Research into side-effects of Bt maize (Cry3Bb1) on non-target organisms

(2005 - 2008) Biological Research Centre for Agriculture and Forestry (BBA) (since 2008 Julius Kühn Institute (JKI)), Institute for Biological Control, Darmstadt

Topic

This aim of this project is to investigate potential side-effects of pollen from transgenic Bt maize of the MON88017 line on leaf beetles that are found in the vicinity of maize fields. Since this maize produces the Bt toxin Cry3Bb1, which is effective against the Western corn rootworm (Diabrotica virgifera virgifera), a member of the leaf beetle family (Chrysomelidae), there is a possibility that other leaf beetles could be harmed as well.

Summary

Toxin biotest: The larvae from three different species of leaf beetle proved sensitive to neat Cry3Bb1 toxin on their food plants. Young larval stages in particular displayed a significantly higher mortality rate and significantly lower weight gain and pupation rate. By contrast, larvae of the European corn borer (Ostrinia nubilalis) and of the diamond-back moth (Plutella xylostella) were not sensitive to the Cry3Bb1 toxin.

From laboratory experiments and data in the literature on maize pollen disposition, it was possible to estimate that the tested leaf beetle larvae had probably not been able to ingest more than 1.6 per cent of the LD50 dose of Cry3Bb 1 toxin, as determined in assays, via the Bt maize pollen each day. The three tested species are therefore not expected to suffer any acute damage as a result of ingesting pollen from the MON88017 maize variety under field conditions. Neither were any indications of possible chronic toxicity or sublethal damage found as a result of ingesting pollen from MON88017 maize.

Pollen biotest: When maize pollen from MON88017 was fed to larvae of the green dock leaf beetle (Gastrophysa viridula) no negative effect was observed on larval mortality, weight gain or pupation rate compared with the isogenic line and the untreated control variant.

Risk analysis: A risk analysis based on data from the literature was unable to rule out a potential risk to 37 species of leaf beetle as a result of the Cry3Bb1 toxin produced by the MON88017 maize line. However, there is not enough basic data available to draw more specific conclusions or to make a more detailed analysis.

Experiment description

Green dock leaf beetles mating

Green dock leaf beetle eggs

Green dock leaf beetle larvae

Photos: Martin Felke, BBA Darmstadt

Laboratory experiments were conducted with various leaf beetles (Chrysomelidae). The larvae for the laboratory tests were bred on their host plants:

Green dock leaf beetle (Gastrophysa viridula) on common dock,
Colorado potato beetle (Leptinotarsa decemlineata) on potato plants,
Mustard beetle (Phaedon cochleariae) on Chinese cabbage, and
Chrysolina varians on St. John’s wort.

Toxin biotest: A biotest was developed for feeding neat Cry3Bb1 toxin to larvae of the different species of leaf beetle.

The test examined the effect on mortality, weight gain and pupation rate of the larvae in the first (L1) and second (L2) larval stages. In addition, LD50 values (toxin concentration at which 50% of trial organisms die) were calculated for the larvae.

Furthermore, the reaction of larvae of the European corn borer (Ostrinia nubilalis) and the cabbage moth (Plutella xylostella) to the Cry3Bb1 toxin was investigated in feed trials.

Pollen biotest: First or second-stage larvae of the green dock leaf beetle (Gastrophysa viridula), and mustard beetle (Phaedon cochleariae) were used in pollen biotests. The larvae were given pollen from the Bt maize line and the isogenic line to eat. Their weight gain (after two days), pupation rate (on day 6 or 7) and mortality (after a week) were measured. Both fresh and frozen pollen was used.

Determining the amount of food eaten by the green dock leaf beetle during larval development

In another laboratory experiment, the area of leaf eaten by the green dock leaf beetle during larval development was measured in order to estimate the amount of pollen consumed by the larvae in the field, using data from the literature on Bt maize pollen density on associate field flora, and to compare it with the laboratory data.

Research into chronic toxicity and possible sublethal effects of Cry3Bb1

The chronic toxicity of Cry3Bb1 on the development and lifespan of the green dock leaf beetle was also investigated. Around one-tenth of the LD50 dose (0.041 µg Cry3Bb1 protein) was administered throughout the larval development stage.

Food intake was measured. The weight of the larvae and pupae, the pupation time and the mortality of the larvae and pupae were also recorded. Once the adult beetles had emerged, the number of eggs laid by each female was counted each day and the lifespan of the beetles was recorded.

Risk analysis

A study of the literature was conducted to determine which of the 518 species of leaf beetle documented in Germany have a high probability of coming into contact with maize pollen or stamens. The following selection criteria were used to select the species that could be at risk from the cultivation of Bt maize line MON88017:

Habitat preference and food plant spectrum
Larval diet
Concurrence of larval activity and maize-flowering period

Results

Laboratory populations of four different species of leaf beetle were reared and biotest methods established for these species. One of the species chosen was the green dock leaf beetle (Gastrophysa viridula) which can also be used for biological control of dock. The other model organisms chosen were the mustard beetle (Phaedon cochleariae), the Chrysolina varians leaf beetle and the Colorado potato beetle (Leptinotarsa decemlineata).

Toxin test: Mortality (in %, + SD) of L1 larvae of the green dock leaf beetle after 7-day toxin biotest, based on toxin quantity applied in µg per larva. Differences between columns with the same letters are not significant.

Mortality (in %, + SD) of L2 larvae of the green dock leaf beetle after 7-day toxin biotest, based on toxin quantity applied in µg per larva. Differences between columns with the same letters are not significant.

Pollen test: Mortality (in %, + SD) of L1 larvae of the green dock leaf beetle after 7-day pollen biotest with deep frozen pollen from field samples. Differences between columns with the same letters are not significant.

Toxin biotest: Larvae of the green dock leaf beetle (L1-L3), Colorado potato beetle (L1-L2) and L1 larvae of the mustard beetle are sensitive to neat Cry3Bb1 toxin on their food plants. They display significantly higher mortality, significantly lower weight gain and, in the case of the green dock leaf beetle, a significantly lower pupation rate.

There were not enough Chrysolina varians beetles in the biotest to enable a clear evaluation of the results.

LD50 and LD90 values were determined for the L1 larvae of the green dock leaf beetle, mustard beetle and Colorado potato beetle. The larvae of the mustard beetle showed the lowest sensitivity (0.3 µg of toxin per larva), followed by those of the green dock leaf beetle (0.03 µg of toxin per larva). The larvae of the Colorado potato beetle showed the greatest sensitivity (0.018 µg of toxin per larva).

No significant differences were found between the larvae that received Bt toxin and those that did not for the European corn borer or the diamond-back moth. The mortality rate in the European corn borer was slightly higher, but not significantly so.

Pollen biotest: A biotest was established for feeding maize pollen to leaf beetle larvae. After feeding high doses of isogenic or transgenic maize pollen (470 or 750 pollen grains per larva) no effect was observed on mortality, weight gain or the pupation rate.

Among the green dock leaf beetles, the mortality rate, weight gain and – in the L1 larvae – pupation rate were significantly higher in the trial variants with pollen than in the control without pollen. There were no significant differences between the two pollen variants. For the larvae of the mustard beetle, pupation rates in all variants varied significantly.

Based on information provided by Monsanto regarding the toxin content of the pollen, it was calculated that a single pollen grain from the transgenic line Mon88017 contains 3.018 x 10-6 micrograms of Cry3Bb1 toxin. This means that green dock leaf beetle larvae (L1) have to ingest 9,907 pollen grains to reach the LD50 value for this species. L2 larvae of the green dock leaf beetle have to ingest 85,190 pollen grains and L1 larvae of the mustard beetle need to ingest 98,113 pollen grains.

In view of the quantity eaten and the expected pollen density on the food plants, it seems relatively unlikely that the larvae of the three leaf beetle species tested could ingest the necessary amount of pollen under natural conditions.

Research into chronic toxicity and possible sublethal effects of Cry3Bb1

Continuous toxin intake by the larvae of the green dock leaf beetle did not increase the mortality rate or alter the time that pupation began or the duration of pupal dormancy, or reduce the number of eggs laid. The sex ratio was even. The weight of the larvae was higher in the Cry3Bb1 variant than in the control. However, this difference was not statistically significant and could be the result of differing starting weights. The pupa and adult weight in both variants was practically identical. The average lifespan of the females was slightly higher in the control group than in the toxin variant. The average lifespan of the males was almost identical in both variants.

Risk analysis

The risk analysis, which looked at the worst case scenario, found 37 leaf beetle species for which it is not possible to rule out damage from the cultivation of MON88017 maize. These species were assigned to three different risk groups (high, medium and low potential risk: 12, 21 and 4 species respectively). However, the risk assessment can make only a rough estimate of the risk situation. The basis data needed for more specific conclusions and a more accurate analysis are not available. In particular, laboratory tests would need to be carried out on other species of leaf beetle. Based on current knowledge, it seems unlikely that larvae of the leaf beetle species examined in the laboratory could ingest a sufficient quantity of toxin through pollen on their food to result in acute mortality. Neither did the results of this project provide any indication of potential chronic toxicity or sublethal effects of Cry3Bb1 on the tested species.

Scenarios for the intake of Bt toxin by leaf beetles