Characterization of exposure and effects of insecticidal proteins on nontarget species


Numerous Cry proteins (Cry1Ab, Cry1Ac, Cry1F, Cry9C) have been expressed in commercial corn hybrids to control the European corn borer and the southwestern corn borer (Diatraea grandiosella Dyar). However, only hybrids using either Cry1Ab or Cry1F are currently used for control of lepidopteran pests in corn. Recent EPA risk assessments have considered their possible nontarget effects, in part by confirming the relatively narrow range of toxicity for Cry1 proteins.27,28 Results from a spectrum of studies conducted on nontarget species not closely related to target pests (earthworms, daphnia, springtails, honeybees, ladybird beetles, parasitoids, lacewings) showed that ingestion of extremely high doses of Cry1Ab or Cry1F was not harmful to nonlepidopteran organisms (Tables 4.1 and 4.2).24,27,28,47,48

Historical data, however, suggest that the spectrum of toxicity for Cry1 proteins in Bt corn includes some nontarget lepidopterans.49,50 But because only moths feeding on corn tissues (primary or secondary pests) should be exposed to the Bt toxins produced by corn,51,52 little risk was perceived for nontarget moths and butterflies. However, an unanticipated route of exposure was noted for larvae of the Monarch butterfly (Danaus plexippus Linnaeus); Cry1Ab-expressing pollen and anthers naturally drift from Bt corn onto leaves of the Monarch's host plant, common milkweed, which grows as a weed in and around agricultural fields. Initial studies revealing the potential harm to Monarch larvae by Bt corn pollen53,54 resulted in a comprehensive investigation.

Several coordinated studies indicated that exposure of Monarch larvae to the Bt corn pollen should be low for Monarch larvae under field conditions,48,55 and toxicity had likely been overestimated. In particular, Hellmich et al.24 showed the acute toxic effects to Monarch larvae were produced largely due to pulverized anther contamination in pollen, a collection artifact. Results also depended on the Cry protein and event considered, and the growth stage at the time of initial exposure.24,56 Investigation of the potential effects of anthers from Bt corn indicated anthers did not pose a significant risk to Monarch butterflies based on the relatively low exposure of larvae to anthers on milkweed plants.57

Research subsequent to the findings of Losey et al.53 and Jesse and Obrycki54 illustrates the flexibility of the tiered process of testing for nontarget effects; subsequent studies both clarified the results of previous laboratory studies and extended testing to more realistic field conditions. Similarly, overall assessments of risk to Monarch butterfly populations have been iteratively revised. Screening level risk assessment for Monarchs showed the potential adverse effects of Cry1 protein exposure via corn pollen were limited to the Bt cornfield and near field edges.58 A higher-tier ecological risk assessment showed minimal impact from short-duration exposure on Monarch populations throughout the U.S. Midwest.59 Both assessments highlighted the importance of environmentally relevant exposure estimates. A subsequent regional assessment of risks from long-term exposure of Bt corn pollen to

o era


summary of Nontarget Invertebrate testing for corn expressing the Oy^b Protein24





common name

Protein source


effect endpoint


Apis mellifera

Honeybee (larvae)

bacterial derived

20 |g CrylAb per mL honey water

mean survival to

no effect


Folsomia candida


lypholized leaf

0.253, 2.53, and 25.3 |g CrylAb per

adult survival and

no mortality at 4 x fresh

tissue (MON810)

g diet


tissue expression

Chrisoperla carnea

Green lacewing

bacterial derived

16.7 |g CrylAb per g moth eggs

mean survival to

no effect



Brachymeria intermedia

Parasitic wasp

bacterial derived

20 |g Cry1Ab per mL honey water

mortality at 30 d

no effect

Hippodamia convergens

Ladybird beetle

bacterial derived

20 |g Cry1Ab per mL honey water


no effect

Danaus plexippus

Monarch (larvae)

corn pollen

dose-response to M0N810 pollen

growth reduction

no effect level > 5 x in-field


on milkweed leaves

after 4 d


Eisenia fetida


bacterial derived

200 |g Cry1Ab per g dry soil

mortality at 14 d

no effect

Daphnia magna


corn pollen

dose-response to Event176 pollen


no effect at > 20 x expression


on milkweed leaves

after 2 d

in M0N810 or Bt11

Pr ot ei n


Summary of Nontarget Invertebrate Testing for Herculex™ Corn Expressing the CrylF Protein2427'2848

Species Common Name Protein Source

Apis mellifera Honeybee (larvae) bacterial derived


640 |g CrylF per larva

Effect Endpoint mean survival to emergence

Result corn pollen (Tcl507) 2 mg pollen per larva no effect at > 200 x corn pollen expression no effect

Folsomia candida Springtail

Chrisoperla carnea Green lacewing (larvae)

Brachymeria intermedia

Parasitic wasp Ladybird beetle bacterial derived bacterial derived bacterial derived

0.63, 3.1, and 12.5 |g Cry1F adult survival and no mortality at > 79 x field exposure per g diet reproduction, 28 d

480 |g Cry1F per g diet mean survival to pupation, 13 d

320 |g Cry1F per mL diet mortality at 12 d bacterial derived 480 |g CrylF per g diet mortality at 29 d

Hippodamia convergens

Danaus plexippus Monarch (larvae) corn pollen (Tc1507) dose-response to Tc1507

no effect at > 15 x corn pollen expression no effect at > 10 x corn pollen expression no effect at > 15 x corn pollen expression growth reduction no effect level > 5 x in-field exposure

Eisenia fetida Daphnia magna

Earthworm Daphnid bacterial derived pollen on milkweed leaves after 4 d 2.26 mg Cry1F per g dry soil mortality at 14 d corn pollen (Tc1507) 100 |g pollen per mL bacterial derived 100 |g Cry1F per mL

immobilization after 2 d no effect at > 100 x field levels no effect no effect at > 104 x aquatic exposure

era i ot e

o era y

Monarch larvae showed that although the chronic effect to Monarch was significant, there remained minimal impact at the population level.60

Studies also have investigated the possibility that nontarget species might be exposed to and adversely impacted by Bt toxins through consumption of herbivorous insects in Bt corn. Even though negative indirect effects on beneficial species not susceptible to plant-incorporated Cry1 toxins have been shown, they appear to be a result of consuming poor-quality prey intoxicated from feeding on Bt corn61,62 and not related to the predators' secondary exposure to Bt toxin. Further, potential for secondary exposure through predation is reduced by evidence that feeding by some herbivores does not result in a meaningful transfer of the Bt toxin. For instance, only negligible residues of Cry1Ab protein are found in aphids feeding on Bt corn.63,64

Nontarget organisms in the soil are potentially exposed to Bt toxins and their breakdown products over extended periods;65-67 this route of exposure may differentially impact soil organisms in comparison with Bt used as a microbial insecticide.65 Cry1 toxins from Bt corn may enter the soil ecosystem through incorporation of plant residues after harvest or release to the rhizosphere during active stages of growth.68 Therefore, long-term effects of Bt corn production on the soil ecosystem are evaluated both in terms of Cry toxin persistence and effects testing on earthworms and springtails — groups that reflect integrated soil health. In the case of Cry1Ab and Cry1F proteins, there is limited persistence in soils characteristic of corn production systems,69,70 and these proteins do not appear to accumulate in field environments.71 As stated previously, toxicity testing has shown no adverse effects of Cry1Ab or Cry1F on either earthworms or springtails (Tables 4.1 and 4.2).27,28

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