Bt-maize and non-target organisms Basel, October 1999 1. SUMMARY . 4 2. INTRODUCTION . 4 3. EFFECTS OF BT-MAIZE ON NON-TARGET INSECTS - REGULATORY AND NON- REGULATORY REQUIRED STUDIES . 10 STUDIES ON THE IMPACT OF BT MAIZE TO NON-TARGET INSECTS.10
48-hour static renewal toxicity of pollen from the geneticallymodified maize to water fleas (Daphnia magna) .11
Single dose test evaluating toxicity to earthworms (Eisenia foetida)using CryIA(b) enriched maize leaf protein .11
Effect of pollen from the genetically modified maize on lady beetle(Coleomegilla maculata) larval development.12
Effect of pollen from the genetically modified maize on lady beetle(Coleomegilla maculata) larval development.13
Effect of Bt maize pollen on larval honeybee (Apis mellifera L.)development .13
Effect of Bt maize pollen on larval honeybee (Apis mellifera L.)development .14
28-day survival and reproduction study in collembola (Folsomia candida,common name springtails) using CryIA(b)-enriched maize leaf protein .14
Impact of transgenic maize expressing truncated CryIA(b) protein onseveral non-target insect populations: Diptera, Hymenoptera, andColeoptera (Coccinellid family) as well as Homopterans.15
Effects of CryIA(b) protein on several insect populations:Diptera, Hymenoptera, Coleoptera and Lepidoptera .16
Effect of Bt maize on the development of aphids (Rhodopalosiphum padi)and of green lacewings (Chrysoperla carnea), their natural predators .16
Oviposition of European corn borer (Lepidoptera: Pyralidae) and impactof natural enemy populations in transgenic versus isogenic corn.17
Effects of Bt maize on non-target arthropods under field conditions.17
Field and laboratory evaluations of transgenic Bacillusthuringiensismaize on secondary lepidopteran pests (Lepidoptera: Noctuidae).17
Preimaginal development, survival and field abundance of insectpredators on transgenic Bacillus thuringiensis maize. .18
STUDIES ON THE IMPACT OF BT MAIZE TO THE NON-TARGET INSECTS MONARCH BUTTERFLY AND GREEN LACEWING.19 3.2.1. EFFECT OF BT-MAIZE ON MONARCH BUTTERFLIES.19
Future studies on monarch butterflies .22
3.2.2. EFFECTS OF BT-MAIZE ON GREEN LACEWING .23
Study on various insect predators on Bt maize .23
Effects of Bt maize on various arthropods, including green lacewings .23
Effects of Bt maize on the aphid Rhodopalosiphum padi and of the greenlacewing Chrysoperla carnea feeding on it.24
Effect of the Bt protein (CryIA(b)) on green lacewing (Laboratory feedingstudy “Hilbeck et al.” No.1).24
Effect of the Bt protein (CryIA(b)) on green lacewing (Laboratory feedingstudy “Hilbeck et al.” No.2).26
4. STUDIES ON THE IMPACT OF BT MAIZE TO OTHER ANIMALS - REGULATORY REQUIRED STUDIES . 26
Study 1: Effect of the genetically modified maize on rodents/small mammals .26
Study 2: Effect of the genetically modified maize on birds (Bobwhite quail).27
Study 3: Evaluation of transgenic event 176 Bt-maize in broiler chickens .27
5. USEFUL WEBSITES . 28 6. FURTHER READING . 29 7. UPDATING… … … … … … … … … … … … … … … … … … … … … … … … … … … … … … … … .30 1. Summary
Bt maize represents a new, environmentally friendly way to control devastating insect pestsand ensures yield to the farmer. Novartis has developed two different types of genetically modified maize plants containingthe truncated CryIA(b) protein. CryIA(b) is an endotoxin with highly specific insecticidaleffects on certain Lepidopterans and protects the maize plants against corn borer larvae. Theprimary target insect is the European corn borer (Ostrinia nubilalis) and the pink stem borer(Sesamia spp.), devastating maize pests that, when left uncontrolled, cause an estimated $1billion in lost U.S. corn yields annually. Some types of Bt maize also provide protectionagainst other Lepidopteran pests that feed on maize, including the Southwestern corn borer,corn earworm and fall armyworm. Non-target insects are those that donot feed on maize,including other Lepidopterans such as the Monarch butterfly.
Novartis Seeds stands by the quality and safety of its Bt maize. Extensive and rigorousstudies, undertaken for review by regulatory authorities and other bodies over the past yearshave concluded that Bt maize is as safe as conventional maize. These results have beenconfirmed by more than 30 scientific committees. The overwhelming body of scientific evidence published so far supports the view that non-target species (including beneficial insects) are unaffected by the presence of Bt maize in thefields.
This compilation of data gives an overview of the field and laboratory studies that wereconducted or are currently ongoing. To get a quick overview, the organisms examined and the results of the various studies, as well as the corresponding authors are given in a summary table on the following pages (5-7).
Novartis has conducted several studies with third parties to monitor insect populationsassociated with maize cultivation since 1993, in the context of hybrid registration in Spainand France. In a continuing effort to monitor the effect of Bt maize on the environment,Novartis is planning a monitoring programme which looks at the impact of Bt maize on insectpopulations, among other parameters. Novartis is evaluating research protocols to deliverdata that can provide answers to the theoretical issues raised by individual laboratorystudies.
Additional field trials have compared the populations of insects in plots of Bt maize and non-Bt maize, as well as the impact of a conventional chemical insecticide commonly used onmaize. Results of these studies indicated no difference in the number of total insects or thenumbers in each of the specific groups, such as Coleoptera (e.g., lady beetles), Homoptera(e.g., aphids) and Hymenoptera (e.g. bees). No effects on Diptera, Lycosidae, Linyphiidae,Opiliones, Staphylinidae, Carabidae, Cicadellidae, Thysanoptera, Anthocoridae, Nabidae,Coccinellidae, Chrysopidae and Chalcoidea could be found. In contrast, treatment with theconventional insecticide had dramatic effects on the total numbers of insects and on thenumbers within specific groups, including beneficial insects, compared to the untreated plots. No effect of the Bt protein was detected on birds, broiler chickens, rodents and smallmammals.
These studies found no evidence that exposure to Bt maize protein expressed in maizeplants or pollen resulted in any toxic effect on the organisms tested. To the contrary, due toits selective toxicity to specific Lepidopteran pests (e.g., European corn borers), Bt maize willhelp preserve populations of beneficial insects that might otherwise be threatened by use ofchemical insecticides. Summary table of studies conducted to assess the effect of Bt- Maize on non-target organisms Organism studied CryIA(b) protein applied as Study design
CryIA(b) protein preparation from Laboratory study
Bt-pollen (decrease of vitality for more University*
than 50% compared to optimal diet inboth groups)
No differences in the average number California
Springtails (Collembola, Folsomia CryIA(b)-enriched maize leaf
mg protein/kg soil [0.088 ppmCryIA(b)] to 250 mg protein/kg soil[0.175 ppm CryIA(b)]
ra, Coleoptera and LepidopteraAphids (Rhodopalosiphum padi)
and green lacewings (Chrysoperla plant
Summary table of studies conducted to assess the effect of Bt- Maize on non-target organisms Organism studied CryIA(b) protein applied as Study design
Natural enemy populations of Whole transgenic Bt-176 maize
sloughing of eggs of the Europeancorn borer; no difference in densitiesof predators
Lycosidae, Linyphiidae, Opiliones, Whole transgenic Bt-176 maize
Neither reduction in quantity, nor any Candolfi
changes in population development in et al*
Coccinellidae, Chrysopidae andChalcoideaSecondary lepidopteran pests
No effects on A. ipsilon and P. nebris. Pilcher et al
reared on Bt- leaf extract were slightlylighter in pupal weight, delayed inpreimaginal development and showedtrends for lower survival
preimaginal development, fieldabundance and survival
Higher mortality rate in larvae fed with Losey et al
Higher mortality rate in larvae fed with Hansen/
Summary table of studies conducted to assess the effect of Bt- Maize on non-target organisms Organism studied CryIA(b) protein applied as Study design
and green lacewings (Chrysoperla plant
appropriate control preparation,or the native bacterial CryIA(b)
Introduction Selective mode of action of CryIA(b) protein
Since 1993, in a continuing effort to monitor the effect of Bt maize on the environment,Novartis has studied the impact of Bt maize on insect populations, among other parameters. Several studies were conducted to assess the environmental safety of the maize containingthe CryIA(b) protein. Novartis has sponsored or conducted a number of studies on the effectsof Bt maize on non-target insects, including beneficial insects as well as on birds,earthworms, aquatic organisms and mammals. The overwhelming body of scientific evidence supports the view that non-target species(including beneficial insects) are unaffected by consumption of or exposure to CryIA(b)protein.
The CryIA(b) protein in Novartis Seeds’ maize plants is selective in its insecticidal activity; itis only effective against a narrow range of insects within the order Lepidoptera (caterpillars),such as the European corn borer, the Southwestern corn borer and Sesamia. The insect species are susceptible to the protein due to the presence of unique binding sitesin their guts that ‘recognise’ CryIA(b) and allow it to exert its toxicity. By eating the maizeplant, the corn borer larva takes up the Bt protein in its digestive tract, where it binds to thelining of the corn borer’s gut. This leads to holes in the intestinal lining, which is followed byparalysis of the gut. Ultimately the corn borer stops feeding and starves. Only a few mothlarvae other than the ECB larvae have the unique binding sites that recognise the CryIA(b)protein. Therefore, the Bt protein is expected not to be toxic to other organisms, which willdigest the Bt protein in the same way they digest other proteins. This conclusion is supportedby decades of research and widespread field use of Bt-based microbial products. Mode of action of Bt-sprays
Bt sprays are generally considered to be safe for beneficial insects. They have been used inbiological pest control by organic growers and home gardeners for 40 years. However,spores (and in older sprays, β-exotoxins), which are found only in Bt sprays (i.e. not in Bt-plants which only contain the CryIA(b) protein), can contribute to adverse effects onbeneficial insects. Scientific background
Novartis has developed two different lines of genetically modified maize plants:a) Bt 176 Bt-176 maize was developed by the former CIBA Seeds AG. This maize contains three newgenes, cryIA(b) from Bacillus thuringiensis,bar from Streptomyces hygroscopicus, and bla,the gene for bacterial resistance to ampicillin. The genetically modified maize plants containtwo new proteins, a truncated CryIA(b) protein and the bar gene product, phosphinothricin-acetyl-transferase (PAT). CryIA(b) is an endotoxin with highly specific insecticidal effects onlyin lepidopterans and protects the maize plants against corn borer larvae. PAT leads totolerance against the herbicide glufosinate. Although a bacterial beta-lactamase (bla) genefor bacterial resistance to the antibiotic ampicillin is also present in Bt-176 maize, it is notfunctional in the plants, does not produce a new protein, and cannot confer antibioticresistance on the plants or organisms that consume the plants. b) Bt 11Bt-11 maize was developed by Northrup King, of the former Sandoz Inc. It contains two newgenes, cryIA(b) from Bacillus thuringiensis var. kurstaki and pat from Streptomycesviridochromogenes. The genetically modified maize plants contain two new proteins, atruncated form of CryIA(b) protein and the pat gene product, the phosphinothricin-acetyl-transferase (PAT). CryIA(b) protects the maize plants against corn borer larvae, PAT leads totolerance against the herbicide glufosinate. (see section a) Bt-176 above)In contrast to Bt-176 maize, there is no bacterial beta-lactamase (bla) gene for bacterialresistance to the antibiotic ampicillin present in Bt-11 maize.
The gene product of the bar or pat gene, PAT, can inactivate phosphinothricin, also referredto as glufosinate ammonium. This substance is a glutamine synthetase inhibitor that hasbeen developed by Hoechst Inc. as a broad spectrum herbicide. Exposure tophosphinothricin causes accumulation of ammonia to cytotoxic levels in all plant cells. Thepresence of the herbicide tolerance gene facilitates tracking of transgenic plants in breeding.
The genetically modified maize is morphologically indistinguishable from non modified maize. Molecular tools are available to identify all parts of the modified maize plants. The CryIA(b)protein in the genetically modified maize is only active against specific insects. All parts ofthe plants are safe when fed to animals, or used as base for human nutrition in any form. Nohazards relating to health consideration could be identified in the genetically modified maize. For example, extensive side-by-side analyses of the composition of Bt maize grain andconventional grain have shown that they are nutritionally equivalent. Difference of Bt protein expression in different Bt hybrids
Novartis Seeds developed and sells two lines of genetically enhanced Bt maize hybrids. Those hybrids sold as NK Brand Bt maize with YieldGard insect protection (Bt-11) expressthe Bt protein in all critical areas of the maize plant. In contrast, hybrids sold as NK Brand Btmaize with KnockOut insect protection (Bt-176) primarily express the Bt protein in theplant's green tissue and pollen. Effects of Bt-Maize on Non-target Insects (i.e., other than corn borers and other lepidopteran pests of maize) 3.1. Studies on the impact of Bt maize to non-target insects – regulatory and non- regulatory required studies
Novartis has sponsored or conducted a number of studies on the effects of Bt maize on non-target insects, including beneficial insects as well as on birds, earthworms, aquaticorganisms and mammals. In fact since 1993, Novartis has conducted field evaluations ofinsects in maize fields. These ongoing studies, which include research by independent thirdparties, monitor the effect of Bt maize on non-target insects.
• Field studies comparing the populations of non-lepidopteran insects in plots of Bt maizeand non-Bt maize, as well as the impact of a conventional chemical insecticide commonlyused on maize. Results of the Bt maize studies indicated no difference in the number of totalinsects or the numbers in each of the specific groups, such as Coleoptera (e.g., ladybeetles), Homoptera (e.g., aphids) and Hymenoptera (e.g., bees). In contrast, treatment withthe conventional insecticide had dramatic effects on the total numbers of insects and on thenumbers within specific groups, including beneficial insects, compared to the untreated plots. • Other studies evaluated the effect of Bt maize pollen on lady beetles. No differences insurvival or development were observed between lady beetles reared on Bt maize pollen andthose reared on non-Bt maize pollen. • Still more research evaluated the effect of Bt maize pollen on larval honeybees and foundthe pollen had no effect on larval honeybee development.
These studies found no evidence that exposure to Bt maize protein expressed in maizepollen resulted in any toxic effect on the organism tested. To the contrary, due to its selectivetoxicity to Lepidopteran pests (e.g., European corn borers), Bt maize will help preservepopulations of beneficial insects that might otherwise be threatened by use of chemicalinsecticides. Design of the Novartis Seeds regulatory and monitoring studies
The toxicity of the Bt maize CryIA(b) protein to non-target organisms was examined. Twoprimary test materials were used in these studies: (1) a CryIA(b)-enriched leaf proteinpreparation (referred to as Bt maize protein), obtained by extracting Bt maize leaves,enriching the protein for the CryIA(b) fraction, and lyophilising the material to yield a fineprotein powder, and (2) pollen collected from Bt maize plants (referred to as Bt pollen) thatwere homozygous for the transgenes. In addition, for certain tests comparing the activity ofBt maize protein and native CryIA(b), a cell paste containing the CryIA(b) crystal proteinproduced by fermentation of Bacillus thuringiensis var. kurstaki strain HD1-9 was used. Thiswas referred to as native CryIA(b). The specific material selected for a study was based onthe most likely route of exposure for the organism being tested (e.g., aquatic organisms wereexposed to pollen because that is the most likely part of a maize plant expressing CryIA(b) toenter an aquatic environment).
In addition to testing for potential effects of the transgenic plant products against non-targetorganisms by comparison to negative control groups, non-transgenic maize controls werealso used in most of the ecological effects studies. These controls consisted of the same testmaterial (i.e., pollen or maize protein) produced by isogenic (non-transformed) maize plantsgrown under the same environmental conditions as the transformed maize. By includingthese controls, effects of the test substance per se could be distinguished from effectsattributed to the presence of CryIA(b) in the test materials from transformed maize. Study 1: 48-hour static renewal toxicity of pollen from the genetically modified maize to water fleas (Daphnia magna) Study design
Springborn Laboratories, Inc. conducted a 48-hour static-renewal test with pollen from thegenetically modified maize (homozygous for the truncated cryIA(b) gene) and isogenic pollenon Daphnia magna. Daphnids were <24 hours old at the time of study initiation. For thedefinitive test, dose levels of 19, 32, 54, 90, and 150 mg pollen/L (containing 5.87 ? gCryIA(b)/g pollen) were employed. In addition, isogenic controls at the same pollenconcentrations as the treatment group were tested along with a negative control group. Eachtest or control concentration consisted of two replicates of 10 daphnids each for a total of 20daphnids/ concentration or control group. Daphnids were exposed for 48 hours with completerenewal of the test solutions after 24 hours. Results of the study
Mean survival was 100 percent for each of the genetically modified, isogenic, and negativecontrol groups. All daphnids in the genetically modified, isogenic, and negative controlgroups appeared normal during the study. No immobilisation or sublethal signs of toxicitywere observed. The only effect noted was a decrease in dissolved oxygen in the higher testconcentrations of both pollen groups. Dissolved oxygen concentrations were inverselyrelated to the concentration of pollen tested and were similar in equivalent concentrations ofthe genetically modified and isogenic groups. The decrease in dissolved oxygen had noeffect on the survival of the daphnids. Higher concentrations for both types of pollen werecloudy and some daphnids were observed to be coated with pollen. At 48 hours, the EC50based on immobilization was >150 mg pollen/L for both the genetically modified and isogenicgroups. Based on these results, the NOEC was 150 mg genetically modified or isogenicpollen/L (the highest concentration tested). Study 2: Single dose test evaluating toxicity to earthworms (Eisenia foetida) using CryIA(b) enriched maize leaf protein Study design
Springborn Laboratories, Inc. completed a 14-day study on the toxicity of the geneticallymodified maize to earthworms based on OECD earthworm testing guidelines. Test groupswere exposed to the protein preparation from leaves of the genetically modified maize[0.07% CryIA(b)], isogenic protein, or represented a negative control group. A preliminary 14-day study was conducted at 455, 90.9, and 18.2 mg total leaf protein/kg soil. There were noadverse effects on growth or survival of worms at any concentration in the pilot. Based onthese results, a single high concentration of 500 mg maize protein/kg soil (0.35 mg CryIA(b)protein/kg soil) was selected for the definitive study. An isogenic control at 500 mg maizeprotein/kg soil was used in addition to a negative control. Each test or control groupconsisted of four replicates containing ten worms per replicate (40 worms/concentration). Observations for mortality, toxicity, and behaviour were made on day 7 and day 14. Earthworm body weight was recorded on days 0 and 14. A reference test usingchloroacetamide was also used to verify the health of the earthworm culture and the propersensitivity of the test design. Results of the study
The LC50 for genetically modified maize protein was determined to be >500 mg protein/kgsoil (>0.35 mg CryIA(b) protein/kg soil) and the NOEC was 500 mg protein/kg soil (0.35 mgCryIA(b)/kg soil). Conclusions of the investigators
No effects on survival or signs of toxicity were noted in the worms exposed to proteinpreparations of the genetically modified maize, isogenic protein preparations, or in thenegative controls on the day 7 observations. After 14 days, no mortality or signs of toxicitywere noted in either maize protein group or the negative control. No adverse effects onearthworm body weight after exposure to transgenic protein as compared to worms exposedto isogenic protein or the negative controls occurred. Results of concurrent testing withchloroacetamide verified satisfactory performance of the test design. Study 3: Effect of pollen from the genetically modified maize on lady beetle (Coleomegilla maculata) larval development Study design
A study assessing the toxicity of Bt maize pollen (homozygous for the cryIA(b) gene) to ladybeetle larval development was conducted at Iowa State University (Ames, IA), according toEPA Guideline No. 154A-23. C. maculata larvae were reared on either transgenic maizepollen, isogenic (control) pollen, or an optimal diet of pea aphids (Acyrthosiphon pisum). Noother food source was available to the lady beetles during the study. Testing was initiatedwith first instar larvae and these were followed until adult emergence of survivors. Prior to theinitiation of the definitive study, a pilot study using hybrid Bt maize pollen, which washemizygous for the transgenes, was conducted. In this pilot study, five out of five C. maculata larvae successfully completed development to adults. In the definitive study therewere three replicates of 15 larvae/replicate in each test or control group. Larval survival,development times of the different life stages, and adult weight were measured. Results of the study
Results of the definitive study indicated that survival of C. maculata larvae reared on peaaphids was 91%, survival of larvae raised on isogenic control pollen was 43%, and survivalof larvae raised on transgenic pollen was 47%. Conclusions of the investigators
Pea aphids are an insect prey species considered to be an optimal diet for C. maculatadevelopment and the good survival observed for this group indicates that appropriateenvironmental conditions for larval development were maintained during the test. The patternof mortality observed in the two pollen groups was similar, with most mortalities occurring inthe later developmental stages (fourth instar through eclosing adult life stages). Theobserved mortalities were not attributed to the presence of CryIA(b) in the transgenic pollengroup because of similar effects in the isogenic group; rather it is suspected that the pollen(transgenic and isogenic) may not have provided sufficient nutritional factors for optimaldevelopment. Development time in both pollen diet groups was significantly longer in firstthrough fourth instars compared to development time in the pea aphid diet group. There wasno significant difference in development time between the two pollen groups. There was nosignificant difference among the three treatments in pupal development times. The meanweight of emerged adults in the isogenic pollen diet group was significantly less than themean weight of emerged adults in the transgenic pollen and pea aphid diet groups. Therewas no significant difference in the weight of adults raised on transgenic pollen compared tothose raised on pea aphids. Due to the high mortality in the isogenic and genetically modified pollen groups, NovartisSeeds is currently developing an improved testing protocol for this study. The low survival oflarvae in both pollen groups complicates interpretation of the results; however, severalresults of this study suggest that the genetically modified nature of the pollen does not affectC. maculata larval survival and development. These results include the similar survivalpatterns and similar developmental times for larvae raised on isogenic and genetically
modified pollen, and the absence of weight differences between surviving adults in thegenetically modified pollen group compared to the pea aphid control group. The goodsurvival of the group raised on pea aphids (>90%) indicates that satisfactory environmentalconditions were maintained for larval development. The results suggest that a nutritionaldeficit in maize pollen (both isogenic and genetically modified) may relate to the lowersurvival of C. maculata larvae reared to adult stage on these pollens. Study 4: Effect of pollen from the genetically modified maize on lady beetle (Coleomegilla maculata) larval development Study design
Pilcher et al published the results of a repeat lady beetle study using Bt-176 pollen in asimilar protocol, wherein higher survival was obtained by supplementing the diet with peaaphids. Results of the study
No statistically significant differences in survival or development time were observedbetween the groups reared on Bt-176 pollen and control pollen, and satisfactory overallsurvival of pollen (+aphid supplement) groups was observed. Average survival among thelady beetles reared on Bt-176 pollen (supplemented with aphids) was 89%, compared with69% survival on control pollen (supplemented with aphids) and 64% survival on aphidsalone.
Pilcher, C.D.; Obrycki, J.J; Rice, M.E.; Lewis. L.C. Preimaginal development, survival and field abundance of insect predators on transgenic Bacillus thuringiensis corn. Environm. Entomol. 26 (2), 446-454 (1997). Study 5: Effect of Bt maize pollen on larval honeybee (Apis mellifera L.) development Study design
Pollen produced by event 176-derived maize plants contains CryIA(b) protein, and ingestionof this genetically modified pollen is anticipated to be the primary route by which honeybeeswill be exposed to Bt maize. A study conducted by California Agricultural Research (Kerman,CA), evaluated whether ingestion of Bt maize pollen from event 176-derived maize plantshad any measurable effects on larval honeybees (Apis mellifera L.) maturing withinhoneycomb brood cells.
A single dose study was conducted in which approximately 1 mg of Bt maize pollen and adrop of water was administered as sole source of food of three to five day old honeybeelarvae. For comparative purposes, one control treatment included larval bees that receivedno treatment while a second control group was administered a 1 mg dose of non-transgenicpollen and a drop of water. A positive control involved the incorporation of the carbaryl
insecticide Sevin® and non-transgenic pollen into the larvae’s diet. Each treatment includedfour replicates of 25 larvae each. Following treatment, the bees were allowed to be cappedinside their source hives and later moved to a growth chamber. Those bees surviving toemergence were counted and all treatments were statistically compared to ascertaintreatment effects on larval bee survival as well as time to adult emergence. Results of the study
The studies have been performed twice. Three statistically significant groups were identified:(1) larval bees administered the genetically modified pollen had an average emergence
frequency of 95% (92.5% in the second study), while the untreated group’s value was 96%(95% in the second study), (2) a 65% (92.5% in the second study) emergence frequencyfrom the group receiving non-transgenic pollen, and (3) 4% emergence from the carbaryltreated group (second Study: 6.25% from the potassium arsenate treated group). Conclusions of the investigators
The cause of the reduced emergence frequency in the non-transgenic pollen group isunclear. Relative differences in hive vigour or genetic variability may have contributed. Therewere no differences observed among any of the groups in the average number of days toemergence, nor were any behavioural effects observed. Based upon these results, there are no measurable detrimental effects of ingestion ofCryIA(b)-containing pollen on larval honeybee development. To address the reduced emergence in the control pollen group in this study, a second similarstudy was conducted, at the request of the Canadian authorities. In this study higheramounts of pollen were used (2 mg per cell), a different positive control compound was used(potassium arsenate) and, of most relevance, the treatment groups were represented equallyamong all the hives, circumventing the problem that arose in the first study. The averagesurvival in this study was 93% among larvae that received control pollen, 95% among larvaethat received Bt maize pollen, 95% among untreated larvae, and 6 % among larvae exposedto potassium arsenate (a known insect gut toxin). Study 6: Effect of Bt maize pollen on larval honeybee (Apis mellifera L.) development Study design
Novartis is currently evaluating potential effects of Bt maize on honeybees under semi-fieldconditions in the south of Germany near Pforzheim. Mortality, foraging activity and brooddevelopment of small colonies originating from the same breeding line is examined. The beecolonies are held in cages placed over plots of Bt-maize and an isogenic non-Bt-maize at thetime of pollination of the maize plants. Bees are fed with an auxiliary food source and “apicandy” ad libitum. Results of the study
Monsanto did studies on larval and adult honeybees that showed no effect when feedingpurified CryIA(b) protein. Study 7: 28-day survival and reproduction study in collembola (Folsomia candida, common name springtails) using CryIA(b)-enriched maize leaf protein Study design
A 28-day chronic toxicity and reproduction study was conducted by Springborn Laboratories,Inc., using the collembolan, Folsomia candida. Collembolans were exposed to one of threesoil concentrations of protein extracted from Bt maize (corn) leaves ("Bt maize leaf protein"),protein extracted from nontransgenic maize leaves ("control maize leaf protein"), or untreatedsoil. Each test or control group consisted of forty 10-12 day-old collembola subdivided intofour replicates of 10 animals each. The concentrations of Bt maize leaf protein used were125 mg protein/kg soil, 250 mg protein/kg/soil, and 500 mg protein/kg soil. The CryIA(b)delta-endotoxin content of the Bt maize leaf protein was 0.07%, therefore the testconcentrations in terms of CryIA(b) were 0.088, 0.175, and 0.35 mg/kg soil. A concentrationof 500 mg control maize leaf protein/kg soil was used as a control for possible effects frommaize protein per se. The collembolans in each replicate of each treatment group were
provided with 2 mg yeast as food on days 0 and 14. Since collembolans live within soilsubstrate, it was not possible to observe the condition of the animals until study terminationwithout unduly disrupting the test system. Adult survival and reproduction were analysed atstudy termination. Results of the study
The NOEC (no effect concentration) value was found to be 125 ppm, the maximumacceptable toxicant concentration (MATC) range being 125 mg protein/kg soil [0.088 ppmCryIA(b)] to 250 mg protein/kg soil [0.175 ppm CryIA(b)], with a point estimate (geometricmean of the NOEC and LOEC) of 180 mg protein/kg soil [0.126 ppm CryIA(b)]. Points to be considered
Collembolans prefer to feed on saprophytic fungi found on decaying plant matter, rather thanon living plants per se (Klironomos, J.N. et al, (1992) soil. Biol. Biochem. 24: 685-692.). thescenario of pre-harvest soil incorporation of genetically modified maize is most unlikely. Therefore, collembolans would not be likely to ingest significant quantities of fresh maizeplant tissue but rather ingest partially digested plant tissue that has been colonised by fungi. It is likely that the fungi would have degraded some of the CryIA(b) protein present in theplant tissue. Therefore the actual safety margins will likely be greater since the data for theabove values assume that the organisms will be exposed to 100 percent of the CryIA(b)protein per hectare of Bt-maize.
Monsanto Company did a collembola study using a different exposure system. Instead ofadding the Bt maize leaf protein to artificial soil, they provided it to the organisms on theirbreeding substrate, a 9:1 (wt:wt) mixture of plaster of Paris and neutralised activatedcharcoal. The results showed no toxicity to Collembola.
Sims, S.R. and Martin, J.W. Effect of the Bacillus thuringiensis insecticidal proteins CryIA(b), CryIA(c), CryIIA and CryIIIA on Folsomia candida and Xenylla grisea (Insecta: Collembola). EPA Guidelines, Subdivision M, Microbial and Biochemical pest control, Registrant submitting data. January 18, 1996.
In a second study, conducted by Ricerca Inc. on behalf of Monsanto, collembola were fed amixture of up to 50% lyophilised material of Bt-maize plants mixed with yeast for 28 days. The results showed no toxicity to Collembola.
Halliday, W.R. Chronic Exposure of Folsomia candida to corn tissue expressing CryIA(b) protein. Monsanto study number XX-97-064, Monsanto Company, St. Louis, MO 63167. (1997). Study 8: Impact of transgenic maize expressing truncated CryIA(b) protein on several non-target insect populations: Diptera, Hymenoptera, and Coleoptera (Coccinellid family) as well as Homopterans Study design
Ciba Seeds conducted a small plot field study in Bloomington, IL during the summer of 1993to evaluate the impact of maize expressing the CryIA(b) endotoxin on associated populationsof insects. The study focused on beneficial predators and parasites in the orders Diptera,Hymenoptera, and Coleoptera (Coccinellid family) as well as Homopterans, which representan important food source for beneficial predators. Insect populations in transgenic hybridmaize plots were compared to populations in isogenic hybrid maize and wild type maizeplots. The study also evaluated the impact of a conventional chemical insecticide,permethrin, on insect populations in maize. There were three replicate plots of each type of
treatment. Insect populations were monitored weekly over a 10 week period from mid-June
through early September using Scentry Multigard® yellow sticky traps. Two traps wereplaced in each plot (plots were approximately 7 m long by 3 m wide). Traps were coded atcollection and sent to an independent laboratory (Ricerca, Inc) for scoring. Results of the study
Results of the monitoring study indicated no difference in the number of total insects or thenumbers in specific Orders between the transgenic maize plots and either the isogenic orwild type control maize plots. There was no shift in the taxonomic distribution of insectsassociated with the Bt maize compared to the control maize. In contrast, treatment withpermethrin had significant effects on the total numbers of insects and on the numbers withinspecific groups compared to the untreated plots. Conclusions of the investigators
The beneficial lady beetle predators (coccinellids) were particularly susceptible to permethrin. Coccinellids, dipterans, and hymenopterans represent the majority of beneficial predatorsand parasites associated with maize. The results of this monitoring study suggest thatexpression of CryIA(b) in maize should not adversely effect insects in these groups. Study 9: Effects of CryIA(b) protein on several insect populations: Diptera, Hymenoptera, Coleoptera and Lepidoptera Study design
The study was carried out in Italy, in the Po valley, in summer 1994, with a different maizehybrid carrying the same genetic modification. There were three replicate plots at twolocations for both the genetically modified hybrid and the isogenic control hybrid, which weremonitored four times. Results of the study
Insects belonging to the orders Coleoptera, Diptera, non-target Lepidoptera andHymenoptera were observed during the whole season in all plots. No significant differenceswere observed between the plots with the genetically modified maize hybrid and the plotswith the non modified maize hybrid. Only the aphid Rhopalosiphum maidis, present up to theend of July, was no longer detected later. This disappearance is most likely due to thepresence of aphid predators such as Coccinellids and was observed on both the geneticallymodified maize and the non modified control maize. Conclusions of the investigators
The results of this monitoring study suggest that expression of CryIA(b) in maize does notaffect insects in these groups, except for the expected impact of CryIA(b) protein on thetarget organisms.Study 10: Effect of Bt maize on the development of aphids (Rhodopalosiphum padi) and of green lacewings (Chrysoperla carnea), their natural predators Study design
In a two years study the influences of Bt maize on the development of aphids(Rhodopalosiphum padi) and of green lacewings (Chrysoperla carnea), their naturalpredators fed on them, was reported. Two different experiments were conducted to separatethe impact of insecticidal plants on sucking insects from the prey – predators interaction. Thespecimens of R. padi L. used for the experiment were field collected in spring and
subsequently bred separately in isolation on Bt maize Event 176 or on a nontransgenichybrid isogenic to it. Stage-specific development times until maturity were recorded. Neonateaphids were placed in isolated cells on the maize plant leaves in the laboratory. The datesafter which the exuviate were found were reported, as well as the date of the first birth. Toassess fecundity and longevity of R. padi viviparous females were isolated on parts of leavesand the number of births or dead viviparous females was recorded daily. Mortality of C. carnea exclusively fed on R. padi who had fed upon transgenic and non-transgenic maize was recorded. In daily observations the times relative to pupation andemergence of the adult lacewings were recorded, as were the deaths that occurred beforecompletion of development. Results of the study
No detrimental effects of transgenic Bt maize on postembryonic developmental time,fecundity or survival of R. padi were recorded. Moreover, no influence on preimaginaldevelopment or mortality of C carnea were observed when reared on R. padi that had fed onBt maize.
Lozzia, G.C.; Furlanis, C.; Manachini, B. and Rigamonti, I.E.; Effects of Bt corn on Rhodopalosiphum padi (Rhynchota Aphidiae) and on its predator Chrysoperla carnea Stephen (Neuroptera Chrysopidae). Boll. Zool. Agr. Bachic. Ser II, 30 (2): 153-164. Study 11: Oviposition of European corn borer (Lepidoptera: Pyralidae) and impact of natural enemy populations in transgenic versus isogenic corn Study design
In a 1994 field experiment, oviposition, predation and parasitism of the European corn borerwere recorded in transgenic and isogenic maize plants. Results of the study
No adverse impact could be detected in the transgenic plants with respect to egg masspredation, parasitism of egg masses and sloughing of eggs of the European corn borer. There was no difference in densities of predators of the European corn borer throughout theoviposition period. Parasitism of European corn borer larvae by Eriborus terebrans andMacrocentus grandii was not significantly different in transgenic and non-transgenic plots.
Orr, D.B. and Landis, D.L, Oviposition of European Corn Borer (Lepidoptera: Pyralidae) and Impact of Natural Enemy Populations in Transgenic versus Isogenic Corn J. Econ. Entomol. 90(4): 905-909 (1997) Study 12: Effects of Bt maize on non-target arthropods under field conditions Study design
In a field trial with normal-sized agricultural maize plots in the Burgundy (France) thearthropod fauna (including Lycosidae, Linyphiidae, Opiliones, Staphylinidae, Carabidae,Cicadellidae, Thysanoptera, Anthocoridae, Nabidae, Coccinellidae, Chrysopidae andChalcoidea) in Bt-176 maize and an isogenic non-transgenic control field was compared withfields, where Bt-sprays or synthetic insecticides were used. Soil-living animals were caught intraps, arthropods on the leaves were counted directly, or the number was determined withknock-off assays. Flying arthropods were caught in so-called yellow traps and counted.
Parasitism of the European corn borer larvae was determined throughout the growingseason of the maize plants. Results of the study
Bt-maize efficiently protects itself against European corn borer. Neither reduction in quantity, nor any changes in population development could be detected in the soil and plant dwelling fauna. The analysis of the flying fauna has to be completed. Candolfi, M. et al, manuscript in preparation. Study 13: Field and laboratory evaluations of transgenic Bacillusthuringiensis maize on secondary lepidopteran pests (Lepidoptera: Noctuidae) Study design
Field maize, genetically engineered to produce a protein derived from Bacillus thuringiensiskurstaki strain HD-1, was evaluated for its effects on lepidopteran larvae of the noctuidspecies Agrotis ipsilon (Hufnagel), Papaipema nebris (Geuneee), Pseudaletia unipuncta(Haworth), and Helicoverpa zea (Boddie). Results of the study
No effects were observed on larval survival, pupal weight, or days of adult emergence for A. ipsilon and P. nebris; however, Pseudaletia unipuncta (Haworth) reared on Bt maize leafextract were 0.068 lighter in pupal weight, delayed in preimaginal development (from 31 to41 days) and showed trends for lower survival ( 11.to 25%), as could be expected for targetorganismns of the CryIA(b) protein.
Pilcher, C.D.; Rice, M.E.; Obrycki, J.J; Lewis. L.C. Field and laboratory evaluations of transgenic Bacillus thuringiensis corn on secondary lepidopteran pests (Lepidoptera: Noctuidae) J Econ. Entomol; 90 (2), 669-678 (1997). Study 14: Preimaginal development, survival and field abundance of insect predators on transgenic Bacillus thuringiensis maize. Study design
Laboratory studies determined the effects of feeding transgenic maize pollen to threedifferent predatory species: 13-spotted lady beetle (Coleomegilla maculata DeGeer;Coleptera: Coccinelidae), insidious flower bug (Orius insidiosus Say; Heteroptera:Anthocoridae), and green lacewings (Chrysoperla carnea Stephens; (Neuroptera:Chrysopidae). Results of the study
No acute detrimental effects of the transgenic pollen were observed on preimaginaldevelopment and survival.
Pilcher, C.D.; Obrycki, J.J; Rice, M.E.; Lewis. L.C. Preimaginal development, survival and field abundance of insect predators on transgenic Bacillus thuringiensis corn. Environm. Entomol. 26 (2), 446-454 (1997). 3.2. Studies on the impact of Bt maize to the non-target insects Monarch butterfly and green lacewing 3.2.1. Effect of Bt-maize on Monarch butterflies
The monarch butterfly (Danaus plexippus) is an abundant insect. It is one of the most recognised butterflies found from central Mexico to Southern Canada. The monarch is totally dependent on the common milkweed for feeding during the larval stage. Southern Ontario and the northern United States is the traditional summer breeding ground for the monarch while winter migration takes them to the Sierra Chincua mountains near Mexico City, Mexico, to overwinter. Declining Monarch butterfly populations have been a concern for decades. It is known that many factors play a role in these declines. The most critical is the loss of vital over-wintering habitat in the butterflies' southern winter ranges. Other factors include the effect of insecticides on non-target insects, weed management practices that affect their exclusive milkweed host (mowing of highway right-of ways, ditches and pastures, which destroy the milkweed), urban sprawl, which destroy the habitat where the milkweed will grow, automobile-related mortality and, conceivably, the use of topical Bt sprays. General remarks on Monarch and the potential effects of Bt maize
A particular variety of maize sheds pollen only during a 5-10 day period. Even within a smallgeographic area, different maize hybrids will shed pollen at different times due to geneticdifferences as well as agronomic and environmental factors. It is not known whether femaleMonarchs would actually chose to lay eggs upon milkweed plants in the presence of maizepollen under field conditions, where pollen-free plants in other locations are available. Formany areas of the US, the peak time when larvae will be actively feeding on milkweed plantswill occur prior to the peak times of maize pollen shed. For example, the first generation ofMonarchs produced in the midwest occurs in June, whereas maize in this area typicallysheds pollen during mid-July to mid-August. Moreover, the location of milkweed is outsidethe range of most pollen drift, since the Iowa State study found that pollen density decreasesby 70 percent at the edge of a cornfield, and by 90 percent three meters away from the edgeof the cornfield. For these reasons it is likely that the vast majority of monarch larvaethroughout their range over a growing season are never exposed to maize pollen in nature atall. Study 1: The Losey study
Dr. J. Losey et al. of Cornell University completed a preliminary laboratory feeding study toassess the potential for Bt maize pollen to affect Monarch butterfly larvae.
Losey, J., L. Rayor and M. Carter. Transgenic pollen harms monarch larvae. Nature, 399 (6733), p 214, (1999) Study design
In a laboratory setting, Monarch butterfly larvae were placed on milkweed leaves artificiallycoated with pollen derived from Bt maize and non-Bt maize along with leaves free of pollen. Milkweed consumption, larval survival and final larval weight were recorded over a four-dayperiod. The study used pollen from a Bt-11-derived maize hybrid. Results of the study
Losey et al. found a higher mortality rate in larvae fed milkweed leaves coated with Bt maizepollen as compared to larvae fed leaves coated with non-Bt maize pollen and leaves free ofpollen. Conclusions of the investigators
Losey reported that "larvae of the Monarch butterfly reared on milkweed leaves dusted withpollen from Bt corn ate less, grew more slowly and suffered higher mortality than larvaereared on leaves dusted with untransformed corn pollen or leaves without pollen." Theprinciple investigator acknowledges that the findings are preliminary. “Our study wasconducted in the laboratory and, while it raises an important issue, it would be inappropriateto draw any conclusions about the risk to monarch populations in the field based solely onthese initial results.“ (News Release, Biotechnology Industry Organization, June 21, 1999)Indeed, Losey goes so far as to say ”Bt-corn and other transgenic crops have a hugepotential for reducing pesticide use and increasing yields. This study is just the first step, weneed to do more research and then objectively weigh the risks versus the benefits of this newtechnology.” (Professor John E. Losey, Cornell Assistant Professor of Entomology, NewsRelease, Cornell University News Service, 19 May, 1999). Points to be considered
The Losey study discounts that Bt pollen could affect milkweed's palatability, resulting inreduced consumption and/or starvation among the Bt pollen-exposed larvae. Indeed, Losey'sdata suggests that milkweed without pollen is a preferred food source for Monarch larvae.
The results of the study conducted by Dr. Losey need to be put into perspective. Among ourconcerns:
• Palatability. The results could indicate the Monarch larvae find milkweed coated in maizepollen unpalatable and thus starvation and/or malnutrition caused the increase in mortality. The author acknowledges this fact saying, "the reduced rates of larval feeding on pollen-dusted leaves may represent a gustatory response of this highly specific herbivore to thepresence of a 'non-host' stimulus." • Avoidance. Given this preference, in the wild, where Monarchs encounter milkweedplants both with and without maize pollen, females may avoid laying eggs on milkweedplants laden with pollen. • Dose-Response. Contrary to common scientific protocol, no attempt was made in thisstudy to establish a dose-response relationship. This means we cannot correlate the amountof Bt pollen the larvae consumed with the degree of larvae mortality observed. Indeed, therewas no confirmation that the Bt protein was detectable in the plant pollen. • Pollen Differences. The study did not consider differences in pollen sources caused byenvironmental conditions or genetic background. For example, pollen from different maizehybrids is known to vary in moisture level, susceptibility to fungal contamination and amountof natural plant defence compounds. As a result, differences in pollen sources may havecontributed to the observed effects. Additionally, following collection, the pollen sampleswere stored refrigerated in paper bags (instead of the standard storage at –20 to –80°C) foran unspecified period of time. • Negligible Effect. For many areas of the U.S., June is the peak time when Monarchlarvae actively feed on milkweed plants. In contrast, the peak time of maize pollen shed istypically mid-July through early August. Therefore, any potential effect would have anegligible influence on Monarch populations. • Milkweed Population and Pollen Spread Patterns. Milkweed is not typically prevalent incornfields, because broadleaf herbicides effectively control the weed. As pollenconcentrations are highest in areas immediately adjacent to cornfields, the farther the plantgrows from a cornfield, the lower the incidence of pollen. Further, a cornfield typically sheds
pollen only during a five to 10 day period. Given these factors, any potential adverse effects would be confined to those few larvae feeding on milkweed growing in very close proximity to the Bt maize during or immediately following the five-to-10 day window when the cornfield pollinates. Study 2: The Hansen/ Obrycki study
Dr. L. Hansen and Dr. J. Obrycki of Iowa State University completed a similar preliminary laboratory feeding study to assess the potential for Bt maize pollen to affect Monarch butterfly larvae.
Hansen, L. and Obrycki, J. (1999) "Non-Target Effects of Bt Corn Pollen on the Monarch Butterfly (Lepidoptera: Danaidae)," 54th Compiled Proceedings, Annual Meeting, North Central Branch of the Entomological Society of America, abstract. Study design
Researchers collected milkweed leaf samples from within a Bt maize field and at the edge ofthe field. Then in a laboratory setting, Monarch butterfly larvae were placed on leaves toassess effects. The study used pollen from maize hybrids derived from Bt-176. Results of the Study
Hansen/Obrycki found that when first instar Monarch larvae were placed on the collectedmilkweed samples in the laboratory, 19 percent mortality occurred within 48 hours on leavescontaining Bt pollen, as compared to 0 percent mortality on leaves containing non-Bt pollenand 3 percent mortality among larvae not exposed to pollen. Also of note, the researchers reported finding only low levels of maize pollen on milkweedplants three meters from the cornfield. Conclusions of the investigators
The study suggests that exposure to Bt pollen may be associated with lower survival inMonarch larvae. The experiment represents a single, unreplicated laboratory trial intended asa preliminary study. The authors intend to do further experiments. Points to be considered • No attempts were made to quantify or verify the presence of the CryIA(b) protein in the Bt pollen source, or to confirm the identity or bioactivity of the pollen samples, e.g. by immunoassay or by bioassay against a known CryIA(b)-sensitive lepidopteran pest such as the ECB larvae. • Contrary to common scientific protocol, no attempt was made in this study to establish a dose-response relationship. This means we cannot correlate the amount of Bt pollen the larvae consumed with the degree of larvae mortality observed. Indeed, there was no confirmation that the Bt protein was detectable in the plant pollen. No data were provided to indicate that equivalent amounts of Bt – and non-Bt pollen were deposited on the milkweed plants representing each pollen-exposure condition. • The study did not consider differences in pollen sources caused by environmental conditions or genetic background. For example, pollen from different maize hybrids is known to vary in moisture level, susceptibility to fungal contamination and amount of natural plant defence compounds. As a result, differences in pollen sources may have contributed to the observed effects.
• The study cannot distinguish between the possibility that the Bt pollen may have causedor contributed to a true toxic effect, or whether the presence of Bt pollen affected thepalatability of the milkweed or had any anti-feedant properties, possibly resulting inavoidance behaviour, reduced milkweed consumption and/or starvation of among the Bt-pollen exposed larvae. • No attempt was made to use pollen from a non-Bt hybrid that was an isogenic control forthe Bt hybrid, and no information is provided to indicate whether the Bt and non-Bt plantswere grown under comparable environmental conditions, or whether the pollen was shed orcollected under comparable conditions. Different maize hybrids are known to vary in thetiming of pollen shed, moisture levels, susceptibility to fungal contamination and in the levelsof natural plant defence compounds. Fungal contamination is known to be a limiting factor inthe conduct of laboratory insect bioassays with maize tissue (Novartis Seeds data)
Future studies on monarch butterflies
The major companies selling Bt-maize technology today, including AgrEvo USA, Dow AgroSciencesLLC, Monsanto Company, Novartis Seeds, Inc. and Pioneer Hi-Bred International, Inc., have joined together with the American crop Protection Association and the Biotechnology Industry Organisation to sponsor the following important research objectives: • Distribution of monarch larvae on milkweeds in agricultural and non-agricultural areas
• Pollen deposition and monarch distribution in milkweeds in and around corn fields
• Behaviour of monarch larvae when exposed to Bt- and non-Bt-pollen in choice and no-
• Overlap of monarch phenology and corn anthesis
• Development of a Bt pollen risk model with sensitivity analysis for key variables, and Assessment of the relative risk of monarch exposure to Bt pollen in the context of the fullrange of risk factors that impact monarch populations. 3.2.2. Effects of Bt-maize on green lacewing
Lacewing larvae (common green lacewing, scientifically known as Chrysoperla carnea andrelated species) feed on a large number of soft bodied pests, mites and insect eggs. Withhollow tusks, called mandibles, they use to pierce it's prey and suck out body fluids. After 14to 21 days the larvae pupates into a cocoon for about 14 days to emerge as an adult. Adult lacewings are green with long translucent wings and large golden eyes. Adults feed onnectar, pollen and honeydew to stimulate their reproductive process. An adult female will layabout 200 eggs. After a few days the eggs hatch and a tiny predatory larva emerges ready to eat the pests. Lacewing larvae are also known as aphid lions. Several studies, including laboratory studies, have not detected harmful effects on beneficialinsects. Study 1: Study on various insect predators on Bt maize Study design
In a two-year field study, combined with an additional laboratory feeding study, beneficialinsects, including green lacewings, were exposed to Bt maize pollen and non-Bt maizepollen. Results of the study
Pilcher et al of the Iowa State University concluded that no detrimental effects were observedin the abundance of, among others, the common green lacewing in Bt maize compared withnon-Bt maize. In addition, no difference in mortality was found.
Pilcher, C.D.; Obrycki, J.J.; Rice. M.E.; and Lewis, L.C., Preimaginal Development, Survival, and Field Abundance of Insect Predators on Transgenic Bacillus thuringiensis Corn Environ. Entomol. 26(2): 446-454 (1997) Study 2: Effects of Bt maize on various arthropods, including green lacewings Study design
Several field studies were conducted at the Università degli Studi in Milan. Results of the study
The results indicated no difference in the number or type of insects, which included greenlacewings, between the transgenic maize plots and the control maize plots.
Lozzia, G.C and Rigamonti, I.E.; Prime Osservazioni Sull’Artropodofauna Presente in Campi di Mais Transgenico ATTI Giornate Fitopatologiche, 223-228; (1998), and
Lozzia, G.C.; Rigamonti, I.E.; and Agosti, M.; Metodi di Valutazione degli Effetti di Mais Transgenico sugli Artropodi Non Bersaglio
Notiziario sulla Protezione delle plante, 8:
Study 3: Effects of Bt maize on the aphid Rhodopalosiphum padi and of the green lacewing Chrysoperla carnea feeding on it Study design
In a two-year study the influences of Bt maize on the development of aphids(Rhodopalosiphum padi) and of green lacewings (Chrysoperla carnea), their naturalpredators fed on them, was reported. Two different experiments were conducted to separatethe impact of insecticidal plants on sucking insects from the prey – predators interaction. Results of the study
No detrimental effects of transgenic Bt maize on postembryonic developmental time,fecundity or survival of R. padi were recorded. Moreover, no influence on preimaginaldevelopment or mortality of C carnea were observed when reared on R. padi that had fed onBt maize. This study is also described in section ‘Effects of Bt maize on non-target organisms: Study 8,page 16)
Lozzia, G.C.; Furlanis, C.; Manachini, B. and Rigamonti, I.E.; Effects of Bt corn on Rhodopalosiphum padi (Rhynchota Aphidiae) and on its predator Chrysoperla carnea Stephen (Neuroptera Chrysopidae). Boll. Zool. Agr. Bachic. Ser II, 30 (2): 153-164 (1998). Study 4: Effect of the Bt protein (CryIA(b)) on green lacewing (Laboratory feeding study “Hilbeck et al.” No.1)
Hilbeck et al have completed a laboratory feeding study to assess the potential forgenetically modified corn (Bt maize) to indirectly affect a non-target insect species. Thepurpose of the study was to analyse the effect of the Bt protein (CryIA(b)) on a non-targetinsect when it eats corn borers, the target of the Bt insecticidal properties.
Hilbeck, A.; Baumgartner, M. Fried, P.M.; Bigler, F; Effects of transgenic Bacillus thuringiensis corn-fed prey on mortality and development time of immature Chrysoperla carnea (Neuroptera: Chrysopidae). Environm. Entomol. Vol. 27 (2): 480-487 (1998). Study design
Green lacewing larvae (C. carnea), a natural predator of the European corn borer (ECB),were provided with ECB larvae that had fed on either a Bt or a non-Bt hybrid maize plant. Ina second part of the experiment (the ”control”), green lacewing larvae were provided with adiet of Egyptian cotton leafworm, that had also fed on either Bt or non-Bt hybrids. When European corn borers eat Bt maize, they become sick and are, therefore, a poor foodsource for the green lacewing. Egyptian cotton leafworms were included as a ”control”, based on the assumption that theywere not sensitive to the CryIA(b) protein produced by the Bt maize plants and, therefore,those which fed on the Bt plants would not be any sicker than those which fed on non-Btplants. Because of the differences in Bt toxin sensitivity between the ECB and the cotton leafworms,the authors hypothesised that any differences in effect on the green lacewing predators couldbe attributable to the Bt toxin. The study used the Bt protein found in Bt-176 (the former CibaBt maize). Results of the study
Hilbeck et al found a higher mortality rate (67%) in lacewings reared on Bt-exposedEuropean corn borer larvae as compared to lacewing larvae reared on non-Bt-exposedlarvae (37%). Conclusions of the investigators
The authors conclude that ”this was probably due to a combined effect of Bt-exposure via Bt-fed O. nubilalis (ECB) and nutritional deficiency caused by sick larvae of poor nutritionalvalue.”
Relevance of the findings of the first „Hilbeck et al.“ study
The relevance of the data from the artificial laboratory conditions of the „Hilbeck et al.“ studyto an actual field situation is questionable due to the protocol adopted. For example, the lacewings in the study were provided with a single primary food source, insome cases, only sick, dying prey larvae. In a natural field situation, green lacewing larvaefeed on a variety of insect (mainly aphids) and plant species. They would not rely upon asingle food source for their development and would not feed exclusively on either Bt-fed ornon-Bt-fed ECB larvae. Points to be discussed
The relevance of the data from the artificial laboratory conditions of the „Hilbeck et al.“ study to an actual field situation is questionable, as the authors themselves conclude: ”No conclusion can be drawn at this point as to how increased mortality and differences in development time detected in laboratory trials might translate in the field.” The results could be consistent with lacewings that have been fed only ”sick” larvae, that is, starvation, malnutrition and/or reduced palatability of the diet could be the cause of increased mortality. Mortality among the lacewing groups fed non-Bt exposed prey was 39% during the course of the study. The unaffected ”control” part of the trial assumes that Egyptian cotton leafworms are not sensitive to the CryIA(b) protein and would, therefore, all remain healthy. However, a recent paper by Müller-Cohn et al. describes CryIA(b) protein as having low but measurable insecticidal activity in Egyptian cotton leafworm larvae. Therefore, it appears likely that the control group consisting of Bt-exposed Egyptian cotton leafworm larvae were ”sick” larvae and also represented a sub-optimal diet.
No data are provided to compare the amounts of larvae consumed by each group, to assesswhether decreased palatability of the exposed species may have been a factor. Since thelacewings were not given an alternative diet, starvation among with lacewings provided onlywith exposed larvae is a reasonable possibility.
There was no attempt to establish a dose-response relationship; i.e. it was not possible tocorrelate the amount of Bt-exposed prey consumed with the degree of lacewing mortalityobserved. It is common scientific practice to establish a correlation between the ”dose” andthe ”response” when making claims that an agent is toxic or harmful. Indeed, there was noconfirmation that CryIA(b) protein was detectable in the species exposed to Bt plants.
Müller-Cohn, J.; Chaufaux, J.; Buisson, C.; Gilois, N.; Sanchis, V.; and Lereclus, C.; Spodoptera littoralis (Lepidoptera: Noctuidae) resistance to CryIC and cross- resistance to other Bacillus thuringiensis crystal toxins, J. Econ. Entomol. 89(4): 791- 797 (1996). Study 5: Effect of the Bt protein (CryIA(b)) on green lacewing (Laboratory feeding study “Hilbeck et al.” No.2) Study design
A second laboratory feeding study, conducted at the Swiss Federal Research Station forAgronomy found an increased mortality rate in green lacewing larvae fed with purified andtrypsinised Bt toxin as compared to lacewing larvae not fed with Bt toxin. Laboratory studieswere carried out to determine the effects of CryIA(b) on developmental time and mortality ofChrysoperla carnea larvae. The CryIA(b) protein was incapsulated into small paraffinespheres together with a liquid diet. After reaching the second instar, all larvae received thisartificial diet with or without CryIA(b) protein.
Hilbeck, A.; Moar, W.J., Pusztai-Carey, M., Filippini, A., Bigler, F; Toxicity of Bacillus thuringiensis CryIA(b) Toxin to the predator Chrysoperla carnea (Neuroptera: Chrysopidae). Environm. Entomol. Vol. 27 (5): 1255-1263 (1998). Results of the study
When reared only on artificial diet containing CrylA(b) protein, total immature mortality was57% compared to 30% in the untreated control. 29% of the Chrysopid larvae that receivedthe CrylA(b) protein later during their larval development, died, compared to 17% in therespective control group. No differences were detected in developmental time between thetwo groups. Conclusions of the investigators
CrylA(b) protein is toxic to C. carnea larvae at 100µg/ml diet by using encapsulated artificialdiet. Points to be considered
The relevance of the data from the artificial laboratory conditions of the “Hilbeck et al.” studyto an actual field situation is questionable due to the protocol adopted. The lacewings in the study were provided with a single primary food source, a speciallydesigned diet leading to up to 40% higher mortality even in the control group without Btprotein. This was most probably due to the high concentration of protein in the paraffinspheres. Lacewing larvae in both groups required significantly higher developmental timeswhen reared on this artificial diet, regardless of exposure to CrylA(b) protein. In a natural fieldsituation, green lacewing larvae feed on a variety of insect and plant species. They would notrely upon a single food source for their development. Novartis therefore believes thatrelevance of the data from the narrow laboratory conditions of the second “Hilbeck et al”study to an actual field situation is questionable. Studies on the impact of Bt maize to other animals – regulatory required studies Study 1: Effect of the genetically modified maize on rodents/small mammals Study design
An in vitro digestibility study, and two acute oral toxicity studies in mice were carried out byStillmeadow Inc., using either a CryIA(b)-enriched maize leaf protein preparation (0.07%CryIA(b) protein by weight) and an appropriate control preparation, or the native bacterialCryIA(b) protein of B. thuringiensis var. kurstaki strain HD1-9 (65% CryIA(b) protein byweight). Results of the studies
There were no significant differences in clinical findings or body weight gain in the twotreatment groups. The CryIA(b) protein was quickly degraded in simulated mammaliandigestive fluid. A complete discussion on the safety of the CryIA(b) protein for ingestion isgiven in the part of the dossier dealing with food and feed safety. Conclusions of the investigators
These results indicate that the CryIA(b) protein as produced in the genetically modified maizewill be of no toxicological significance to the mammalian wild fauna. Study 2: Effect of the genetically modified maize on birds (Bobwhite quail) Study design
Wildlife International Ltd. conducted an acute oral toxicity on birds (Bobwhite quail) using asingle dose of 1.4 mg/kg CryIA(b) protein from enriched leaf preparation from Bt-maize andcorresponding isogenic control preparations on 8-week old Bobwhite quail. The dose wasdetermined in a rangefinder study for toxicity. Birds were examined twice daily for 14 days. Results of the studies
No mortality occurred. There were no remarkable necropsy findings in both groups. Noadverse effects on body weight or feed consumption could be observed. Conclusions of the investigators
The acute oral LD50 was established to be >2000 mg CryIA(b) containing leafpreparations/kg equalling 1.4 mg/kg CryIA(b) protein/kg and the NOEL was 2000mg totalprotein (1.4 mg CryIA(b) protein). Study 3: Evaluation of transgenic event 176 Bt-maize in broiler chickens Study design
A 38 day feeding study evaluated whether standard broiler diets prepared with transgenicevent 176-derived Bt-maize had any effects on broiler chickens of each sex. Results of the study
No statistically significant effects in survival, feed efficiency, or body mass were observedbetween birds reared on diets prepared from transgenic or isogenic non-transgenic maize.
Brake, J.; Vlachos, D.; Evaluation of transgenic event 176 Bt-corn in broiler chickens. Poultry Science77 (5), 648-653 (1998). Useful websites Novartis homepage: Information on Monarch butterflies:
www.ipm.iastate.edu/ipm/icm/1999/6-14-1999/monarchbtwww. monarchwatch.org
General information on genetic engeneering: Pharma Information service for newsgroups: [email protected] Further reading
Barrett K.L., Grandy N., Harrison E.G., Hassan S. & Oomen P. (1994) Guidance Document on Regulatory Testing Procedures for Pesticides with Non-Target Arthropods. From the ESCORT Workshop (European Standard Characteristics of Beneficials Regulatory Testing). Wageningen, Holland, 28-30 March, 1994. SETAC-Europe. 51 pp.
Bay T; Hommes M., Plate H P. (1993) The Green Lacewing Chrysoperla carnea (Stephens). Review of Systematics, Distribution, Biology, Rearing and Use. (Die Florfliege Chrysoperla carnea (Stephens). Ueberlick ueber Systematik, Verbreitung, Biologie, Zucht und Anwendung), Mitt.Biol.Bundesanst.LandForstwirtsch No. 288, 1-175)
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Pilcher, C.D.; Rice, M.E. (1998) Management of European corn borer (Lepidoptera: Crambidae) and corn rootworms (Coleoptera: Chysomelidae) with transgenic corn: a survey of farmer perceptions. American entomologist, Vol. 44, No. 1. p. 36-4 Updating 7.1 Newest studies on monarch butterflies ( see also chapter 3.2.1) 7.1.2. Potential impact of pollen from Bt -maize Nov. 15/99 from Douglas Powell
Scientists convened in Chicago on November 2, 1999, to share preliminary results of research conducted this summer on the possible effects of genetically-engineered Bt-maize on the Monarch butterfly.
Bt-maize has proven effective at controlling European corn borer, increasing yields and lowering mycotoxin levels since commercial planting began in 1996 (for summary, see Bhatia, et al., 1999). Bt-maize, manufactured by Novartis AG, Pioneer Hi-Bred International Inc. and Monsanto Co., accounted for more than 25 per cent of the 80 million acres of maize planted in the United States in 1998 (Currie, 1999), and about one-third of the field-maize planted in Ontario in 1999 (AgCare, 1999) .
The Bt toxin used in Bt-maize is active against the Lepidoptera family of moths and butterflies, including the Monarch butterfly. However, when Bt-maize was approved in the U.S. and Canada, regulators and scientists reasoned that the impact of Bt-maize -- or more correctly the pollen from Bt-maize containing active toxin -- on Monarch populations would be minimal, given that milkweed, the desired food of Monarch larvae, is rarely found in maize fields, but in adjacent fields, that the toxin is rapdily inactivated by ultraviolet light and drought conditions, and that non-discriminate spraying for other corn pests may present a significantly higher risk to the Monarch population through chemical drift.
In May 1999, a study by Cornell University researchers published in the journal Nature (Losey, et al., 1999) indicated that pollen from Bt-maize could kill Monarch caterpillars in laboratory tests. The authors correctly recognized that the study was limited in apllicability, and that field tests would be required to determine the significance of this finding in an artificial environment. Upon publication, Dr. John Losey was quoted as saying, "We can't forget that Bt-maize and other transgenic crops have a huge potential for reducing pesticide use and increasing yields. This study is just the first step, we need to do more research and then objectively weigh the risks versus the benefits of this new technology."
Shelton and Roush (1999) responded that although the recent short correspondence in Nature reporting a laboratory study in which pollen from Bt-transgenic maize was fed to Monarch butterflies (Losey, et al., 1999) has attracted considerable coverage in the popular press, it has also widespread rebuttals and criticisms in the scientific press (Beringer, 1999; Fumento, 1999; Hodgson, 1999). Shelton and Roush (1999) also state that a previous and more relevant and realistic field study (Hansen and Obrycki, 1999) has been largely overlooked, whereby the authors examined Bt-maize pollen deposition on milkweed plants within, and adjacent to, field maize and then assayed the leaves with first instar larvae. Pollen levels were highest within the field (where Monarchs are scarce), but even there Monarch mortality was only 16 per cent.
In response to the Cornell report, a consortium of biotechnology and pesticide companies -- the Agricultural Biotechnology Stewardship Working Group (ABSWG) -- funded 17 studies to quantify the risk of Bt-maize to Monarchs (Weiss, 1999; Currie, 1999). The research was conducted during the summer of 1999 at universities in maize-producing regions of North America (BIO, 1999). Data presented at the meeting indicated that not all strains of Bt-maize are equally toxic (Brower and Zalucki, 1999); some varieties of Bt-maize may, in a theoretical or laboratory setting, harm the butterfly, while other types may not (Currie, 1999). Furthermore, it was suggested that the amount of pollen migrating to milkweeds was "likely to be dangerous to only those Monarchs feeding on milkweeds within or close to the edges of the maizefields" (Brower and Zalucki, 1999).
Although researchers have much to learn about the ecological consequences of Bt-maize on Monarchs, the findings of the meeting were, according to media accounts and discussions with some participants, generally positive. Stuart Weiss, a Stanford University expert in ecological modeling, was quoted as saying, "the worst-case scenario of this toxic cloud of pollen saturating the maize belt is clearly not the case."
Mark Sears, chair of the department of environmental biology at the University of Guelph and chair of the Ontario Corn Borer Coalition, reported that virtually all pollen grains land within 10 yards from the field, 90 per cent of which travel less than five yards (Weiss, 1999). Sears postulated that the risk of the hazard to Monarch larvae is minimal, especially after discovering that at least 500 grains of pollen per square centimeter of milkweed leaf are necessary to sicken caterpillars. After three days of accumulation during pollination season, Sears found this concentration was barely attained on nearby milkweed leaves.
Iowa State University's John Pleasants found that wind direction, rainfall and other factors significantly affect pollen concentrations on
milkweed. Pleasants found that "88 per cent of milkweed within one meter of a maize field would fall below the level where they could hurt the caterpillars and 100 per cent of the milkweed just two meters from a Bt-maize field would be Monarch-safe" (Kendall, 1999). Such findings on pollen dispersion are especially significant when coupled with planting preferences. Powell et al. (1999) found that planting the borders of a maize field to non-Bt maize was the second most prevalent implementation of Bt-refugia guidelines among 400 Ontario maize producers who planted Bt-maize in 1999, and the most common practice among those with more than 100 acres of maize .
John Losey, author of the original Cornell study, was cited as saying he believed that Monarchs might avoid milkweed near maize plants (Kendall, 1999), but also indicated during the meeting that it is "too early to be reassured, or more alarmed, based on this data" (Weiss, 1999).
Various accounts have described different conclusions from the November 2, 1999, meeting of the ABSWG, though all agreed that results were preliminary and studies were far from complete (Yoon, 1999). Brower and Zalucki (1999) identified three key areas of the problem. The effects of Bt maize on Monarch butterflies will depend on distribution and abundance of milkweed within and around the edges of maize fields, oviposition on the milkweeds, and temporal coincidence between susceptible Monarch life stages and pollen shedding from the maize crop. Review of data indicated that basic Monarch biology and ecology were poorly understood, and that data from toxicity bioassays were too preliminary to draw any conclusions. Brower and Zalucki (1999) encouraged researchers to conduct field research during the summer of 2000, exposing cohorts of Monarchs to pollen on field plants within maize fields using various Bt-maize strains and non-Bt maize and wild controls. Toxic and chronic effects of Bt also need to be determined. Oher participants, quoted in media accounts and e-mail summaries of discussions at the meeting, quantified the risk to Monarchs, based on preliminary data, as extremely remote.
European corn borer, Ostrinia nubilalis, is the most damaging insect pest of maize throughout the United States and Canada. Entomologists estimate that losses resulting from ECB damage and control costs exceed $1 billion each year (Alstad, 1997; Dekalb, 1998; Andow and Hutchison, 1998; Haag, 1999). ECB typically go through two life cycles during the maize growing season, the 2nd generation usually causing the most damage. In 18 tests over the last six years, Iowa State University researchers saw losses of 4 bu/acre or more from 94 percent of the fields they examined due to ECB (Dekalb, 1998). Very conservative estimates place the value of Bt- maize at $7-10 million annually in improved maize yields in Ontario in 1998, when about 20 percent of the crop was planted to Bt varieties. ECB damage also causes human health concerns. Maize kernel feeding by ECB often leads to infection by fungi in the genus Fusarium, including the fumonisin-producing species (Munkvold et al., 1999). Fumonisins are a class of mycotoxins. Esophageal cancer in humans has been associated with consumption of maize with high concentrations of the fumonisins (Munkvold et al., 1999). References:
AGCare. 1999. Bt Corn Effective in Destroying Corn Borer: Impact on Monarch Butterfly a Concern to Farmers. Guelph, Ontario, May 20. http://www.agcare.org/updates.html
Alstad, D.N., J.F. Witkowski, J.L. Wedberg, K.L. Steffey, P.E. Sloderbeck, B.D. Siegfried, M.E. Rice, C.D. Pilcher, D.W. Onstad, C.E. Mason, L.C. Lewis, D.A. Landis, A.J. Keaster, F. Huang, R.A. Higgins, M.J. Haas, M.E. Gray, K.L. Giles, J.E. Foster, P.M. Davis, D.D. Calvin, L.L. Buschman, P.C. Bolin, B.D. Barry,and D.A. Andow & D.N. Alstad. [Editors: K.R. Ostlie, W.D. Hutchison, & R. L. Hellmich]. 1997. Bt-corn & European Corn Borer- Long-Term Success Through Resistance Management. University of Minnesota. http://www.extension.umn.edu/Documents/D/C/DC7055.html Andow, D.A. and W.D. Hutchison. 1998. Now Or Never: Serious New Plans to Save a Natural Pest Control: Chapter 3-Bt-Corn Resistance Management. Union of Concerned Scientists
Beringer, J. E. 1999. Nature 399, 405.
Bhatia, J., Grant, S.E. and Powell, D.A. 1999. Backgrounder: genetically-engineered Bt-containing field corn. Agri-Food Risk Management and Communication Technical Report No. 11. http://www.oac.uoguelph.ca/riskcomm/plant-ag/bt-survey/bt-backgrounder.htm
BIO, 1999. Scientific symposium to show no harm to monarch butterfly. Biotechnology Industry Organization (BIO) press release. Washington, November 2.
Brower, L.P. and Zalucki, M.P. 1999. Bt-corn and its effects on Monarch butterflies: a note of caution. November 11. e-mail listserve.
Currie, B.M. 1999. Altered corn-butterflies. Associated Press, November 3.
Dekalb. December 15, 1998. Corn borers and Bt-corn. http://www.dekalb.com/dktraits/BTcorn.htm
Fumento, M. 1999. The Wall Street Journal June 25: p. 4.
Haag, Ed. March 1999. Is Bt-corn right for silage? Farm Journal - Dairy Today.
Hansen, L and Obrycki, J. 1999. http://www.ent.iastate.edu/entsoc/ncb99/prog/abs/D81.html
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Kendall, P. 1999. Monarch butterfly so far not imperiled; gene-altered corn gets an early OK in studies. Chicago Tribune November 2: p 4
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Munkvold, G.P., Hellmich, R.L., and Rice, L.G. 1999. Comparison of fumonisin concentrations in kernels of transgenic Bt maize hybrids and non transgenic hybrids. Plant Disease. 83:130-138.
Powell, D.A., Grant, S.E. and Lastovic, S. 1999. A survey of Ontario corn producers to assess compliance with refugia recommendations to manage development of resistance to genetically engineered Bt-corn in the
European corn borer, 1999. Agri-food Risk Management and Communication Technical Report No. 10. http://www.oac.uoguelph.ca/riskcomm/plant-ag/bt-survey/bt-survey.htm
Shelton, A.M. and Roush, R.T. 1999. False reports and the ears of men. Nature Biotech.: 17(9): 832
Weiss, R. 1999. Gene-altered corn's impact reassessed; studies funded by biotech consortium find little risk to monarch butterflies. The Washington Post, November 3: A3.
Yoon, C.K. 1999. No consensus on the effects of engineering on corn crops. New York Times. November 4, 1999
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Bt-maize and non-target organisms