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Summary Articles published in "Plant Protection Days"

Darvas et al. (2003)



Hungarian Academy of Sciences, Plant Protection Institute, Ecotoxicology Department, Budapest

Insect larvae are most vulnerable to toxic substances in the first phase of their development. The reason for this is that the enzyme systems involved in detoxification are induced in the case of feeding. The range of their isoenzymes is growing through this process, enabling increased efficiency of xenobiotic treatment. The L1-L2 stages are most vulnerable to Bt toxin as well. Our experiment was carried out on the second larvae generation of the Inachis io, a protected species in Hungary The second generation imagos Inachis io - together with Vanessa atalanta, another species of similar biological features - prefer to feed on privet flowers in their maturing process, and this is the pollination period of maize varieties maturing in the middle of the maturing period. The butterflies place their eggs in clusters of hundreds of eggs, on the abaxial surfaces of the leaves. The young larvae stay together, and they move on to new shoots after eating all of the edible parts of a nettle shoot. The larvae do not consume the large quantities of maize pollen settling in the veins of the leaves. In our experiment a few hundred larvae consumed, over a 12-day period after hatching, Urtica dioica contaminated with a large quantity of maize pollen (in the case of a variety producing 35 kg pollen/ hectare, the 800 pollen/cm2 is found only inside the field, around the female plants, while the 300 pollen/cm2 is found at the edges of fields; see Darvas, Gharib, Csóti, Székács, Vajdics, Peregovits, Ronkay and Polgár, Abs. Plant Protection Days 2002). Thereafter the larvae fed on untreated nettles, for maize is shedding pollen over a 1-2 week period. One gram of the fresh dry pollen of the variety we used contained 38 ± 2 ng CryIAb. The pollen of the quantity that was characteristic of the edge of the field (in our experiments we used pollen of 31 ± 1 ng CryIAb stored for a year at 5 ºC) significantly reduced the weight of the larvae. One week after the termination of the treatment, the deficiency was still perceptible, but the difference had diminished. The differences had disappeared by the time of pupation. This is an indication of the fact that the smaller weight of the larvae originates from development and growth shortage in the early stage. Early larvae mortality was found - under conditions characteristic of the edges of the fields - which was estimated at some 20% of the Monsanto MON 810 event variety. The pollen of this variety contains a quarter of the Bt toxin that is contained in the Novartis varieties whose effects were assessed on Danaus plexippus. Accordingly, I. io was found to be similarly sensitive as was the butterfly D. plexippus by Hansen and Obryczki (1999), which is a protected butterfly species in the USA. Our experiment was repeated on other Nymphalidae thriving in several generations on nettles. The rare Polygonia c-album also showed similar vulnerability to that of I. io. On ruderal weeds suitable for catching pollen, along edges of fields an increasing mortality may occur during the pollination period and the young stages of the Nymphalidae. This may involve - on nettle species - the larvae of I. io, V. atalanta, P. c-album, Araschnia levana and Aglais urticae. In the case of the varieties originating from the MON 810 event, the hazard exists but the likelihood of its realisation is low, owing to the numerous criteria to be met. The testing of the pollen output of varieties originating from other genetic events (e.g., Bt 11, Bt 176) (some of them produce up to 120 kg pollen/hectare) and their Bt toxin contents should definitely be carried out - prior to licensing - for the toxin quantity produced in pollen may be an order of magnitude larger than in the case we have tested.

Our experiments are supported by the Ministry of Education (BIO-00024/2000) and the Ministry of Environmental Protection and Water Management (K-36-01-00017/2002).




1 Hungarian Academy of Sciences, Soil Science and Agrochemical Research Institute, Rhizobiology Research Unit, Budapest 2 Eszterházy Károly College, Botany Department, Eger 3 Szent István University, Zoology and Ecology Department, Gödöllo

We participated in complex research relating to the domestic production of genetically modified Bt maize, covering the largest possible number of parameters of the ecosystem, within the framework of a project supported by the Bio-technology programme of the Ministry of Education. Since Bt toxin appears not only in the leaves of maize but has also been proven to be excreted in the rhizosphere of the modified plant, there seemed to be a need for a comparative study of the biological activity of the soils based on a variety of indicators. We assumed that in addition to the direct effects of the toxin, the development of an adequate level of risk analysis methods also necessitates knowledge of its indirect effects, i.e. those on the decomposition of plant residues. The background for the research was provided by the research fields of the Plant Protection Institute of the Hungarian Academy of Sciences. The speed of the decomposition of the leaf and root residues in the soil of the Bt toxin containing (DK-440-BTY) and the maternal line maize (DK-440) was studied using the 'litter bag' method following the instructions of House and Stinner (1987) and Kiss and Jáger (1987). The bags of known weights, containing the plant residues, were placed in the soil after the autumn harvest and until the sowing in the spring we checked the quantity of the decomposed plant materials on seven occasions. At the same time, the C:N ratio and the phosphorous macro-element were also established from the samples. The data were analysed using a correlation/ regression method. The residues of maize containing Bt toxin were found to be decomposing slower - probably owing to genetic manipulation. In contrast to the leaves, the root residues did not fully decompose by the next sowing period. The C:N ratio also modified in the residues containing Bt toxin, showing a different curve in comparison to the control materials. Having consulted technical literature this is probably a result of the increased lignin content of the residues of Bt maize. The deceleration of the speed of decomposition was, however, also caused by other changes in the soil biology as well, which were triggered by the Bt toxin content, according to soil biota assessments. In the future, an assessment of the involvement of the various components of the soil biota in the decomposition process using nets of various hole sizes should yield interesting results.

Our experiments are supported by the Ministry of Education (BIO-00024/2000)

House, G.J.; Stinner, R.E. (1987): Pedobiologia, 30: 351-360. Kiss, I.; Jager, F. (1987) Bull. of the Univ. of Agric. Sci. at Gödöllo, 1:99-104.




1 Szent István University, Zoology and Ecology Department, Gödöllo 2 Hungarian Academy of Sciences, Soil Science and Agrochemical Research Institute, Rhizobiology Research Unit, Budapest 3 Hungarian Academy of Sciences Plant Protection Institute, Budapest

Very little information is available on the soil-biological effects of genetically modified maize varieties that produce Bt toxin. Data published so far have all shown that these maize varieties have no effects on the biological activity of soil and on the life history and activity of the populations of non-target species of soil zoology. In our research we tested whether the maize variety producing CryIAb Bt toxin (DK-440-BTY) really has no effect on the biological activity of the soil and on the choice of territory and food of the collembolan Folsomia candida, Heteromurus nitidus and Sinella coeca. The field experiments were carried out on the Julia Major site of the Plant Protection Institute of the Hungarian Academy of Sciences while the laboratory tests were performed at the Zoology and Ecology Department of Szent István University in Gödöllo. The field biological activity was assessed by Törne's 'bait lamina' test. The territory selection of the animals was assessed by a test of our own development while food selection was tested by the dual test. In the course of the field experiments in August, the roots of Bt maize contained 205.5 ± 2.6 ng/g toxin. Biological activity was significantly lower in this soil than in the soil under isogenic maize a few metres away. A difference was also found between the two soils when the test trays were arranged from the inside row of the Bt maize towards the soil without Bt toxin. In the soils of isogenic maize germinating for two weeks under laboratory conditions, a larger average number of collembolan were found than in the soil of the seedlings producing Bt toxin. (The roots of the seedlings contained 438.0 ± 6.0 ng/g toxin.) The difference was statistically significant. In the paired food choice tests the collembolan more frequently chose the isogenic maize than the Bt toxin containing pair. The above findings confirm that the DK-440-BTY maize has a substantial impact on soil biology. The effect of the toxin on the soil organisms is assumed to be responsible for the decline of biological activity. Accordingly, the collembolan are capable of recognising and avoiding Bt toxin containing plant residues as has been confirmed by laboratory experiments, as a consequence of which the operation of the decomposition system may be altered.

Our experiments are supported by the Ministry of Education (BIO-00024/2000)


Csóti et al. (2003)


Attila Csóti2, László Peregovits3, László Ronkay3 and Béla Darvas1

1 Hungarian Academy of Sciences Plant Protection Institute, Ecotoxicology Department, Budapest 2. Szent István University, Horticulture Faculty, Budapest 3. Hungarian Museum of Natural History, Zoology Collection, Budapest

The different maize varieties produce 20-120 kg/ha of pollen. The pollen of Bt maize contains more or less (40-160 ng/g) CryIAb toxin. The criteria of the interaction of this substantial quantity of toxin (1-20 g/ha) with sensitive butterfly caterpillars were assessed. For comparison: by delivering a permitted Dipel treatment approx. 5g/ha mixed Bt toxin is administered. A. When does maize shed pollen? At an average of two years, pollination of the DK-440-BTY (YieldGard) maize occurred in the second half of July. In the case of the varieties available in Hungary, pollination occurs in July and August. The species Zerynthia polyxena living on birthwort is therefore saved since its sensitive young larval stage precedes the pollination of maize. B. How is this quantity of pollen distributed? At a maximum of 20 metres from the edge of the field (in our case: 35 kg/ha pollen output, 38 ng CryIAb toxin/g pollen; 1.3 g/ha toxin) it drops to below 50 pollens per cm2. (See Darvas, Gharib, Csóti, Székács, Vajdics, Peregovits, Ronkay and Polgár, Abs. Plant Protection Days, 2002). The butterfly species living in or around maize fields on herbs may be affected. C. Does the same pollen density measured on the different herbs mean the same dose? Pollen sticks in the largest quantities on leaves. The extreme values of the leaf surface/weight ratio (mg/cm2) among the weed species living in our area - Urtica dioica : maize : senecio were 1: 2: 3. If the same amount of leaves is consumed, the highest Bt toxin dose is consumed by consuming Urtica dioica resulting from the same quantity of pollen on a unit of leaf surface. D. Does maize pollen stick equally to the leaves of the various weed species? Pollen sticks to dicotyledonous weeds with broad horizontal leaves with glandular hairs. The caterpillar species living on plants with glossy, smooth and waxy leaves (such as Euphorbia, Chondrilla, Daucus etc.) may avoid the effects. E. How many butterfly species are protected? 191 species in Hungary, 17% of which may feed on ruderal herbs. F. Are the various species and their different larval stages equally exposed? For the effects to materialise, the young caterpillars have to consume the surface of the leaf. Some species avoid poisoning by skinning the abaxial surfaces of the leaves when young. The larvae of some protected butterflies, though they hatch at the time of pollination, live in the flowers (see Schinia cardui, Schinia cognata) and are not affected as a consequence. Only the outstanding vulnerability of stages L1 and L2 were noted (see Darvas, Kincses, Vajdics, Polgár, Juracsek, Ernst and Székács, Abs. Plant Protection Days, 2003). G. Are the above criteria met? One plant/insect community was found to be affected: the group of Nymphalidae living on nettle species in the drainage ditches of the fields.

Our experiments are supported by the Ministry of Education (BIO-00024/2000) and the Ministry of Environmental Protection and Water Management (K-36-01-00017/2002).





Hungarian Academy of Sciences Plant Protection Institute, Ecotoxicology Department, Budapest

Owing to the Lepidoptera-specific effect of the Bt toxin (CryIAb) produced by the DK-440-BTY transgenic maize variety, no direct toxicity is - in general - expected on Hymenoptera parasitoids. Bt toxin consumed in sub-lethal doses, however, does have an effect on the post-embryonic development of a susceptible host animal, which in turn may affect the parasitoid living inside. A laboratory test method has been devised to test this food chain effect in the Plodia interpunctella and its parasitoid, Venturia canescens (Ichneumonoidae) host/parasitoid system. The host larvae were kept on standardised laboratory feed to which was added the ground product of the leaves of the maternal line (DK-440) and the transgenic maize variety (DK-440-BTY), which were collected at the same time (in 10% and 20% ratios). The Bt toxin content of the DK-440-BTY leaves was 491.0 ± 16.0 ng/g, established by an immunoanalytical method (ELISA). 21 days after the 24-hour egg-laying period of the P. interpunctella imagos, the weight of 30 larvae selected at random was measured - in each treatment - and then two newly hatched V. canescens females were placed among the larvae whose treatment continued (V. Canescens reproduces through parthenogenesis). This experiment was repeated several times. At the end of the experiments, the wing length of the parasitoid imagos that hatched in the various treatments was measured. The weight of the host larvae differed significantly in each treatment, as in the case of our previous experiments (see: Darvas, Gharib, Csóti, Székács, Vajdics, Peregovits, Ronkay and Polgár, Abs. Plant Protection Days 2002). While the differences between the treatments containing ground maize leaves is explained by the different quantities (the allelochemicals of maize had a small negative impact on the development of P. interpunctella), the difference between the treatments without maize leaves and those containing Bt maize is explained by the effect of the CryIAb toxin. The length of the wings of the V. canescens imagos did not show significant difference in the case of the host animals kept on feed with the maternal line. Nevertheless, there was a significant difference between the lengths of the wings of the parasitoid imagos grown in host animals kept on feed containing Bt toxin and those on feed not containing Bt toxin. This shows that the biological changes caused by sublethal doses of the Bt toxin resulted in reduced size in the parasitoid growing inside the host animals. Further experiments are planned to be carried out to find the effects of the quality change in the host animal as a result of the Bt toxin on the rest of the biological features of the parasitoid in addition to reduced size, such as reproduction, length of life, and the manner in which these change in the case of the exposure of successive generations.

Our experiments are supported by the Ministry of Education (BIO-00024/2000) and the Ministry of Environmental Protection and Water Management (K-36-01-00017/2002).




Directive 2001/18/EC of the European Union on genetically modified organisms contains the following statements of specific importance for us:

F No GMOs, as or in products, intended for deliberate release are to be considered for placing on the market without first having been subjected to satisfactory field testing at the research and development stage in ecosystems which could be affected by their use. F It is necessary to establish a common methodology to carry out the environmental risk assessment based on independent scientific advice. It is also necessary to establish common objectives for the monitoring of GMOs after their deliberate release or placing on the market as or in products. Monitoring of potential cumulative long- term effects should be considered as a compulsory part of the monitoring plan.

As a result of the new EU directive, the domestic regulations also have to be amended:

A two-phase procedure is required for a proper solution. The tests required for initial release and those required for the state recognition of a variety have to be distinguished from one another. In addition to releases, the above EU directive always specifies placement on the market as the next step. As a matter of course, this is only possible if the variety has been recognised by the state. The latter step after release is the one with real major environmental impacts, for in this case there is less scope for influencing and controlling circumstances than in the case of the variety experiments, which are also regarded as release. The point is that the variety experiment permit does not necessarily provide proper guarantees for production without hazards. For this reason, an objective procedure needs to be developed which @ complies with the requirements of the relevant EU directives, @ applies the 'prudence' principle in respect of a variety of issues entailing uncertainties, @ develops conditions and criteria that can be implemented in practice, @ is capable of managing uncertainties and the difficulties of interpretation stemming from the results of the estimation of risks that may make it difficult for decision makers to make correct and responsible decisions. The necessary steps in the licensing process F The first decision to be made by the authority is whether there is any reason prohibiting release. F The next question to be decided by the authority is whether there is a need for a standardised licensing process or can a simplified procedure be followed. F Performance of the necessary expert work for considering the license for release. F Based on the expert documentation concerning release, the authority decides whether the variety may be released for variety experiments, and, if the first permit is given, it also decides whether field tests will be required for subsequent permits. F The performance of field experiments by GKV on the basis of which a summary study is produced, containing a risk analysis as well. F Follow-up of long-term or cumulative effects with the aid of a monitoring system.

Our experiments are supported by the Ministry of Education (BIO-00024/2000) research program.




1 Hungarian Academy of Sciences, Soil Science and Agrochemical Research Institute, Rhizobiology Research Unit, Budapest 2 Eszterházy Károly College, Botany Department, Eger 3 Szent István University, Zoology and Ecology Department, Gödöllo 4. BI(r)OTOP Bt., Érd

The increasingly widespread use of genetically modified plants may justify their examination in respect of their effects on the elements of the non-targeted soil biota. Examination of the issue is particularly justified by the fact that, according to the literature, non-targeted microbiota is not susceptible to Bt toxin; indeed it may use it as a carbon and nitrogen source. For this reason we examined how the most important micro-biological features of the rhizosphere of the transgenic maize (Zea mays, DK-440- BTY), which produces the Bacillus thuringiensis Cry1Ab endotoxin, and the control maternal line maize (DK-440) develop over the course of two successive growing seasons. Samples were taken at the Julia Major site of the Plant Protection Institute of the Hungarian Academy of Sciences, where at three seasonal points in time within each growing season soil and root samples were collected for laboratory analysis. The effects of the Bt and the isogenic maize root exudates on the abundance of the most important microbes that can be bred (heterotrophic, oligotrophic, spore growing, microscopic fungi etc.) were studied using a selective food plate system that we had modified (Angerer et al. 1998). Furthermore, the differences in the species spectrum and morphology of the Trichoderma fungi were also checked, parallel with the colonisation values of the symbiotic arbuscular mycorrhizal fungi (AMF). The total microbe soil biology activity was checked by fluorescein diacetate (FDA) hydrolysis. It was found that in the first growing season the abundance of the microbe groups involved in the test did not differ between the two types of maize. The colonisation differences of the symbiotic endophite fungi were influenced more by the variability of soil conditions than any effects of the Bt toxin. Nor were any statistically confirmed changes found in the species spectrum or morphology of the Trichoderma fungi. By contrast, in the second year of the test period some characteristic groups of the microbiota that could be bred showed increased activity in the soil taken from the transgenic maize root zone in August. The same trend was also shown by the tests on total microbiological enzyme activity, which developed in the first year of the experiment and then remained unchanged. These results are indicative of the effects of the change of the nutrient conditions that had already manifested in the short run and that lead to a change in the ratios between the groups of microbes tested, i.e., to a change in the composition of the soil biota. Our results point out the necessity of longer term experiments and of the comparison of different lines of maize.

Our experiments are supported by the Ministry of Education (BIO-00024/2000) research program.

Angerer, I., Bíró B., Köves-Péchy K., Anton A., Kiss E. (1998): Agrokémia és Talajtan, 47:297-305.





1 Hungarian Academy of Sciences Plant Protection Institute, Ecotoxicology Department, Budapest 2. Faculty of Agriculture, Minia University, Minia, Egypt 3. Szent István University, Horticulture Faculty, Budapest 4. Hungarian Museum of Natural History, Zoology Collection, Budapest

According to Losey et al. (Nature, 399: 214), the CryIA toxin containing pollen of Bt maize lines reduces the populations of the caterpillars of the protected Danaus plexippus. The criticism of the above assertion argued that the dose of the pollen applied (i.e., the truncated and modified cryIA gene-dependent toxin of Bacillus thuringiensis) had not been established. As a model, D. plexippus pointed to the potential endangerment of protected butterflies. The toxin content may vary in the pollens of Bt maize varieties. In the case of a 135 pollen/cm2 density on Asclepias sp. in the case of the Novartis varieties originating from the Bt11 line/event (Alpha Bt, Pelican Bt) 46%, in the case of Bt176 (Occitan Cb, Furio Cb) 65% of the D. plexippus caterpillars were killed (Hansen and Obrycki, 1999). Our experiment was carried out on the YieldGard Bt variety originating from the MON 810 line, which is resistant to Scolytus multistriatus, as well as with its maternal line (DK 440; FAO number: 330). The pollen of YieldGard is CryIAb toxin positive. Answers were sought to the following questions: (A.) What period is the pollen shedding of maize limited to? In our case, in 2001 this started on the 74th day after sowing, and pollen rearrangement took place even on the 88th day. On the 81st day - 25 July - 60% of the pollen sacks were open. As a result of the differences between maize varieties in terms of sowing dates and growing seasons, the period of pollen shedding varies between July and August in Hungary; (B.) What is the pollen output of maize and what is its vertical distribution like? The variety concerned produced 34-37 kg/ha dry pollen (to be compared to 4.07 ± 0.66 mg dry pollen/10 pollen sacks; 403± 108 flowers / tassel; approx 1209 pollen sacks / tassel, approx 492 mg dry pollen / plant; 70-75,000 plants per ha), most of which ended up on the hairy maize leaves. At the end of July still green (below which 1-2 yellow and 3-4 brown), on the 1st-4th leaf levels from below, in the case of silage maize density (100,000 plants per hectare) only a few pollens are found (29 ± 21 pollen/cm2). On the leaf levels containing the female flowers (5-7) a substantial quantity of pollens accumulated (e.g., on the 5th leaf level: 641 ± 79 pollens/cm2); (C.) Can an 'effective' quantity (estimated between 100 and 500 pollens/cm2) of pollen be stuck on the leaves of the herbs along the edges of the field. The quantity of the pollens of maize (globules not easily carried by the wind) dramatically declines in the 20 m zone along the edge of the field, which may, however, be substantially modified by the prevailing wind direction. The shape of the leaf and its hairiness also influences the sticking of pollens (compare: Urtica spp. > Aristolochia clematitis > Euphorbia spp. > Picris hieracioides > Chondrilla juncea); (D.) Where does the protected Lepidoptera species live? Most of them are not on ruderal areas. The caterpillars of several species living on the edges of the fields (e.g., Schinia spp.) do not eat leaves in a substantial part of their development; (E.) Do the larval states of the species concerned coincide with the pollen shedding period of the Bt varieties? A time series analysis was carried out on the MTM collection; (F.) How vulnerable are the non-targeted Lepidoptera species? The basic experiments were carried out on Plodia interpunctella larvae. On feed containing approx. 50% of the CryIAb concentration of green maize originating from above-ground parts of YieldGard in mid-June (16% dry mass) 56 ± 15% of the caterpillars reared from hatching, were killed. The growing time of the survivors increased (by 44 ± 5% until appearance of imago), and their pupa weight dropped (by 48 ± 21%). A 0.5% YieldGard pollen content of the feed had no measurable side-effect in the case of 1% (of 35% dry matter, wet pollen; approx 3% CryIAb) only the development period increased by 7 ± 1%.

Our experiments are supported by the Ministry of Education (Bio-00024/2000)