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Does Bt protein persist or break down during the agricultural cycle?

When genetically modified Bt maize is cultivated, Bt protein enters the soil via root exudates, harvest residues and pollen deposits. If Bt maize is used as cattle feed, Bt protein could also enter the soil through liquid manure spread on the fields. Scientists from the Bavarian State Research Centre for Agriculture (Bayerische Landesanstalt für Landwirtschaft) and the University of Technology in Munich (TUM) have for the first time investigated what happens to the Bt protein throughout the agricultural cycle – from cultivation to animal feed, to the spreading of liquid manure and the following crop. They were able to gain important insights into the breakdown and persistence of Bt protein in the soil following long-term Bt maize cultivation.

Dr Martin Müller, Bavarian State Research Center for Agriculture (LfL), head of the working group on gene transfer and GMO safety research at the Institute for Crop Science and Plant Breeding, and scientific project director.

Ten sampling points were marked on each plot on the trial fields.

Soil samples were taken at various points on the Bt maize long-term monitoring sites before sowing and after the maize harvest.

Ten soil samples were extracted using the probe rod for each depth and combined to produce representative mixed samples.

Ten soil samples were extracted using the probe rod for each depth and combined to produce representative mixed samples.

Liquid manure was collected over the course of six days from cows fed on Bt maize in a long-term feeding experiment.

Liquid manure was collected over the course of six days from cows fed on Bt maize in a long-term feeding experiment.

Predefined quantities of liquid manure were taken from the slurry tanks and spread on the trial fields. At the same time, slurry samples were taken for analysis to investigate the effect of slurry storage on the Bt protein.

Predefined quantities of liquid manure were taken from the slurry tanks and spread on the trial fields. At the same time, slurry samples were taken for analysis to investigate the effect of slurry storage on the Bt protein.

Marking out the Finsing grassland plot on which slurry from cows fed on Bt maize and from cows fed with the closely related isogenic parent variety was spread.

Liquid manure from the cows fed on Bt maize was applied to the grassland areas after mowing.

Liquid manure from the cows fed on Bt maize was applied to the grassland areas after mowing.

Soil samples were taken from the sites treated with slurry to trace what happens to the Bt protein in the slurry management process of the trial farm using Bt maize as fodder.

Bt maize plants and the isogenic, Bt-free control variety grown on the slurry-treated parts of the field were harvested separately and prepared for further analyses.

The cut grass from the plots treated with slurry from cows fed on Bt maize is harvested to test for Bt protein residues.

Maize plants of the same variety treated with the same type of slurry (four repetitions) were processed at the same time.

The soil is the basis of production for farming and is a complex ecosystem, in which the individual components are closely interrelated. Organic substances, like proteins compete for binding sites in the soil or are broken down by chemical and microbiological processes. This includes the Bt protein Cry1Ab from MON810 Bt maize, which is effective against the European corn borer. Bt protein is known to enter the soil, particularly through rotting plant remains after harvesting. But it is only now that Helga Gruber, a PhD student at the LfL and TUM has investigated the extent to which this occurs and whether Bt protein can accumulate in the soil as a result of long-term cultivation. She was able to use trial fields on which, during her project, MON810 Bt maize was being grown for the eighth and ninth year in succession. These sites were therefore extremely suitable for investigating the potential accumulation of Bt protein. As a control, the isogenic (not genetically modified) parent variety was also grown on the trial fields.

The plants were harvested in the autumn. Remains of stems and roots and maize stubble were left on the field and turned under again. Soil samples were taken after harvesting and before the new crop was sown. The protein was extracted and the Bt protein analysed with a highly sensitive, specific protein detection method (ELISA).

“Our results show that Bt protein that enters the soil through harvest residues breaks down quickly. We did not find any accumulation of the protein on the long-term trial fields. In the spring before the next crop of maize was sown, we were no longer able to detect any Bt protein on any of the plots,” says Helga Gruber.

The aim: Comparable measurements

The sensitive ELISA detection test was developed beforehand for animal specimens from a feeding experiment with Bt maize at TUM’s Physiology Department (under Prof. Heinrich H.D. Meyer), and validated in line with EU guidance. The test was then checked and used for the detection of Cry1Ab in other sample materials, including animal feed, slurry, soil and harvested crops. “This validation ensured that the data would be comparable and we were able to trace the breakdown of the Bt protein through the various types of sample material,” Helga Gruber explains.

Bt proteins, like other proteins, are bound in the soil and it is possible that, even in this form, they could have an insecticidal effect on non-target organisms. “But the only way to discover the actual insecticidal effect of any Bt protein bound in the soil is to conduct a bioassay. So, in collaboration with Dr Sebastian Höss (Ecossa, Starnberg) we conducted nematode bioassays using soil from our Bt maize sites, including soil samples in which we had detected Bt protein. The evaluation of these trials will be published shortly.”

No Bt protein or genetically modified DNA detected in milk

Maize is used to feed animals that supply us with food. In another project, Dr Patrick Gürtler therefore investigated the potential effects of feeding dairy cows with Bt maize over the long term. Eighteen cows were fed GM maize for 25 months, while another group of 18 cows was fed non-GM maize. The milk yield of the two groups was compared over this period and various metabolic parameters were analysed, as well as the health of the animals. “The use of Bt maize had no impact on feeding behaviour, milk yield or animal health, or on the performance and metabolic parameters,” says Dr Gürtler, summarizing the results.

As well as these parameters, samples of blood, dung, urine and milk were taken and examined for genetically modified DNA and Bt protein. However, no elements of these were found in either the blood or the urine. Neither was any Cry1Ab DNA detected in the dung, but the animals excrete the Bt protein in their dung, so the protein does enter the slurry. “In terms of the milk, we can summarize our results by saying that no Bt protein or genetically modified DNA was detected in the milk. This means that we were unable to detect any transfer of these Bt maize components from the animal feed to the milk”.

“No indication of genetically modified DNA entering the soil via slurry”

Since Bt protein and Cry1Ab DNA could also enter the soil through liquid manure, the researchers investigated this agricultural route of entry as well. The aim was firstly to find out whether Bt protein does in fact enter the soil via slurry. Secondly, it was important to find a way of measuring the Bt protein throughout the entire agricultural process in order to be able to say to what extent the Bt protein is broken down at each stage.

During the long-term feeding study with MON810 Bt maize, a field trial was conducted using the liquid manure from the cows fed on Bt maize and from the control group. The liquid manure from the different groups was collected at different times, stored in tanks and spread on grassland and trial maize fields at predefined times that are usual in farming practice. The feed, the liquid manure from the cows, the soil of the fertilized plots and the plants were then analysed for both Cry1Ab DNA and for Bt protein.

Helga Gruber and her team were not able to detect any Cry1Ab DNA in the slurry, but did find very small amounts of the Bt protein. “This was because of Bt maize plant material that had not been fully digested,” Helga Gruber explains. Neither was this Bt protein completely broken down while the slurry was in storage.

However, the scientist was able to show that more than 95 per cent of the Bt protein is destroyed when the maize plants are processed to make animal feed, which means that the feed contains much less of the insecticide protein than the maize plants on the field.

Once the liquid manure had been spread on the fields, Bt protein could no longer be detected in the soil because the slurry was quickly broken down in the biologically active soil. Nor was any Bt protein detected in the harvest (cut grass and isogenic maize).

“Through this project we have for the first time traced the breakdown and persistence of the Bt protein throughout the cycle of slurry management on a farm growing MON810 Bt maize and feeding it to cattle. Our most important finding was to show that the Bt protein does not accumulate in the soil as a result of long-term Bt maize cultivation, and that only minimal residual quantities of Bt protein are brought onto fields in the slurry. Here, the remaining Bt protein breaks down so fast that it does not enter the feed again via the harvested crop,” says Helga Gruber summarising the results.

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