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Genetically modified Bt maize and non-target organisms

‘Escape test’ with earthworms

It is the end of October 2010. Most of the maize on the trial field has been harvested and there are only a few rows of maize left on the field. The stems and leaves have lost their moisture, and with it most of their green colour. The ripe cobs are clearly visible and are to be harvested as grain maize in the next few days. Working slowly through the stubble, scientists from RWTH Aachen University are digging holes in the ground one last time to count earthworms. They are investigating whether genetically modified Bt maize has an impact on these beneficial soil-dwelling creatures.

Eva Schultheis of RWTH Aachen University and her colleague dig for earthworms

Most of the maize trial field has been harvested. Only a few rows of maize, which are due to be harvested as grain maize, are still on the field at the end of October.

On each maize plot on the trial field earthworms are dug up and counted down to a depth of 10 cm on an area measuring 45 cm x 45 cm.

Eva Schultheis prepares the earthworm escape test. Soil from the trial field is mixed with the various Bt proteins – individually and in combination. One half of a pot is filled with soil and Bt proteins, while the other half is filled with untreated soil.

Ten earthworms are placed in the middle of the pots.

After 72 hours the researchers check the pots…

… and record how many earthworms are found in each of the two halves.

Intact earthworms and worm fragments are collected in separate containers.

Earthworms tunnel through the soil, eating soil and dead plant remains, which they digest and excrete. This mixes the soil, loosens it and ventilates it, which helps the plants grow better and helps with the breakdown of dead plant material. Earthworms are therefore very important for the soil ecosystem. If genetically modified Bt maize is grown, earthworms will ingest the remains of Bt maize plants in the soil, so will come into contact with the Bt proteins. A research group at RWTH Aachen University is taking a closer look at the possible impacts of Bt maize on earthworms.

Preliminary earthworm samples were taken at the start of the project in 2008 from various parts of the trial field, before the first crop was sown in order to obtain a benchmark value for the earthworm density on the field. “We found that the density of earthworms is very low all over the trial field,” says Eva Schultheis, running her hands through the heap of soil that she has dug up. She and her colleague find only six intact earthworms in the whole mound of earth taken from around a quarter of a square metre of the field. The volume of soil to be analysed is defined by a metal frame 45 cm square and 20 cm high, which is pressed into the ground. “In autumn 2009 we found so few earthworms that we stopped collecting samples after twenty plots,” Eva Schultheis says, pulling a fragment of earthworm out of the soil. She also collects earthworm fragments, which can be the result of worms being cut in half by the spade. These are counted separately. The earthworms are placed in alcohol on site and are identified according to species later on in the laboratory. The laboratory tests will also measure the level of any Bt proteins detected in the worms. So far, following the evaluation of the earthworm samples from the first two years of the project, it is not possible to detect any differences between the different varieties of maize, either in terms of earthworm density or the composition of the species community.

Do earthworms avoid soil containing Bt protein?

In the laboratory Eva Schultheis prepares an earthworm escape test. This is a standard method of investigating toxic effects e.g. of chemicals on soil organisms. She transported the soil for this experiment to Aachen from the maize trial field. Now she fills medium-sized flowerpots with the soil. Each pot is divided into two halves by a separating wall of cardboard. Untreated soil is placed in one half, while the other half is filled with soil to which the various Bt proteins have been added. To do this, some of the soil is passed through a fine sieve and then carefully mixed with the protein solution.

The Bt maize tested in the project produces three different Bt proteins. They are tested in this experiment individually and in all possible combinations. “The concentration of the proteins that we add to the soil is ten times as high as in the field,” says Eva Schultheis, tipping a soil/protein mixture into a measuring cup containing soil. She mixes it well and fills one half of the pot with it. “We repeat the test five times with each Bt protein,” she explains.

Once both halves of the pot are full, the separating cardboard is removed and ten earthworms are placed on the soil along the separating line. Over the next 72 hours they are free to tunnel through the soil. Then the researchers look to see how many worms are in each half of the pot. Do they avoid the soil containing Bt proteins and ‘escape’ to the other half? “We have not observed any difference so far,” says Eva Schultheis as she carefully digs in the pot with a spoon. Here too, injured or fragmented worms are counted separately and the number of dead worms is also recorded. It is too early to draw any final conclusions – the researchers still have some spadework to do for the planned repeat experiments.