, lacewings, have increased mortality when fed on larvae of a maize pest, the corn borer, which had been fed on B[11]. Numbers of beneficial ladybeetles were found to be lower in Bt maize plots than in non-Bt maize. Ladybeetles feed on many food sources including on aphids, pollen, European corn borer eggs and other pest eggs[12], so have several routes of exposure to the Bt toxin. Non- target, beneficial species that may feed on eggplant could be similarly affected.

Changes in populations of both pests and of natural enemies have been documented in Bt cotton. Data from China show that use of Bt crops can exacerbate populations of other secondary pests, including aphids, lygus bug, whitefly, Carmine spider mite and thrips[13]. Studies there have shown significant reductions in populations of the beneficial parasites Microplitis sp. (88.9% reduction) and Campoletis chloridae (79.2% reduction) in Bt cotton fields[14]. “Single-species studies of non-target effects represent a narrow approach to assessing the positive and negative ecological impacts of non-target effects. Understanding the ecological consequences of non-target effects also depends on accurately identifying what physical and biological processes a transgenic organism may alter, and understanding what impacts these alterations have on ecosystems.” Ecological Society of America (2004)

The disturbing conclusion is that Bt toxins from GE plants can kill non-target species and be passed higher up the food chain, an effect that has never been observed with the Bt toxin in its natural form.

 Mahyco’s comparative studies between a Non Bt and Bt Brinjal effects on non-target insects for one season in just one location, is inadequate to come to any scientific conclusion.  A comparative study needs to be conducted between the beneficial insect number and composition in a NPM/organically grown Brinjal field as compared to a specific Bt Brinjal hybrid field so as to asses the impacts on non- target organisms scientifically.  Study needs to be conducted on the toxicity of Cry proteins on lepidopterans and coleopterans that interact with wild Solanum members, as there is every possibility of gene flow and subsequent Cry protein expression in the wild, related specieii of Solanum melongena. 3. Contamination of the center of diversity The document states “Brinjal plant is usually self-pollinated, but the extent of cross- pollination has been reported as high as 48% and hence it is classified as often cross- pollinated crop. Brinjal is often cross-pollinated due to heteromorphic flower structure called as heterostyly. Outcrossing primarily takes place with the help of insects. ..Pollen flow studies at two locations show that at Jalna (Maharashtra) maximum distance that the pollen traveled was 20 meters, 10 out of 681 progenies showing the presence of the gene giving a outcrossing percentage of 1.46%. At Ranebennure (Karnataka), maximum distance that the pollen traveled was 15 meters and 18 progenies out of 663 show outcrossing (2.7%).”

Any field trial of GE brinjal will undoubtedly lead to contamination. It is known that for foundation seed stock, an isolation distance of 200 m[15] is recommended. This, therefore should be regarded as an absolute minimum. However, isolation distances will not prevent contamination of brinjal as it is insect pollinated and insects can travel long distances. As one UK scientist said, “distance will not protect us; if cross-pollination can occur, it will. A bee that gets on a train could deliver its cargo of pollen to far-flung places”[16]

If contamination of neighboring brinjal occurs, it will be very difficult to contain it. Brinjal contains many seeds, and each seeds produces many fruits. If just one seed from one GE contamination event is grown, it would multiply into tens or even hundreds of GE seeds from just one plant. If any of these seeds are themselves sown, they will grow into GE brinjal plants and again many GE seeds will be produced. If people grow brinjal in their back yards, and save seeds, then the contamination will persist, and even get worse, just from the field trial. Such contamination was seen from GE papaya in Thailand[17], from field trials.

Even more seriously is that India contains wild and weedy species of brinjal. There are landraces, weedy forms and allied wild species in India although the evolutionary relationships and classification are not well understood[18]. GE contamination could reach these wild and weedy forms. Once in these populations, it cannot be eradicated. In addition, the GE trait in Bt brinjal is insect resistance. This has an evolutionary advantage so is likely to spread (transgress) through a wild and weedy population Ellstrand (2001).

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 Extent of pollen flow also depends on the pollen load and also on the insect activity[19] in the area. The 2 studies conducted at Rannebennur and Jalna are only conclusive of pollen flow in those locations and this data cannot be extrapolated for the whole country.

 No studies on the effects of the presence of the Cry 1 Ac gene in wild verities have been done so far. The surety of gene flow along with the absence of the knowledge on the impacts of the insertion of such genes in the wild verities creates a dangerous situation. 4. Presence of Antibiotic resistance marker genes The document states “NPTII and AAD proteins are used as a selectable marker and have no pesticidal activity and are not known to be toxic to any species.”

The GE brinjal contains not one but two antibiotic resistance genes, which could render certain diseases untreatable. Importantly, this brinjal would not be acceptable to be put on the market in the EU because of presence one of the antibiotic resistance genes.

Eating foods derived from GE crops containing these marker genes therefore poses a risk that the antibiotic resistance genes could be transferred to bacteria living in the digestive tract of humans and animals and render them immune to antibiotic drug treatments. Antibiotic resistance can also be transferred to soil bacteria from decomposing parts of the plants.[20]

Proponents of genetic engineering claim that there is little likelihood that such gene transfer would actually occur, but scientists and regulatory authorities have expressed the view that even the slightest risk would be unacceptable. Producers of GE plants containing antibiotic resistance genes argue that, even if these genes were transferred to human or animal gut bacteria, this would make little difference to the already high levels of antibiotic resistance[21]. Such an attitude is irresponsible, since any increase in antibiotic resistance could be disastrous for human and veterinary medicine.

However, importantly, the European Food Safety Authority, which advises European Union governments on food issues, said that marker genes conferring resistance to streptomycin and spectinomycin "should be restricted to field trials and not be present in genetically modified plants placed on the market"[22]. And the Codex Alimentarius Commission, the international food-standards body, has urged the agricultural biotechnology industry to use alternative methods to refine genetically modified strains in the future.

 The use of the AAD marker goes against the codex guidelines that states, “Antibiotic resistance genes used in food production that encode resistance to clinically used antibiotics should not be present in foods” [23]

 The presented data does not show a study done to verify whether the DNA sequence for antibiotic marker resistance never reaches the gut of human beings where it can get conjugated in to the bacteria present in the human gut.

5. Impact on Soil Health

Soil organisms play a crucial role in soil health. GE Bt brinjal could be problematic for long-term soil health, as the crop express proteins that are known to be toxic to certain insects and are suspected of being toxic to a range of non-target organisms as well, including earthworms [24]. An unknown number of species make up the soil food web and could be affected by Bt – yet tests have been conducted on very few, in very few soil types and ecosystems.

If, under field conditions, the Bt deposited in the soil by brinjal residues have an impact on soil organisms – bacteria, fungi, insects, worms – there will necessarily be downstream effects. If Bt brinjal kill or otherwise reduces the activity of any of these soil organisms, it will disturb the web of relationships necessary for carrying out essential ecosystem functions, such as decomposition and nutrient cycling.

Further studies are needed to determine whether the persistence of Bt would cause problems for non-target organisms and the health of the soil ecosystem. This highlights the need for long-term studies on the impacts of Bt crops.

Because of the crucial role that soil organisms play in soil health, it is necessary to understand how different agricultural practices affect them. Bt crops may be problematic for longterm soil health, as they express proteins that are known to be toxic to certain insects known as lepidopterans (moths and butterflies) and coleopterans (beetles) and are suspected of being toxic to a range of non-target organisms as well, including earthworms[25]. An unknown number of species make up the soil food web and could be affected by Bt – yet tests have been conducted on very few, in very few soil types and ecosystems. If the Bt deposited in the soil by these crops has an impact on soil organisms – bacteria, fungi, insects, worms – there will necessarily be downstream effects. If you kill or otherwise reduce the activity of any of these soil organisms, you disturb the web of relationships necessary for carrying out essential ecosystem functions, such as decomposition and nutrient cycling. According to the US Environmental Protection Agency’s (EPA) scientific advisory panel[26], Bt proteins “are likely to be present in the rhizosphere soil not only throughout the growth of the crop, but perhaps long after the crop is harvested.” Therefore researchers and regulators must assume “that continuous exposure to Cry [Bt] proteins is likely within the soil system.” The Panel concluded that “it would be prudent to determine under operational field conditions in different geographical regions and soil types, the extent to which Cry [Bt] proteins accumulate in soil.” The Panel drew attention to studies that showed Bt could persist in certain soil types for up to 234 days[27], and recognized that further studies needed to be done to determine whether the persistence of Bt would cause problems for non-target organisms and the health of the soil ecosystem.

As noted by agricultural expert Charles Benbrook[28], in addition to long-term research on impacts of Bt in soils: “Research is needed on the short-term soil microbial community impacts of a big dose of Bt as corn trash and other crop residues break down in the spring and early summer. One might hypothesize that under some circumstances, Bt entering the soil will impact soil microbial communities in ways that lead to complex, multi-tier impacts on microbial and soil insect bio-control, pathogen pressure, immune response and nutrient cycling. Even if the impacts last only 4 to 8 weeks, that is ample time to leave a lasting mark on the performance of the cropping system, both in one season and over many years as microbial communities evolve to a new steady state.” Post-harvest decomposition of these plants may therefore result in the accumulation of Bt toxins in soil at concentrations high enough to constitute a hazard to non-target organisms such as beneficial insects (e.g. pollinators, parasites, and predators of insect pests) and other animal classes[29]. Studies have also showed that Bt crops may secrete the toxin from the root into the soil.[30]

In the Mahyco study state that “ANOVA analysis of the microbial population showed no significant difference between Bt & non-Bt soil samples”. Normally soil samples with less of humus content and where large quantities of fertilizers and pesticides tend to have a very low microbial presence. The same is the case with soil insects and earthworms. Presence of earthworms is very lows in soil where there is high use of chemical fertilizers and pesticides. A comparative study needs to be conducted between the composition of soil micro flora in a NPM/organically grown Brinjal field as compared to a specific Bt Brinjal hybrid field so as to asses the impact of the Bt toxin on the soil microflora. There is also a need for an intergenerational study to be done. ADOPTING THE PRECAUTIONARY APPROACH.

The Precautionary Principle The regulation of GMOs requires a precautionary approach to be followed. In situations where serious harm may arise, lack of evidence of the harm arising should not prevent action being taken to prevent harm. In giving the biotechnology industry, rather than the environment, the benefit of the doubt , we will be failing to implement the precautionary principle enshrined in the Cartegena principle to which we are a signatory. There is a need to prioritise environmental protection, not the biotech industry, in its interpretation of the implications of uncertainties and gaps in knowledge.

Several countries around the world have prohibited the release of these products due to concerns about their environmental impact.  Austria, Greece, Hungary and Poland [31] prohibited cultivation of maize MON810 in view of its potential impact on the environment.  The South African government introduced a moratorium on approvals of all new varieties of transgenic maize due to growing concern about the long-term environmental effects and the economic impact of transgenic maize for South African farmers.  South African governments ban on any GM study in Sorghum owing to Africa being the center of diversity for Sorghum and the Mexican ban on Maize since Mexico is the center of origin of maize are significant decisions that need to be followed in any region that is the center of origin/diversity of any crop.  The Minister of Agriculture for the Greek Republic through Decree Number 8080/30-1- 06 prohibited sales of genetically modified MON810 maize seeds for cultivation (see the Decree document attached to this petition).

 Zambia has prohibited imports and cultivation of genetically modified maize since 2002. The Zambian government adopted that decision after extensive research into events in many countries throughout the world, including meetings with decision makers and producers in the United States of America.  In the case of Bolivia, the Ministry of Sustainable Development decreed through Administrative Resolution number VRNMA No. 135/05, 14 November 2005 the following: “FIRST.- Reject the application presented by the company Dow AgroSciences Bolivia S.A. referring to trials with genetically modified maize (Trial to test resistance to the fall armyworm and the herbicide ammonium gluphosinate with Bt, Event TC 1507 maize), due to the high probability of genetic contamination of creole maize varieties due to their crossbreeding characteristics and the potential risk which this presents to the genetic diversity of maize in Bolivia and SECOND.- Reject any request to introduce genetically modified maize onto national territory to carry out field trials, sowing, production or deliberate release into the environment.”

The list of countries with prohibitions or moratoriums on sales and/or cultivation of some or all transgenic products include: Algeria, Benin, Uganda (cultivation prohibited), Zambia, Saudi Arabia, Thailand, Albania, Austria, Bulgaria, Croatia, France, Georgia, Germany, Greece, Hungary, Italy, Luxemburg, Spain Switzerland, El Salvador, Bolivia and Venezuela [32]. References [1] Moreno-Fierros, L. García, N. Gutiérrez,R. López-Revilla, R.Vázquez-Padrón, RI..(2000). Intranasal, rectal and intraperitoneal immunization with protoxin Cry1Ac from Bacillus thuringiensis induces compartmentalized serum, intestinal, vaginal and pulmonary immune responses in Balb/c mice. Microbes Infect 2(8): 885-90; . Vázquez-Padrón, R.I, Moreno-Fierros, L. Neri-Bazán, L, de la Riva, G.A & López-Revilla, R. (1999). Bacillus thuringiensis Cry1Ac protoxin is a potent systemic and mucosal adjuvant. Scand J Immunol 49: 578-584; Vázquez-Padrón, R.I Moreno-Fierros, L. Neri-Bazán, L, de la Riva, G.A & López-Revilla, R. (1999). Intragastric and intraperitoneal administration of Cry1Ac protoxin from Bacillus thuringiensis induces systemic and mucosal antibody responses in mice. Life Sciences 64(21): 1897-1912; Vázquez-Padrón, R. I., Moreno-Fierros, L. Neri-Bazán. L. Martínez-Gil, A.F., de la Riva, G.A. & López- Revilla, R.. (2000). Characterization of the mucosal and systemic immune response induced by Cry1Ac protein from Bacillus thuringiensis HD 73 in mice. Braz J Med Biol Res 33: 147-155; Vázquez-Padrón, R. I., Gonzáles-Cabrera, J., García-Tovar, C. Neri-Bazán, L., López-Revilla, R., Hernández, M., Moreno-Fierros, L. & de la Riva.G.A. (2000). Cry1Ac protoxin from Bacillus thuringiensis sp. kurstaki HD73 binds to surface proteins in the mouse small intestine. Biochem Biophys Res Comms 271: 54-58.

[2] FAO-WHO 2001. Evaluation of Allergenicity of Genetically Modified Foods. Report of the joint FAO/WHO expert consultation on allergenicity of foods derived from Biotechnology, 22-25 January 2001, pp1-26.

[3] GUIDELINE FOR THE CONDUCT OF FOOD SAFETY ASSESSMENT OF FOODS DERIVED FROM RECOMBINANT-DNA PLANTS ,CAC/GL 45-2003, Point No.11, page no 2. [4] GUIDELINE FOR THE CONDUCT OF FOOD SAFETY ASSESSMENT OF FOODS DERIVED FROM RECOMBINANT-DNA PLANTS ,CAC/GL 45-2003,pointno.35,page 6.

[5] Stanley-Horn, D.E., G.P. Dively, R.L. Hellmich, H.R. Mattila, M.K. Sears, R. Rose, L.C.H. Jesse, J.E. Losey, J.J. Obrycki and L. Lewis. 2001. Assessing the impact of Cry1Ab-expressing corn pollen on monarch butterfly larvae in field studies. Proceedings of the National Academy of Sciences 98: 11931- 11936. [6] Dively, G.P., R. Rose, M.K. Sears, R.L. Hellmich, D.E. Stanley-Horn, D.D. Calvin, J.M. Russo and P.L. Anderson. 2004. Effects on monarch butterfly larvae (Lepidoptera: Danaidae) after continuous exposure to Cry1Ab expressing corn during anthesis. Environmental Entomology 33: 1116-1125. [7] Andow, D.A. and A. Hilbeck. 2004. Science-based risk assessment for non-target effects of transgenic crops. Bioscience, 54: 637-649. Ecological Society of America (ESA) 2004. Genetically engineered organisms and the environment: Current status and recommendations. ESA Position Paper http://www.esa.org/pao/esaPositions/Papers/geo_position.htm. Marvier, M. 2002. Improving risk assessment for nontarget safety of transgenic crops. Ecological Applications 12: 1119-1124. [8] Hillbeck, A. 2001. Transgenic host plant resistance and non-target effects. In: Genetically engineered organisms: assessing environmental and human health effects. Letourneau, D.K. and B.E. Burrows [eds.] Boca Raton, FL: CRC Press. [9] Losey J. E, Raynor, L. & Cater, M.E. (1999). Transgenic pollen harms monarch larvae. Nature 399: 214.

[10] Dively, G.P., R. Rose, M.K. Sears, R.L. Hellmich, D.E. Stanley-Horn, D.D. Calvin, J.M. Russo and P.L. Anderson. 2004. Effects on monarch butterfly larvae (Lepidoptera: Danaidae) after continuous exposure to Cry1Ab expressing corn during anthesis. Environmental Entomology 33: 1116-1125. [11] Hillbeck, A., Baumgartner, M., Fried, P.M. & Bigler, F. 1998. Effects of transgenic Bacillus thuringiensis corn-fed prey on mortality and development time of immature Chrysoperla carnea (Neuroptera: Chrysopidae). Environmental Entomology 27: 480-487; Hillbeck, A., Moar, W.J., Pusztai-Carey, M., Filippini, A. & Bigler, F. (1998) Toxicity of Bacillus thuringiensis Cry1Ab toxin to the predator Crysoperla carnea (Neuroptera: Chrysopidae). Environmental Entomology 27: 1255- 1263. [12] Wold, S.J., Burkness E.C., Hutchinson, W.D.,Venette, R.C. 2001. In-field monitoring of beneficial insect populations in transgenic corn expressing a Bacillus thuringensis toxin. Journal of Entomological Science 36: 177-187. [13] Cui, J. and J. Xia. 1998. Effects of transgenic Bt cotton (with early maturity) on population dynamics of main pests and their natural enemies. Acta Gossypii Sinica 10: 255-262. [14] Cui, J. and J. Xia. 1998. Effects of transgenic Bt cotton (with early maturity) on population dynamics of main pests and their natural enemies. Acta Gossypii Sinica 10: 255-262.

[15] www.avrdc.org/LC/eggplant/eggplantseed.pdf

[16] Crawley, M.J. (1999) Bollworms, genes and ecologists. Nature, 400, 501-502.

[17]http://www.gmcontaminationregister.org/ [18]Sakata, Y. & Lester, R.N. 1997. Chloroplast DNA diversity in brinjal eggplant (Solanum melongena L.) and related species. Euphytica 97: 295–301. [19] Filippini, A. & Bigler, F. (1998) Toxicity of Bacillus thuringiensis Cry1Ab toxin to the predator Crysoperla carnea (Neuroptera: Chrysopidae). Environmental Entomology 27: 1255-1263.

[20]Hoffmann, T., Golz, C. & Schieder, O (1994) Foreign DNA sequences are received by a wild-type strain of Aspergillus niger after co-culture with transgenic higher plants. Curr. Genet. 27: 70-76 [21]See for example, Ciba Seeds (1996) Documentation on Bt-maize from Ciba Seeds. [22]Opinion of the Scientific Panel on Genetically Modified Organisms on the use of antibiotic resistance genes s marker genes in genetically modified plants.Question N° EFSA-Q-2003-109).pinion adopted on 2 April 2004 The EFSA Journal (2004) 48, 1-18 http://www.efsa.eu.int/press_room/press_release/386_en.html

[23] GUIDELINE FOR THE CONDUCT OF FOOD SAFETY ASSESSMENT OF FOODS DERIVED FROM RECOMBINANT-DNA PLANTS ,CAC/GL 45-2003 point No.58 ,page no.10

[24] Zwahlen, C. A. Hilbeck, R. Howald and W. Nentwig. 2003, Effects of transgenic Bt corn litter on the earthworm Lumbricus terrestris. Molecular Ecology 12:1077 –1086. [25] Marvier, M. 2001. Ecology of transgenic crops. American Scientist 89: 160-167.

[26] United States Environmental Protection Agency. 2001a.Report from the FIFRA Scientific Advisory Panel meeting, October 18-20 on Bt Plant-Pesticides Risk and Benefit Assessments.http://www.epa.gov/scipoly/sap/2000/index.htm#October

[27] Koskella, J. and G. Stotzky. 1997. Microbial utilization of free and clay-bound insecticidal toxins from Bacillus thuringiensis and their retention of insecticidal activity after incubation with microbes. Applied and Environmental Microbiology 63: 3561-3568; Tapp, H. and G. Stotzky. 1998. Persistence of the insecticidal toxin from Bacillus thuringiensis subsp. kurstaki in soil. Soil Biology Biochem. 30(4): 471-476.

[28] Benbrook, C.M. 1999. Impacts on soil microbial communities needs further study. AgBioTech InfoNet. June 24. http://www.biotechinfo.net/microbial_communities2.html

[29] Venkateswerlu G. and G. Stotzky. 1992. Binding of the protoxin and toxin proteins of Bacillus thuringiensis subsp. kurstaki on clay minerals. Current Microbiology 25: 225-233. [30] Saxena, D., S. Flores, and G. Stotzky. 1999. Transgenic plants: Insecticidal toxin in root exudates from Bt corn. Nature 402: 480; Saxena, D., S. Flores, and G.Stotzky,2002. Bt toxin is released in root exudates from 12 transgenic corn hybrids representing three transformation events”, Soil Biology & Biochemistry 34:133-137.

[31] Bundesministerium für Gesundheit und Frauen. 2006. Ecological effects of genetically modified maize with insect resistance and/or herbicide tolerance. Austrian Ministry for Women and Health. www.bmgf.gv.at/cms/site/attachments/5/6/2/CH0255/CMS1134457515326/literaturstudie_mais_endbericht .pdf.

[32] Center for Food Safety. Worldwide regulation, prohibition and production of GMOs. www.centerforfoodsafety.org/pubs/World_Regs_Chart.pdf

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