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Ferdinand Copeland
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2 How much Bt toxin do genetically engineered MON810 maize plants actually produce? Bt concentration in field plants from Germany and Spain 1 The variation of Bt concentrations2 2 The risk assessment of the plants2 3 The actual Bt toxin concentrations2 4 The methods for determining Bt concentrations3 Published values of Bt concentrations4 MON810 Bt concentrations according to Monsanto5 Bt concentrations in Germany (BMBF project)6 Bt concentrations of different varieties7 Sampling8 Analysis of the Bt concentration8 Statistical analysis8 ELISA protocol8 Fresh weight or dry weight?9 Variability of Bt concentrations in the field9 Differences between fields and during the cultivation period12 Plants without Bt toxin12 Bt content in relation to fresh and dry weight 13 High variability in the field13 Consider high and low Bt levels14 Is the gene expression unstable owing to environmental factors?14 Controlled test conditions vs variable field conditions15 Standardise the methods15 Publish existing data16 Is MON810 actually effective against pests, or is the Bt toxin more potent than expected?16 Hypothesis 1: Low Bt concentrations fail to control ECB larvae or only do so incompletely16 Hypothesis 2: Even low Bt levels in MON810 control ECB larvae17 Risk assessment studies?18 Inconsistent results18 Materials used20 Sample extraction20 ELISA Protocol20 Literature22

3 1 The variation of Bt concentrations 2 The risk assessment of the plants 3 The actual Bt toxin concentrations

4 4 The methods for determining Bt concentrations

5 MON810 Bt concentrations according to Monsanto

6 Table 1: Original table from Monsanto on the Bt concentration of MON810 plants: Cry1Ab Protein Levels in YieldGard Corn Plants ( g/g fwt issue) 1 US is United States; EU is European Union 2 The mean was calculated from the analyses of plant samples from each field site 3 For the 1994 US trials, values represent the analysis of whole plants; for the remaining trials, values represent the analysis of forage tissue Whole plants were collected two weeks after pollination; forage samples were collected at the soft dough or early dent stage Means were determined from the analysis of plant samples from one site in the US and all sites in the EU A plant sample was a pool of two individual plants 4 Mean of a pooled leaf sample collected at two week intervals, from V4 stage until pollination, at one site Note: The table contains no data on Bt concentrations in either roots or pollen Source: Monsanto 2002 Safety assessment of YieldGard insect-protected event MON810 Published on agbioscom 12

7 Bt concentrations in Germany (BMBF project)

8 Table 2: Bt concentration of MON810 leaves in publicly available literature Bt concentrations of different varieties

11 Table 3: Minimum and maximum Bt concentrations, by region 14 Bt content: Bavaria: fresh w eight, 2 fields pooled, w eekly sam pling high medium medium medium 12 Monsanto 1994: 94 µg Bt/g medium low 10 8 Jehle 2005:: µg Bt/g fw 6 Meant: 22 µg Bt/g fw 4 Median: 13 µg Bt/g fw 2 0 BAY_602 BAY_605 BAY_611 BAY_618 BAY_628 BAY_703 BAY_711 BAY_724 BAY_731 BAY_806 BAY_817 BAY_826 BAY_903 BAY_909 BAY_917 BAY_924 Field/ Week Fig 1: Bt contents of MON810 leaves in Bavaria Every bar shows the levels of six (or three) plants pooled from two fields and sampled onthe same day Dates are indicated as month and day, eg BAY_602 was sampled on 2 June The levels are stacked so that the lowest line (above the dark part of the bar) indicates the lowest Bt content of that day The top end of the bar indicates the maximum value, while additional lines indicate the different levels measured in the other samples See also Figure 3, p 25

12 Borken (North Rhine-Westphalia) 12 plots on one field, sampled three times over three weeks Mean: 05 µg Bt/ g f w 2 0 BOR1,1_728 BOR1,2_728 BOR1,6_728 BOR19,10_728 BOR31,4_728 BOR31,6_728 BOR35,3_728 BOR35,7_728 BOR35,9_728 BOR7,1_728 BOR7,11_728 BOR7,3_728 BOR1,1_803 BOR1,2_803 BOR1,6_803 BOR11,7_803 BOR19,10_803 BOR31,4_803 BOR31,6_803 BOR35,3_803 BOR35,7_803 BOR35,9_803 BOR7,1_803 BOR7,11_803 BOR7,3_803 BOR1,1_817 BOR1,2_817 BOR1,6_817 BOR19,10_817 BOR31,4_817 BOR31,6_817 BOR35,3_817 BOR35,7_817 BOR35,9_817 BOR7,1_817 BOR7,11_817 BOR7,3_817 Week / plot Fig 2: Bt contents in MON810 leaves in Borken (North-Rhine Westphalia) Each bar shows the levels of three plants taken from one of twelve plots in the same field Samples were taken on three dates within three weeks The scale of the graphics is the same as Fig 1 Compared to the results from the other fields, these plans produced a much more uniform Bt content with a mean average of only 05 µg Bt/g fw in these three weeks

13 Table 4: Bt content of MON810 leaves from four different regions in Germany and Spain, by fresh and dry weight (fw, dw) of the plant material

16 Hypothesis 1: Low Bt concentrations fail to control ECB larvae or only do so incompletely

17 Hypothesis 2: Even low Bt levels in MON810 control ECB larvae

20 µ

22 Literature

23 Table 5: Tabulated fresh weight (fw) and dry weight(dw) by sampling month (month fieldsample) and field/country fw dw Month fieldsample country field N Mean Std Dev CV N Mean Std Dev Brandenburg LEB PRA SEL Bavaria FIN POI Brandenburg LEB NTR PRA SEL Spain AL1a AL1b BE BE BE Bavaria FIN POI Brandenburg LEB NTR PRA SEL Spanien AL1a AL1b BE BE BE Borken BOR1, BOR1, BOR1, BOR19, BOR31, BOR31, BOR35, BOR35, BOR35, BOR7, BOR7, BOR7, Bayern FIN POI Borken BOR1, BOR1, BOR1, BOR19, BOR2, BOR31, BOR31, BOR35,

24 fw dw Month fieldsample country field N Mean Std Dev CV N Mean Std Dev BOR35, BOR35, BOR7, BOR7, BOR7, Brandenburg LEB NTR PRA SEL Spanien AL1a AL1b BE BE BE Bayern FIN POI Brandenburg LEB NTR PRA SEL Spanien AL1a AL1b BE BE BE Spanien AL1a AL1b BE BE

25 14 12 Bt content: Bavaria: fresh weight, 2 fields, weekly sampling high medium low µg Bt/g fw POI_602 FIN_605 POI_605 FIN_611 POI_611 FIN_618 POI_618 FIN_628 POI_628 FIN_703 POI_704 FIN_711 POI_711 FIN_724 POI_724 FIN_731 POI_731 Field (FIN & POI)/Week FIN_805 POI_806 FIN_817 POI_817 FIN_826 POI_826 FIN_903 POI_903 FIN_909 POI_909 FIN_917 FIN_924 Monsanto 1994: 94 µg Bt/g fw Jehle 2005:: µg Bt/g fw Mean: 22 µg Bt/g fw Median: 13 µg Bt/g fw Fig 3: B concnetration of MON80 leaves, Bavaria Each bar gives the Bt contents of 3 plants on one sampling event on one of two locations The values are stacked so that the lower line represents the lowest Bt content measured on that day, while the top end of the bar gives the maximum Bt content of that day The second inner line shows the Bt content of the third plant

26 Fig 4: Bt concentration in MON810 leaves, Brandenburg Field( LEB, NTR, PRA &SEL)/Week LEB_523 PRA_523 SEL_523 LEB_529 PRA_529 SEL_529 LEB_606 NTR_606 SEL_606 LEB_612 NTR_612 PRA_612 SEL_612 LEB_619 NTR_619 PRA_619 SEL_619 LEB_626 NTR_626 PRA_626 SEL_626 LEB_703 NTR_703 PRA_703 SEL_703 LEB_710 NTR_710 PRA_710 SEL_710 LEB_718 NTR_718 PRA_718 SEL_718 LEB_724 NTR_724 SEL_724 LEB_801 NTR_801 PRA_801 SEL_801 LEB_808 NTR_808 PRA_808 SEL_808 LEB_814 NTR_814 PRA_814 SEL_814 LEB_823 NTR_823 PRA_823 SEL_823 LEB_830 NTR_830 PRA_830 SEL_830 LEB_904 NTR_904 PRA_904 SEL_904 LEB_912 SEL_912 LEB_921 SEL_ µg Bt/ g fw Bt content: Brandenburg fresh weigt, 4 fields, weekly sampling high medium low Monsanto: 94 µg Bt/g fw Jehle: µg Bt/gfw Mittelwert 13 µg µg Bt/g fw Median: 07 µg Bt/g fw

27 Fig 5: Bt concentration in MON810 leaves, Spanien 2006 Field (AL1a,b, Be1,2 & 3)/Week AL1b_601 BE1_601 BE2_601 BE3_601 AL1a_615 AL1b_615 BE1_615 BE2_615 BE3_615 AL1a_629 AL1b_629 BE1_629 BE2_629 BE3_629 AL1a_713 AL1b_713 BE1_713 BE2_713 BE3_713 AL1a_731 AL1b_731 BE1_731 BE2_731 BE3_731 AL1a_814 AL1b_814 BE1_814 BE2_814 BE3_814 AL1a_824 AL1b_824 BE1_824 BE2_824 BE3_824 AL1a_911 AL1b_911 BE1_911 BE2_911 BE3_ µ Bt/gfw Bt content: Spain Fresh weight, 5 fields, biweekly sampling AL1a_1009 AL1b_1009 BE1_1009 BE2_1009 high medium low Monsanto: 94 µg Bt/g fw M ean: 16 µg Bt/g fw M edian: 06 µg Bt/g fw

MULTIPLE CHOICE. Choose the one alternative that best completes the statement or answers the question. B) Blood type Frequency

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