# Presentation: 029448

## 	Environmental Effects Assessment for WideStrikeTM,  MXB-13 Cotton 

- 	Environmental Effects Assessment for WideStrikeTM,  MXB-13 Cotton

- Zig Vaituzis, Ph. D., Senior Scientist
- Biopesticides and Pollution Prevention Division

## Outline	

- Introduction
- Background of Risk Assessment Process
- Summary of WideStrikeTM Environmental Assessment
- SAP Questions on Environmental Effects Assessment

## INTRODUCTION

- Pesticide:
- WideStrikeTM cotton expressing Cry1F and Cry1Ac to control several lepidopteran pests

## INTRODUCTION

**Assessment includes:**
- Effects on wildlife
- Gene flow to related wild plants
- Development of weediness
- Fate of Cry1F/Cry1Ac in environment
- Effects on endangered species

**Assessment based on:**
- Data submitted to the Agency
-  FIFRA SAP recommendations,
- Consultations with scientific experts, and
- Public comments

## HAZARD ASSESSMENT PROCESS 

- Assess toxicity to representative non-target organisms by a tiered testing system
- Start with Tier I single species, high dose laboratory data
- Mortality as end point
- Maximum Hazard Dose (MHD) approach developed by AIBS
- 1996-approved  by SAP for  naturally occurring and altered MPCAs and  toxins,
- 1999-approved by SAP for protein Plant Incorporated Protectants (PIP).
-  [Cry proteins are bacterial toxins]

## HAZARD ASSESSMENT PROCESS 

- Negative results by Tier I limit testing provide a high degree of confidence that no unreasonable adverse effects are likely to occur
- (PIP) Bt Cry proteins are tested by Tier I for short term hazard assessment
- Long term adverse effects are ascertained by higher Tier long term field testing
- 2000 SAP and the NAS (2000) recommended direct field testing
- 2002 SAP recommended Tier I and field testing with emphasis on Tier I testing of agricultural field invertebrates

## RISK ASSESSMENT PROCESS

- Risk assessment based on:
- Safety Factor
- X
**E**xpected **E**nvironmental **C**oncentration (EEC)
- Data that establishes an LC50, ED50, or LD50 that is > MHD (e.g. LD50 >10 X EEC) is sufficient to evaluate adverse effects
- Levels of concern (LOC):
- Terrestrial: LD50 <  5X EEC  (EEC/LD50  = RQ =  0.2)
- Aquatic: LC50 < 10X EEC (EEC/LC50  = RQ  =  0.1)
- When LD50 is below the safety factor (a LOC), lower dose testing is necessary to establish a definitive LD50 and a Lowest Observable Effect Concentration (LOEC)

## RISK ASSESSMENT PROCESS

- Risk assessment is made by comparing the LOEC to the EEC
- When **EEC is < LOEC**, a “no adverse effect”assessment is made
- 2000 SAP thought that Tier I laboratory tests showing no detrimental effects are sufficient to make a short term hazard assessment and field studies (as higher Tier testing) be conducted when Tier I tests show toxicity
- Bt Cry endotoxins are proteins and do not have the potential to bioaccumulate resulting in delayed adverse effects
- Accumulation through the food chain is not expected to take place and therefore, chronic effects testing of protein substances is not routinely performed.

## NON-TARGET WILDLIFE TOXICITY TESTING

- SAP reports (2000 & 2002 ) recommended:
- testing of species related to the susceptible pest
- focus on invertebrates exposed to the crop
- However, testing was performed to comply with:
- all published Tier I microbial toxin requirements
- Direct field census data on the abundance of invertebrates in the field were also requested, received and evaluated to supplement the findings of the MHD Tier I testing

## NON-TARGET WILDLIFE TOXICITY TESTING

- Mammalian & avian wildlife
- Aquatic organisms
- Plants
- Adult & larval honey bees
- Parasitic wasp

- Green lacewings
- Lady beetles
- Collembola (springtail)
- Monarch butterfly
- Earthworms

**Toxicity has been evaluated for:**

**Reproductive and developmental observations made on:**

- Collembola, honey bee and green lacewing larval maturation studies

## TEST SUBSTANCE

- Form of test substances used in the studies:
- plant material (leaves, pollen)
- purified bacterially-produced Cry1F/Cry1Ac (separately and in combination)
- 2000 SAP guidance:
- plant material preferred
- bacterially-derived protein also valid where:
- the test animals do not consume plant tissue
- large amounts of Cry protein are needed for MHD testing
- plant material toxic, e.g gossypol in cotton
- Adult insect studies are generally of 30 days duration or until negative control mortality reaches 20%.
- Larval studies are  through pupation and adult emergence

## MAMMALIAN WILDLIFE

- No significant toxicity to rodents from acute oral testing at the MHD (Cry1F >600 mg a.i./kg and Cry1Ac >700 mg a.i./kg.)
- No hazard to mammalian wildlife is anticipated

## AVIAN HAZARD

- Acute single MHD oral LD50 for Cry1F/Cry1Ac (7.9:1 ratio) is >128 mg a.i./kg
- Avian 8 day dietary with 10 % cotton seed showed clinical symptoms attributed to gossypol, (8X-10X the maximum allowable in animal feeds) LC50  > 100,000 ppm
- 8 day study insufficient to assess hazards from chronic exposure
- Requested a 6-week broiler study with Cry1F/Cry1Ac-corn (no gossypol)

## AQUATIC SPECIES

- No evidence for sensitivity of aquatic (including endangered) species to anti-lepidopteran Cry proteins
- Aquatic exposure from Bt cotton is extremely small
**Freshwater and Estuarine and Marine Animal Hazard**
- Eight day dietary toxicity to the rainbow trout with a basal fish diet with 100-mg a.i./kg diet of Cry1F/Cry1Ac in a ratio of 7.9:1
-  No mortality or sublethal effects observed
- No fish hazard is expected from the proposed uses of  WideStrikeTM cotton

## AQUATIC SPECIES

**Aquatic Invertebrate Hazard**
- Acute toxicity of microbial-produced 510 g Cry1F/L + 2500 g Cry1Ac/L of test solution to *Daphnia magna* was > 298X and >23,000X the EEC for Cry1F and Cry1Ac protein
- No immobility or other adverse effects were seen during the study
- No hazard to aquatic invertebrates is expected from incidental exposure to WideStrikeTM cotton pollen

## AQUATIC SPECIES

**Aquatic and Terrestrial Plant Hazard**
- The active ingredient is an insect toxin (Bt endotoxin) that has never shown any toxicity to plants
-  Studies have been waived
-  Outcrossing issues will be addressed   separately

## TERRESTRIAL INVERTEBRATE HAZARD

- Soil Invertebrates – General issues
- October 2000 SAP concluded that earthworms and springtails (Collembola) are appropriate indicator species for Cry protein testing despite the specific nature of the Cry protein toxicity to select target species.
- Such soil organisms may be subject to long-term exposure.  However, data received by EPA indicate that such proteins degrade rapidly in field soils.
- Therefore the potential for significant soil buildup and adverse effects to non-target soil organisms are not anticipated.  Post-registration soil persistence confirmatory data, however, are required.

## TERRESTRIAL INVERTEBRATE HAZARD

- Single Species Laboratory Testing – General Comments
- The test material in several of the studies is purified microbial Cry1F and Cry1Ac protein in a ratio equivalent to the Cry1F and Cry1Ac proteins in whole WideStrikeTM cotton plants.
- In Summary:  Toxicity tests conducted with non-target arthropods and soil invertebrates indicate that no adverse effects are expected when exposed to Cry1F and Cry1Ac protein concentrations exceeding the EECs.

## TERRESTRIAL INVERTEBRATE HAZARD 

**Effects on Honey Bee Larvae**
- Methods:
- Exposed to  sucrose solutions containing:
-  genetically modified pollen (Cry1F only)
-  genetically modified pollen (Cry1Ac only)
*Bt* with both Cry1F and Cry1Ac expression
-  Potassium arsenate served as a positive control
- Honey bee larva were scored for survival to capping and adult emergence
- Results:
- Treatment groups were not statistically different from the negative control (sucrose only) which had a survival of 95% to capping and 93.75% to adult emergence

## TERRESTRIAL INVERTEBRATE HAZARD

**Effects on Honey Bee Larvae**
- Results (cont.):
- At 1.98 g Cry1F + 11.94 g Cry1Ac per mL sugar water no effect on survival of larvae to adult emergence was seen
- LC50 is > 4X expression in pollen.
- No hazard to honey bee larvae and adult bee emergence is anticipated.

## TERRESTRIAL INVERTEBRATE HAZARD

**Effects on Parasitic Hymenoptera (wasp)***
- A 40% mortality was observed in the Cry1F + Cry1Ac group at 32X the EEC.
-  The EPA LOC for terrestrial wildlife is an LC50 less than 5X the field exposure (EEC/LC50 = RQ > 0.2)
- The LC50 in this study is greater than 32X the EEC
- No hazard to parasitic Hymenoptera is expected at field exposures (which are minimal to nonexistent
-  * *The August 27, 2002 SAP concluded that parasitic Hymenoptera testing was not appropriate.  Testing another beneficial organism rather than a parasitoid was recommended as  more suitable.*

## TERRESTRIAL INVERTEBRATE HAZARD

**Effects on Green Lacewing Larvae**
- Mortality was increased and pupation was affected in the Cry1F/Cry1Ac at 32X the concentration found in pollen (LC50 > 14X pollen expression)
- No effect is noted at Cry protein levels expressed in pollen that would be encountered by green lacewings in the field
- August 2002 SAP questioned the appropriateness of testing green lacewing and recommended testing an alternate natural enemy such as the minute pirate bug (*Orius insidiosus*) because:
- typically occurs in fields as egg predators
- typically feed on pollen.

## INVERTEBRATE HAZARD ASSESSMENT

**Effects on Lady Beetles**** **
- No adverse effect was seen at limit dose testing (LC50 > 780X Cry1F pollen expression and >8X Cry1Ac pollen expression)
- No hazard to *H. convergens* is expected when feeding on WideStrikeTM  cotton pollen in the field
**Effects on Collembola (spring tail)**** **
- Lyophilized Cry1Ac cotton leaf at 5% or 50% of the diet did not cause  mortality or have adverse effects  on reproduction
- Combination of Cry1F and Cry1Ac showed no effect at up to 10X the anticipated field level of expression.
- No hazard to decomposers represented by Collembola is expected from exposure to WideStrikeTM cotton in the field

## INVERTEBRATE HAZARD ASSESSMENT

**Effects on Earthworms**
- 14-day limit dose study was conducted on earthworms exposed to soils treated with microbial-produced Cry1Ac and Cry 1F, individually and in combination
- There were no overt signs of toxicity to earthworms exposed to soils containing nominal concentrations of Cry1F and  Cry1Ac at 50x the expected worst case EEC
**Monarch Butterfly Risk Assessment**
- Studies published in the Proc. Nat. Acad, Sci. (98 [21]) were used to show that the density of cotton pollen on milkweed leaves (11 grains of MXB-13 pollen per cm2) is 10X less than the minimum pollen density required to elicit subchronic or developmental effects on monarch butterfly larvae
- Therefore monarch larvae are not expected to be adversely affected by exposure to WideStrikeTM cotton pollen in the field

## INVERTEBRATE HAZARD ASSESSMENT

**Activity Spectrum Study**
- The insecticidal activity spectrum of Cry1F and Cry1Ac was determined for nine insect species
- Insects represent taxonomically diverse cotton pests
- Both Cry1F and Cry1Ac activity was restricted to lepidopteran insects,  lending support to the contention that the combination of two Cry proteins did not expand the insect host range

| Lepidoptera | Heteroptera | Coleoptera |
| --- | --- | --- |
| Noctuidae | Miridae | Curculionidae |
| Gelechiidae |  |  |

## INVERTEBRATE HAZARD ASSESSMENT

**Field Evaluation - Effects on Invertebrates**
- Beneficial arthropods in field plots of WideStrikeTM cotton were compared with plots of non-transgenic cotton and those with and without insecticide application in LA and AZ
- Preliminary results show no adverse effect on the numbers of insects from over 50 taxa. (Chemical insecticide treatment reduced the population of some taxa at certain times of sampling).
- Field monitoring supports the the Tier I single species data showing a lack of adverse effects to non-target invertebrates.

## ENVIRONMENTAL FATE

**Soil Degradation**
- The soil DT50 of the plant expressed Cry1F and Cry1Ac was 1.3 days in a laboratory insect bioassay with soil from a cotton growing region (Wayside, MS)
- Soil incorporation rates with cotton leaf tissue were 0.072 mg a.i. Cry1Ac and 0.853 mg a.i. Cry1F per kg of oven dry soil (approximately 3.2 X the EEC for incorporation of defoliated whole plants of MXB-13 into the top 15-cm of soil).
- EPA is requesting additional studies on multi-season persistence of Cry proteins in  soils under  actual field conditions

## EFFECTS ON SOIL MICROORGANISMS

- Published studies performed by the EPA Office of Research and Development on the  impact of transgenic Cry cotton and other plants indicates that adverse effects on soil microorganisms are unlikely
- No effects were seen due to the protein itself, and only a minimal, transient increase in soil microbes attributed to the transgenic cotton plant tissue, rather than the Cry protein expressed in that tissue, was observed

## GENE FLOW

**Horizontal gene transfer (hgt) from***** *****Bt Crops to Soil Organisms**
- Several experiments published in the scientific literature have been conducted to assess the likelihood of hgt and have been unable to detect gene transfer under typical field conditions.
- Hgt has only been detected under laboratory conditions designed to favor DNA transfer.
- The genes that have been engineered into the Bt* *crops have their origin in soil inhabiting bacteria. Transfer of these genes or toxins to other microorganisms or plants is not known to occur.
- In considering these data, the Agency concludes that there is no significant risk of hgt from the transgenes found in Cry1F/Cry1Ac cotton.

## GENE FLOW

**Gene Flow and Weediness Potential**
- In 2001 the Agency reviewed the potential for gene capture and expression of the *Bt* endotoxins by wild or weedy relatives of cotton in the U.S., its possessions or territories.
- Results of review:
-                           1.  Southern Arizona
-                           2.  Hawaiian islands
-                           3.  Southern Florida
-                           4.  Puerto Rico
- There is presently either:
- No production of commercial cotton in these locations, or
- A restriction on commercial cultivation has been placed where outcrossing may occur

- Cultivated cotton has the opportunity to outcross to wild or feral species

## ENDANGERED SPECIES CONSIDERATIONS

- Reviewed non-target data confirm that WideStrikeTM cotton will have no adverse effect on endangered and/or threatened species listed by the US Fish and Wildlife Service, including mammals, birds or terrestrial and aquatic plants and invertebrate species.
- Comparison of the county-level distribution of endangered lepidopteran species relative to cotton producing counties in the US indicate that there will be no overlap with cotton cultivation and endangered lepidopteran breeding habitats.
- No consultation with the USFWS is required under the Endangered Species Act.

## CONCLUSIONS

- This assessment finds no hazard to the environment from cultivation of Cry1F and  Cry1Ac protein expressing cotton for a time-limited registration period.
- However, there are issues that could be looked at in greater depth.  The  Agency believes these issues were not reasonably foreseeable at the time of registration application and initial data development.
- These supplemental, primarily long term effects studies would provide added weight to support the Agency’s conclusions.

## Additional Data Requirements:

-  A six week broiler dietary study (Cry1F/Cry1Ac
-     Corn may be used)
- A Tier I laboratory toxicity test with *Orius insidiosus* (minute pirate bug) -- a non-target insect more appropriate for cotton fields
- Additional invertebrate field surveys on large plots that have been planted with Cry1F/Cry1Ac cotton for at least three consecutive years
- Additional long range soil persistence field studies

## Questions on the Environmental Effects Assessment

**Questions on the Environmental Effects Assessment**

- WideStrike cotton is a product expressing pyramided Cry1F and Cry1Ac Bt proteins.  The submitted non-target effects studies examined the effects of the Cry1F and Cry1Ac proteins separately and in combination to detect any synergistic effects on non-target wildlife.  No synergistic effects or increase in non-target host range were seen as a result of combining these two proteins in the same product.
- The Panel is requested to comment on the need for non-target hazard data development on the combinations of Cry proteins being considered for registration when data on the effects of the individual Cry proteins are readily available and show no adverse effects.

## Questions on the Environmental Effects Assessment

- The weight of evidence from the reviewed data indicates that there will not be a hazard to wildlife from the commercialization of WideStrike cotton. Although the Bt proteins expressed by WideStrike are known to affect only lepidopteran insect species, the Agency evaluated studies of potential effects on a wide variety of non-target organisms that might be exposed to the Cry1F and Cry1Ac protein, i.e., wild mammals, birds, invertebrates, and aquatic species.  EPA concluded that aquatic and terrestrial wildlife was not likely to be harmed and that WideStrike cotton was not likely to threaten the long-term survival of any non-target wildlife populations.
- The Panel is requested to comment on the Agency's analysis of the currently available data on the potential impacts of WideStrike cotton on non-target species.

## Questions on the Environmental Effects Assessment

- The Agency has sufficient information to conclude that there is no hazard  from the proposed uses of WideStrikeTM cotton to non-target wildlife, aquatic and soil organisms.  However, the Agency is requesting additional, primarily long term effects data that were recommended by previous Panels for PIP corn.  The supplementary studies would provide additional weight to support the Agency's conclusions.
- The Panel is asked to comment on:
- 	(a) the scientific value of the proposed additional studies that are identified at the end of the Environmental Assessment section, including avian chronic exposure testing and multi-year field and soil persistence/terrestrial expression studies, and
- 	(b) the applicability of these data to PIP cotton.