Aug 15, 2008

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Persistence of the Bt toxin Cry1Ab in areas where Bt maize is grown

(2004 – 2007) Federal Agricultural Research Centre (FAL)(since 2008 Johann Heinrich von Thünen-Institut (vTI)), Institute of Agroecology; Braunschweig

Topic

In an earlier biosafety research project with Bt maize (MON810) it has already been shown that the Bt toxin (Cry1Ab) can enter the soil via roots and plant remains, and that small quantities of the protein can survive a growing season.

This project now aims to examine how long the Cry1Ab protein can still be detected in areas where Bt maize has previously been cultivated and whether this persistence is influenced by the type of soil, soil preparation or specific crop rotations.

By evaluating the various influencing factors it should be possible to make predictions about the persistence of the Cry1Ab protein in different soils. The findings will provide important insights for drawing up guidelines for the coexistence of GM and non-GM maize cultivation.

Summary

The results provide important findings for developing rules for the coexistence of GM and non-GM maize cultivation.

• On average, the Cry1Ab levels were over 30,000 times lower in the root soil than in the roots. This meant that the Cry1Ab concentrations were several times lower than the known effect threshold.
• A low presence of Cry1Ab in the bulk soil is attributed to pollen or fine roots.
• 15 months after harvest no traces of Cry1Ab were found at any of the sites.
• No movement of the Cry1Ab to deeper soil layers was observed.
• There is an exponential relationship between the Cry1Ab content and the water capacity of the soil.
• It was demonstrated that a part of the Cry1Ab toxin mineralises into CO₂, part of it is incorporated in the biomass and the rest was adsorbed depending on the clay content in the soil.

Experiment description

Fiveteen Bt maize fields have been selected so far for the research into Bt levels. Four fields are former Bt maize fields, while Bt maize was not grown on the other seven fields until 2005. Two of the trial fields were planted with Bt maize two or three years in succession.

Mixed samples of root-free soil, soil attached to roots (rhizosphere soil) and roots were collected from each field from between seven and ten sampling points.

Sampling on the former Bt maize fields took place in the summer. On the other fields, sampling took place for the first time when the Bt maize flowered and then four to six weeks after harvest, followed by sampling twice a year in the spring and autumn.

Quantification of the Cry1Ab protein

Using a detection method optimised in the previous biosafety research project (ELISA), the Cry1Ab content is measured in the bulk soil, the rhizosphere soil and roots (detection limit 0.01 nanograms per gram of soil, dry weight).

Cry1Ab content at various soil depths

The Cry1Ab concentration was measured for all fields four to six weeks after harvest in the bulk soil at various depths to investigate whether the Cry1Ab had moved in the field.

Identifying the soil parameters

At the time of sampling, data on the structure and biology of the soil are recorded. These will be checked to see whether there is any correlation between them and the Cry1Ab levels. In addition to the pH value and water retention capacity, other parameters are also recorded, such as carbon/nitrogen content, grain size and microbial biomass.

Breakdown of Cry1Ab protein in the soil

Soil containers will be used under laboratory conditions to trace the breakdown and movement of the protein in the soil with the help of marked Cry1Ab protein.

Results

Quantification of the Cry1Ab protein

The Cry1Ab levels in the roots were on average 30,000 times higher than in the rhizosphere soil.

A comparison of the Cry1Ab levels in the roots and rhizosphere soil found no correlation in either year. The different concentrations in the rhizosphere soil are probably a result of location factors, which are to be investigated further. There were significant differences between the sites.

In 2005 it was not possible to investigate any more rhizosphere soil on two fields in the autumn because of soil cultivation. In the summer of 2005, Cry1Ab was not detected in the bulk soil, with the exception of one field. In the summer of 2006 low levels of Cry1Ab were found on all fields in the bulk soil, which could be attributable to Cry1Ab transport via pollen or fine roots. Overall, the concentrations of Cry1Ab were several times lower than the known effect threshold, as established in previous research.

Persistence of the Cry1Ab protein in the soil

Four to six weeks after the harvest, the Cry1Ab levels in the bulk soil (0.01 to 0.08 nanograms per gram) were higher than in the summer, which indicates decomposition of the plant material after harvest. Just six months later, low levels of Cry1Ab were found on only three fields. 15 months after the harvest no more Cry 1Ab was found on any of the sites.

Cry1Ab levels at different soil depths

On almost all fields, the Cry1Ab protein was found only in the plough horizon (0-40 centimetres), with the highest concentrations in the top twenty centimetres. Beyond a depth of 40 cm, traces of Cry1Ab protein (less than 0.01 nanograms per gram) were found on only three out of seven fields. This indicates that there is no movement of the Cry1Ab protein to deeper soil layers on the fields under investigation.

Determining the soil parameters

There was no correlation between the pH values and the Cry1Ab levels in the rhizosphere soil in either year.

The exponential relationship between the Cry1Ab content and water capacity observed in previous years was confirmed with the data from 2007. The water capacity depends primarily on the particle size and the amount of organic substance in the soil. A corresponding correlation was found between the Cry1Ab content and the particle size fractions (sand, silt, clay), the cation exchange capacity and the total carbon content.

Breakdown of the Cry1Ab protein in the soil

Overall, the breakdown experiment shows that 30-50% of the Cry1Ab toxin added was broken down within one month and that the breakdown process continues beyond that. Depending on the initial concentration, only 0.007 or 0.01 per cent of the total Cry1Ab amount added was still present in the soil after one month as a bioavailable, biologically active toxin. It was shown that part of the Cry1Ab toxin mineralises into CO₂, part of it is incorporated in the biomass and the rest is adsorbed depending on the clay content of the soil.