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EC-sponsored Research on Safety of Genetically Modified Organisms - A Review of Results
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image Impact and fate of pesticidal proteins from Bacillus thuringiensis spp in tropical soils and waters as influenced by clays

Background and objectives

The microbe Bacillus thuringiensis (Bt) produces a crystal which is commonly used to control the presence of insect larvae on crops. Preliminary studies have shown that Bt toxins (subsp. kurstaki, that protects against a range of butterfly and moth larvae) can bind on clays. Such binding reduces the biodegradation of the toxins but does not eliminate their toxicity to insect larvae. However, few studies on the persistence and toxicity of Bt toxins in soils and sediments have been conducted. We have chosen to study the interaction of Bt toxins with mineral colloids such as reference clays, soil-derived-clays, and natural soils, and to study the effects of such surface interactions on the persistence and pesticidal activity of these toxins in soils and waters. This study will allow us to evaluate any potential effects on tropical soils and waters of toxins produced by genetically modified organisms that contain genes that code for these pesticidal toxins (i.e., transgenic plants and micro-organisms).


Approach and methodology

We prepared Bt toxin/colloidal clay complexes by equilibrium adsorption and binding of the toxins onto test clays. Larvae of insects from the tropics (pests and non-pests) were used in bioassays to assess the toxicity of the free toxins and complexes.

To compare the bioavailability of free and bound toxins, we investigated microbial degradation using inocula from tropical soils. CO2 evolution, enzyme activities and species diversity were measured to evaluate the effects of Bt toxins on the activity, ecology and population dynamics of soil microbiota.

Adsorption and binding assays were performed on soils and their clay fractions to evaluate the persistence and effects of the toxins.
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Main findings and outcome

A multilinear regression equation was found to account for the adsorption of true toxin (63 kDa) of Bt tenebrionis on a reference clay (Na-montmorilonite). The distribution of the toxin between the bulk solution and the clay surface appears to be regulated by the ionic strength of the medium and to a lesser extent by its pH. The toxin has two forms: that adsorbed from the surrounding water solution at very low ionic strength and the additional amount adsorbed at different ionic strengths. The first fraction is not easily desorbed by water alone and can be considered as irreversibly bound. This bound form is not toxic in bioassays with larvae of Leptinotarsa decemlineata (Colorado potato beetle). The second form can be easily desorbed when the ionic strength is lowered and has been shown to be very active.

In vivo, the ionic strength and pH in the midgut of larvae may change and desorb the toxin from the clay. Additionally, the adsorbed toxin can safely reach the receptors, possibly because it may escape proteolysis in the very first part of larvae's alimentary canal. Experiments on soils and clay from soils seem to confirm these findings.

These studies address whether the Bt toxins produced by GMOs can accumulate in soils. The model, with some corrections/ adjustments could be used as a forecasting tool to monitor the behaviour of toxins in soil environments. Early results of microbiological assays indicate that the microbial populations (bacteria, fungi, and actynomicetes) are not quantitatively or qualitatively influenced. Bioassays on non-target Coleoptera species, which spend part of their life cycle in the soil, indicate that the purified Bt tenebrionis toxin (63 kDa) is not effective against earthworms (Eisenia foetida) when mixed with soil. Similar studies will be performed on true toxin produced by Bt kurstaki, as this toxin is often present in transgenic crops such as corn.
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Conclusions

The improvements in the potency and persistence of Bt formulations, as well as the engineering of transgenic organisms able to express the introduced Bt genes, have created the potential problem of the accumulation of Bt toxins in the environment, especially in the residues of transgenic organisms (particularly plants). This could be harmful to non-target species and could result in the selection of toxin-resistant target species.

The accumulation of the toxins is particularly important in soils and sediments where the toxins can bind on colloidal particles (e.g., clay minerals) and, thereby, be rendered less accessible to microbial degradation. The model developed for Bt tenebrionis is a starting point to predict the behaviour of this toxin in soil environments. Moreover, it can account for the high toxicity of adsorbed toxin. Bt toxin was shown to have negligible effects on soil microbiota and non-target organisms such as earthworms.

 

Major publications

Gonzalez P., Fereres A., “Risk assessment of Bt toxins adsorbed or bound to clay minerals”.
Phytoparasitica, 27 (4), 1999, p. 301.

Muchaonyerwa P., Chenu C. and Pantani O.L., Devenir dans les sols de la protéine insecticide de Bacillus thuringiensis var tenebrionis: adsorption sur les fractions argileuses de sols tropicaux, Poster presentation by C. Chenu, Sixièmes journées Nationales de L’Etude des Sols, Nancy, 25-28 April 2000.

Muchaonyerwa P., Chenu C., Pantani O.L. and Calamai L., Adsorption of toxin from Bacillus thuringiensis var. tenebrionis to clay fractions from tropical soils. Accepted, Oral presentation, ISMOM 2000 Soil Mineral - Organic Matter - Micro-organisms Interactions in Ecosystem Health, Naples, 22-26 May 2000.

González P., Pantani O.L., Ristori G.G. and Fereres A., Activity and persistence of Bacillus thuringiensis subsp. israelensis crystals mixed with clays against the mosquito Culex pipiens, Oral presentation, ISMOM 2000 Soil Mineral - Organic Matter – Micro-organisms Interactions in Ecosystem Health, Naples, 22-26 May 2000.

Pantani O.L. and Calamai L., Sorption of the toxin from Bacillus thuringiensis subsp. tenebrionis on na-montmorillonite, as influenced by ph and ionic strength, may also affect its bioavailability, Poster presentation, ISMOM 2000 Soil Mineral - Organic Matter – Micro-organisms Interactions in Ecosystem Health, Naples, 22-26 May 2000.
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imageResearch project
 

Contract number
IC18-CT97-0135

Period
March 1998 – February 2001

Coordinator
G.G. Ristori
Istituto per la Genesi e l'Ecologia del Suolo IGES
Firenze (IT)

 
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Partners


C. Chenu
INRA
Versailles (FR)

A. Fereres
CSIC
Madrid (ES)

O.T. Mandiringana
University of Fort Hare ALICE (ZA)

S. Mpepereki
University of Zimbabwe (ZW)

S. Madoffe Sokoine
University of Agriculture
Morogoro (TZ)

 
 
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