Release and Potential Impacts of Biological Toxins 
Bt Genetically-Modified Corn Crops 
and Biopesticide Application 

F. Gagné, M. Douville C. Blaise, A. Marineau.  St. Lawrence Centre, Environment Canada, Montreal, Quebec, Canada; B. Lachance and G. Sunahara, Biotechnology Research Institute, Montreal, Quebec, Canada.

There is growing concern about the environmental impacts of products derived from biotechnology. The bacterium Bacillus thuringiensis (Bt) produces an endotoxin that is highly toxic to insects from the Coleoptera, Lepidoptera and Diptera families. Bt suspensions are routinely used to control these insects in rivers and on farmland. Moreover, corn has been genetically modified (so-called "Bt-corn") to contain and express the endotoxin gene to increase its resistance to these insects. The purpose of this study is twofold. A first objective is to assess the presence of Bt products in a major tributary of the St. Lawrence River which itself receives river waters from draining areas sprayed with Bt suspensions to control the proliferation of biting insects (e.g. mosquitoes and black flies). A second objective is aimed at studying the release of the endotoxin gene and protein into the aquatic environment from Bt-corn crops. Preliminary results have demonstrated that Bt suspensions are not lethal to earthworms after a two-week exposure period, but these animals did show a decrease in total weight when exposed to concentrations of Bt > 2.4 µg of Bt suspension. A multiple regression analysis revealed that the dose of Bt applied was significantly related to the concentration of endotoxin in earthworm tissues and to the extent of their weight loss (R = 0.953, p< 0.05). In the vicinity of Bt-corn fields, the endotoxin protein was found to be present in surface waters at a mean concentration of 0.03 µg /L and in solid media at a mean concentration of 4.3 ng/g of sediment/soil. Further studies are planned to determine whether these levels change two weeks after pollenization and to detect the presence of the endotoxin gene in the environment.

Bacillus thuringiensis (Bt) is a gram-positive facultative aerobic bacterium normally found in soils. It produces a proteinaceous crystal toxin called -endotoxin that is toxic to insects from the Coleopterus, Dipterus and Lepidopterus orders. It therefore becomes possible to use this bacterium to control larvae infestation of crop-eating insects. Furthermore, the gene coding for this protein complex has been recently introduced into corn thereby allowing it to resist against crop-eating insects. This genetically-modified corn, so-called Bt-corn, is currently cultivated in the vicinity of the St-Lawrence River where many metric tons are produced each year. Cultivation of corn in this region is by far the most important culture in terms of biomass production.

When these insects ingest the bacterium or leaves of Bt-corn, the crystal protoxin is hydrolyzed by the alkaline environment of the gut epithelium which eventually leads to the death of the insect by enteritis and septicemia. The -endotoxin was found to have some effects on non-target organisms such as the monarch butterfly, nematode, earthworms, and Chironomus riparius larvae (Charbonneau et al., 1994 ; Kondo et al., 1992 ; Losey et al., 1999). Continuing use of this biopesticide, either in the form of Bt suspensions or Bt-corn, could contaminate the soil and aquatic environment and cause adverse effects on non-target aquatic invertebrates. Thus, contamination of the environment by the -endotoxin gene and protein is certainly possible and warrants investigation.

This on-going project, which is funded by EMBRR (Environmental Management of Biotechnology for Regulation and Research, Environment Canada), has three main objectives :

1. Identify sectors in the St-Lawrence River likely to be contaminated by Bt biopesticide application practices and Bt-corn cultivation ;
2. Determine the extent of contamination in Bt, gene and expression product (i.e., -endotoxin) to the aquatic environment adjacent to Bt-corn crops or sprayed Bt suspensions;

3. Evaluate the toxicological properties of Bt products to aquatic invertebrates found in the St.-Lawrence River (earthworms Eisenia fetida andrei, nematode C. elegans, amphipod H. azteca, the midge C. riparius and freshwater mussels).

Preliminary results of this project are presented in this poster.

1) Identify tributaries of the Saint-Lawrence River likely to be contaminated by Bt products

Identify the type of application of Bt products

Seek the assistance of the Ministry of Agriculture of the province of Québec to identify sectors of Bt-corn practice and biopesticide application

Identify farmland where Bt-corn is cultivated near rivers that drain directly into the Saint-Lawrence River

Identify sectors likely to be contaminated by Bt biopesticide

Establish a sampling campaign to collect surface waters and sediment/soil samples

Biopesticide application (Gatineau River region) Collect samples 1 week before and 2 weeks after spraying of Bt suspensions (June 2001)	Bt-corn fields (Richelieu River region) Collect samples before seeding (May 14th) and 2 weeks after pollenization of corn (August 16th)

2) Detection of the -endotoxin crystal protein in the environment.

3) Exposure of Bt suspension (biopesticide spray) to earthworms.

Place 10 earthworms in 50 g of soil
(N=3 containers per test concentration)

Add concentration of Bt in each container:
0, 0.02, 0.1, 0.5, 1, 10 mg of Bt suspension

Incubate for two weeks at 20^oC

Retrieve the earthworms and measure their weight

Incubate worms in clean sand for 24 h to purge gut contents

Determine levels of -endotoxin by immunoassay in the whole tissue

and

calculate change in weights
(Initial weight (g) - Final weight (g))

Bt Product      Context      Application                 Sector
Bt suspension   Urban        To limit proliferation      To be determined
                             of biting

                Forestry     To limit proliferation      Outaouais and
                management   of biting insects           Gatineau rivers
                             (mosquitoes, black flies)

                Agriculture  To control larvae           Assomption River
                             infestations of 
                             crop-eating insects

Genetically     Agriculture  Corn, potatoes, soya and    Chateauguay,
modified-corn                canola                      Yamaska and
crops                                                    Richelieu rivers

Figure 1. Geographical distribution of corn growing areas in the St.-Lawrence River sector

Nearly 20 % of agriculture is corn-based and it is estimated that 40 % is Bt-corn. The highest density of Bt-corn biomass occurs on the south shore of the St.-Lawrence River where three major tributaries are located (Chateauguay. Richelieu and Yamaska rivers).

                                                    -endotoxin cry1ab
Sites                                    Surface water Sediment-bound 
                                             (mg/L)        (ng/g)    
I. Incoming drain 1                     not determined      5±1 2
(corn field)
II. Receiving river                       0.03±0.01         4±0.4
III. Connecting river                     0.17±0.02       0.5±0.05
IV. St-Lawrence River                     0.03±0.01        26±3

1. The corn field was located at St-Jean Baptiste de Rouville in the vicinity 
   of the Richelieu River area.
2. The data represent the mean from n=3 samples with the standard deviation.

Figure 2. Change in earthworm weights after exposure to Bt suspension

The mean weights of each treatment group were determined before and after exposure to commercial Bt biopesticide consisting in a mixture of -endotoxin and Bt suspension. The data represent the mean with the standard deviation from n=30 individuals.

The level of the -endotoxin was determined in the total tissues of the earthworms by sandwich immunoassay (Envirologix Inc., USA).

A significant relationship exists between the exposure to Bt suspensions and the levels of -endotoxin in tissues and weight loss in earthworms :

1. Bt products or genetically-modified organisms (GMO) expressing Bt -endotoxin are used at a large scale in the vicinity of the St-Lawrence River. Indeed, Bt pesticides are regularly used to control biting insects in urban, agriculture and in forest areas near rivers to control for the proliferation of mosquitoes and black flies. In addition, it is estimated that nearly 40 % of corn fields are used to grow genetically-modified corn (Bt-corn) in the province of Quebec.

2. Preliminary results indicate that river water and sediments near Bt-corn crops contain trace amounts of -endotoxin at the time of seeding. It is interesting to observe that sediments in the St. Lawrence River contain high levels of the toxin. This could be explained by 1) other sources of -endotoxin exist contaminating this system or 2) amplification of -endotoxin occurring in the environment. Future studies are planned to collect water and sediment/soil samples two weeks after pollenization of corn to determine whether Bt-corn releases the -endotoxin gene and protein into the aquatic environment. It was reported that the roots of Bt-corn release the -endotoxin in soil (Saxena and Stotzky, 2000) and that plasmid DNA could be retained in the clay component in soils and serve as a repository for Bt-DNA (Stotzky, 2000). This could increase the likelihood of horizontal gene transfers to other bacteria (Gonzalez et al., 2000) or other non-target organisms.

3. Although no mortality occurred during the first two weeks of exposure to Bt suspension, a significant decrease in the mean weight of worms was obtained at a threshold of 7.7 µg Bt/g earthworms (Figure 2). Moreover, the worms accumulated significant amounts of -endotoxin in their tissues (Figure 3). Multiple regression analysis revealed that the dose of Bt was significantly correlated to levels of Bt found in tisssues and the weight loss indicating toxic effects of -endotoxin to earthworms.

Charbonneau, C.S., Drobney, R.D., Rabeni, C.F. 1994. Effects of Bacillus thuringiensis var. israelensis on nontarget benthic organisms in a lentic habitat and factors affecting the efficacy of the larvicide. Environ. Toxicol. Chem. 13: 267-279.

Gonzalez, J. M., Barbara, J. B. and Carlton, B. C. 1982. Transfer of Bacillus thuringiensis plasmids coding for endotoxin among strains of B. thuringiensis and B. cereus. Proc. Natl. Acad. Sci. USA 79: 6951-6955.

Kondo, S., Ohba, M. and Ishii, T. 1992. Larvicidal activity of Bacillus thuringiensis serovar israelensis against nuisance chironomid midges (Diptera: Chironomidae) of Japan. Lett. Appl. Microbiol. 15 : 207-209.

Losey, J.E., Ravor, L.S. and Carter, M.E. 1999. Transgenic pollen harms monarch larvae. Nature 399: 214.

Saxena, D. and Stotzky, G. 2000. Insecticidal toxin from Bacillus thuringiensis is released from roots of transgenic Bt corn in vitro and in situ. FEMS Microbiol. Ecol. 33: 35-39.

Stotzky, G. 2000. Persistence and biological activity in soil of insecticidal proteins from Bacillus thuringiensis and of bacterial DNA bound on clays and humic acids. J. Environ. Qual. 29:691-705.