### UNITED STATES ENVIRONMENTAL PROTECTION AGENCY WASHINGTON, D.C. 20460



### MAY 1 42013 OFFICE OF CHEMICAL SAFETY AND POLLUTION PREVENTION



### MEMORANDUM SUBJECT:

FROM:

THRU:

TO: Environmental risk assessment for the FIFRA Section 3 registration of Bacillus thuringiensis subsp. galleriae strain SDS 502 (EPA File Symbols 88347-R, 88347-E, and 88347-G); DecisionNos. 452525,453161, and 472696; SubmissionNos. 900477, 909917, 924747, and 933200 (88347- R); 901223, 909915, 924748, and, 933201 (88347-E); and 933202 (88347- G); DP Barcode Nos. 396913, 404845, 406027, and 410690 (88347-R); 396914, 404848, 406028, and 410696 (88347-E); and 410700 (88347-G). Gail Tomimatsu, Plant Pathologis Microbial Pesticides Branch Biopesticides and Pollution Prevention Division, 7511P ShannonBorges, Lead Biologist \\ ' Microbial Pesticides Branch

## ~**,..., ~**  Biopesticides andPollution PreventionDivision, 7511P Jeannine Kausch, Regulatory Action Leader Microbial Pesticides Branch Biopesticides and Pollution Prevention Division, 7511P



### I. Ecological RiskAssessment A. Introduction Phyllom, LLC has applied for FIFRA Section 3 registration ofBacillus thuringiensis subsp. galleriae strain SDS-502 (hereafter "Btg SDS-502") for control ofColeopteran pests in various grain, small fruit and vegetable crops, tree fruit and nut crops, greenhouse/shadehouse and landscape ornamental tree and plant and sod-grass crops. Proposed landscape applications includeturfand ornamental plant sites in recreational (e.g., parks and golfcourses), residential (e.g., ornamental gardens) and commercial areas (e.g. plantscapes and turfscapes in an office park). In addition, there is a proposedpoultrypremise use to control darkling andhide beetles bytreatments to outdoor perimeters and indoor structural areas (e.g., floor, cracks, crevices and supportbeams). A 2-yr Experimental Use Permit (Experimental Use Permit (EUP) No. 88347- EUP-1) was recently approved (May 8, 2013) for efficacy testing to control the Emerald Ash Borer onlimited forest acreage.Btg SDS-502 is the active ingredient (a.i.) inthe manufacturing use product Phyllom SDS-502 MP (EPA File Symbol88347-R) and the end-use products (EPs) Phyllom grubGone! G (EPA File Symbol88347-E), Phyllom beetleGONE! (EPA File Symbol88347-G). The EP Phyllom beetleGONE! (also known as PhyllomboreGONE! under the EUP) is the product intended for use inthe EUP cited above. Phyllom SDS-502 (EPA File Symbol 88347-R), is a manufacturing product that is used to make the end-use products. TheEP Phyllom grubGone!G (EPA File Symbol 88347-E is a granular formulation with 9% a.i., and the EP Phyliom beetleGONE! is a water dispersable powder containing 76

.5% a.i. The MP product contains at least 1 x 1010cfu/g ofBtg SDS-502, which includes fermentation solids, sJ'ores, and insecticidal toxins; the EP Phyllom grubGONE!G contains a minimum of 1 x 10 cfu/g, and the EP PhyllombeetleGONE! contains a minimum of0.85 x 1010cfu/g. Btg SDS-502 is a new a.i., and thus requires a reviewto assess the potential risks to nontarget organisms that may result from its use. This memorandum contains BPPD's ecological risk assessment for this new a.i. and its proposed uses. B. Summary ofNontarget Effects Data Table 1provides the status ofthe datarequirements for the ecological risk assessment ofthe active ingredientBtg SDS-502. Guideline studies and scientific rationales were submitted to satisfy datarequirements for nontarget organisms as published in 40 CFR 

### § 158.2150 and are summarized in Table 1. Information from the studies submitted and cited andthe rationales is included in the discussion ofrisk below (section C). The information provided is sufficient to satisfy the Tier I nontarget organism data requirements and for nontarget organism risk assessment for this new active ingredient and its proposed uses. Table 1. Summary ofdata submitted to comply withnontarget organism datarequirements published in40 CFR 

### § 158.2150 for support oftheregistration ofBacillus thuringiensis subsp. ll

. (B ) t . SDS 502 ga erzae tg, s ram - Data Requirement OPPTS Results Summary and Classification MRID Guideline No. 8 Avian oral 885.4050 The maximum daily dose for Btg SDS- 48553219 toxicity/pathogenicity 502 was determined as 3.0x lOll cfu/kg body 48940107 weight. Classification: Acceptable 49094000 Avian inhalation 885.4100 Scientific rationale was sufficientto satisfy the data 48553220 toxicity/pathogenicity requirement. Btg SDS-502 is not considered toxic or pathogenic to birds. Classification: Acce}ltable Wild mammal 885.4150 Tests required by 40 CFR § 158.2140 are adequate 48553208 toxicity/pathogenicity and appropriate for assessment ofhazards to wild 48553211 mammals. The oral LDso > 2.4x 108 CFU Btg 48940108 SDS-502/rat. Btg SDS-502 was not infective, pathogenic or toxic to laboratory rats. Classification: Acceptable

2Data Requirement OPPTS Results Summary and Classification MRID Guideline No.a 885.4200 Scientific rationale was sufficientto satisfy the data 48553222 Freshwater fish requirement. Btg SDS-502 is not considered toxic toxicity/pathogenicity orpathogenic to fish. Classification: Acceptable Freshwater 885.4240 A 21-day study shows thatthe EC50 for survival, 48553223 invertebrate reproduction, and growth ofDaphnia magna is 48940107 toxicity/pathogenicity > 100 mg/L. TheNOEC for survival is 100 mg!L 49094000 (2.5 x 107 cfu/ml); and theNOEC for reproduction 48553204 and growth is 50 mg!L (8.9 x 1 06 cfulml). Classification: Acceptable Estuarine/marine fish 885.4280 Scientific rationale was sufficientto satisfy the data 48553224 and invertebrate requirement. Btg SDS-502 is not considered toxic testing orpathogenic to estuarine ormarine organisms. Classification: Acceptable Nontargetplant 885.4300 Scientific rationale was sufficient to determinethat 48553225 testing adverse effects are not expected in plants as a result ofexposures to Btg SDS-502

. Classification: Acceptable Nontarget insect 885.4340 The 9-day LC50 and NOEC was > 1700 ppm for 48553226 testing adult ladybird beetles (Hippodamia convergens) 48553204 exposed to artificial diets amended with Btg SDS 48940107 502 Cry8Da protein. Maximum hazard doses were 49094000 confirmed. Classification: Acceptable Nontarget insect 885.4340 The 96-hr LC50 for Btg SDS-502 was> 500**~g** 48553228 testing Cry8Da protein/ml diet for first instars ofColorado 48940107 Potato Beetle (Leptinotarsa decemlineata). The 49094000 maximum hazard dose could not be confirmed. Classification: Supplemental Nontarget insect 885.4340 No mortalities were observed in four species of 48716803 testing adult parasitic wasps (Tetrastichusplanipennisi, 48940107 Oobius agrili,Spathius agrili, Atanycolus 49094000 cappaerti, Order Hymenoptera), whereas 52% mortality was noted for the adult Emerald Ash Borers (Agrilusplanipenis) when exposedto 0.4 IJ.g**Cry8Da/~-tL** honey diet. Maximum hazard doses could not be confirmed

. Classification: Supplemental. Nontarget insect 885.4340 Data and information provided on three 48553227 testing lepidopteran insect species (Bombyx 48553204 mori, Plutellaxylostella, Spodoptera litura) are 48940107 useful for demonstrating potential spectra of 49094000 insecticidal activity. The test duration (2 to 5 days) may have been ofinsufficient length for insect species that showed no mortality (B

. mori, S. litura)

. Maximum hazard doses could not be confirmed. Classification: Supplemental.



### 3Data Requirement OPPTS Results Summary and Classification MRID Guideline No. 3 Nontarget insect 885.4340 No adverse effects were observed when larvae of 48553229 testing Southern corn rootworm (Diabrotica 48940107 undecimpunctatahowardi) were exposed to 49094000 reported concentrations ofpurified Cry8Da protein in artificial diets. There were no quantitative data or information to confirm the concentrations ofthe test material and reported results. Maximum hazard doses could not be confirmed. Classification: Unacceptable Honey bee testing 885.4380 The 20-day NOEC for BtgSDS-502 was 500 ng 48553230 Cry8 Da protein/honeybee larval cell. There is 48940107 uncertainty in the amount ofCry8Da protein that 49094000 might remain in the environment after applications ofthe microbial pesticide. The study provides useful information for a honeybee larval toxicity profile for the Cry8Da protein at the screening level. The maximum hazard dose could not be confirmed. Classification: Supplemental aNote: The top-most MRID citation indicates the actual and original nontarget organism study. SubsequentMRIDs were reviewed for confirmation oftest substances, or for other deficiencies noted in the original study (addressed in 2 resubmissions). C. Ecological Exposure and Risk Characterization The EP, Phyllom beetleGONE! is proposed for applicationto many types ofgrain, vegetable and small fruit crops, including fruit and nut trees, greenhouse/shadehouse and ornamental plants and trees, and poultrypremises. Proposed application methods include ground and aerial sprays, and chemigation. The EP, Phyllom grubGONE!G, is proposed for use onturf, turfgrass, ornamentals plants and trees, and is intended to be applied by ground application equipment. The turfgrass sites include golfcourse greens, residential lawns, commercial grounds, sod farms; and the ornamentals sites include landscapes, interiorscapes, nurseries, fields and greenhouses. Since the proposed applications are expected to result incontact ofthe a.i. with foliar surfaces and soil, and spray drift is expected to occur, exposure to terrestrial and aquatic nontarget organisms is possible. Based on current information concerning the amount ofactive ingredient1in the EP, beetleGONE!, the Agency estimates terrestrial maximum environmental concentrations (EC) of Btg SDS-502**as~** 6,078g/A. An aquatic maximum EC was estimated at 10.0 mg a.i./L ofwater.

1Terrestrial maximum EC calculation: 17.5 lbs/A *454 g/lb * 1 acre* 76

.5%% a.i

. in EP; aquatic maximum EC: 17

.5 lbs/A *454,000 g/lb * 1A/40,468,564 cm2 = 0

.1963 mg/cm2 + 15 em= 0.013 mg/mL * 1000 ml/L = 13

.08 mg/L *76.5% = 10

.0 mg a.i./L.



### 4

### 1. Terrestrial Animals and Plants Birds andMammals Birds and mammals may be exposed to Btg SDS-502 primarily through ingestion or contact with treated surfaces. Informationpresented to BPPD indicates lowrisk to birds and wild mammals as a result ofthe proposed applications. An acute oral toxicity study with birds at the maximum hazard dose (MRID 48553219) showed thatthe acute oral LD50 is 3.0 x 

### lOll cfu/kg ofbody weightper day. Studies with laboratory rats exposed orally (MRID 48553208) showed that Btg SDS-502 is nottoxic, infective, orpathogenic when administered at maximum hazard doses. Other acute toxicity studies (MRIDs 48553211, 48940108) provide additional informationto conclude thattoxicity to mammals is not expected. NontargetInsects andHoney Bees The Agency reviewed submitted studies and information from the open literature. Most ofthe submitted insect studies ("bioassays") provided limited information regarding potential dietary toxicity ofthe Cry8Daprotein, extracted and purifiedfrom BtgSDS-502 (Table 1). Notably, these studies may have been ofinsufficient duration to note potential activity by an insecticide whichmustbe ingested by susceptible insect larvae for toxic effects. Insect bioassays may be influenced byrelative proportions ofthe different proteins within the crystal, thereby obscuring the role an individual toxin may play in insectpathogenesis. Variability in the composition and potency ofcrystals within the same strain may be caused by factors such as mobile genetic elements and nutritional requirements [3]. At least seven different 8-endotoxins were identified in B. thuringiensis subsp. galleriae [2]. Itis not possible to determine whetherthese exposures were conducted at maximum hazard concentrations, based on submitted information about the protein levels inthe test materials for some ofthe studies (MRID 48553204). A couple ofsubmitted studies used differentprotein extraction, purification and quantification and did not report the percentage ofthe Cry8Da protein. Itemized studies (b-f) were determined supplemental, however all studies are useful for the risk assessment ofBtg SDS-502 because they provide a spectrum ofthe tested protein's (Cry8Da) activity. The conclusions ofthese insect bioassays/studies were:

a. No sustained adverse effects are anticipated for populations ofH convergens (Order Coleoptera). b. Potential Cry8Datoxicities were demonstrated for Plutella xylostella, (Order Lepidoptera); defined as "weaklytoxic" (cited by [1], in MRID 48553227). c. No Cry8Datoxicity was demonstrated for Bombyx mori (Oriental silkworm moth), Spodoptera litura (Orientalleafworm moth) d. No Cry8Datoxicity was demonstrated for Leptinotarsa decemlineata (Colorado Potato beetle) e. No Cry8Datoxicityto 4 species ofadult parasitic Hymenoptera



### 5f. High toxicity ofpurified Cry8Da from Btg SDS 502 to the Emerald Ash Borer (Agrilus planipennis orAgrilus marcopoli) and to Scarabaeidae beetle "white grubs" (Anomala cuprea) was demonstrated in another submitted study (MRID 48553204). Asano et al [I] noted that other species ofthe Scarabadiae (e.g., cupreous chafer,Anomola cuprea; Japanese beetle, PopilliajaponicaNewman) are affected by other strains or subspecies ofB. thuringiensis (e

.g., Btgalleriae NRRL B-4027 andBtjaponensis) and other cry8 genes were active against Cotinis sp. (June beetle)

. Although the Cry8Daprotein was reported as weakly toxic to a lepidopteran pest, Plutella xylostella, another formulation withB.t. subspeciesgalleriae was developed to control the wax moth (Galleriae sp) in honeybee hives [4]. Additional text is presentedbelow, Honeybees. Based onthese studies and the information presented above, adverse effects to insect species in the Coleopteran and Lepidopteran Orders are anticipated as a result ofBtg SDS-502 applications. However, refined estimates ofthe potential insecticidal activity to other (untested) species in these orders is not possible because: 

### (1) the majority ofthe studies were not conducted at maximum hazard concentrations, and (2) otheruncertainties associated withbioassays and activity levels associated with different proteinpreparations ofindividual studies

. Notably these bioassays (non-GLP) were conducted primarily to determine the potential insecticidal activity of Cry8Daprotein. Since the Cry8 protein dose(s) tested could not be correlated to potential environmental exposures, it is assumed that any exposures to Btg SDS-502 could cause adverse effects in Coleopteran or Lepidopteran insect species. Honeybees Phyllom also submitted a study with honey bee larvae (Apis mellifera) exposed to Btg SDS-502 Cry8Daprotein. Based onthis study, the NOEC was 500 ng SDS-502 Cry8Daprotein/honeybee larval cell; however, the study was supplemental because ofthe uncertainty in the amount of Cry8Daprotein that mightremain inthe environment after applications ofproducts containing Btg SDS-502. A commercial formulation containingBt subsp

.galleriae (strain not identified) with Lepidopteran activity was discussed in a review paperbyMelin and Cozzi [4]. The formulation (no potency information provided) was developed to control the wax moth (Galleriae spp.) in bee hives; and the bacterium can be sprayed onthe surface ofthe honeycomb or impregnated into the wax. Hives treated withB. thuringiensis subsp. galleriae (strain and viability not provided) in a 40% sucrose solution showed no adverse effect on adult workers or colony life as determined by egg laying, broodproduction, brood capping or honey production [4]. Therefore, adverse risk concerns to honeybees are not expected with this new a.i. Nontarget Plants A scientific rationale was submitted to satisfythe datarequirement for nontarget plants. Bacillus thuringiensis is well characterized with respect to effects onplants, and is not taxonomically



### 6related to knownplant pathogens. Therefore, adverse effects onnontarget plants are not expected to result from the proposed applications Btg SDS-502. 

### 2. Aquatic Animals and Plants Freshwater Fish andInvertebrates A scientific rationale was submitted in lieu ofa study for freshwater fish. Resubmitted information and data confirmedthe taxonomic classification as Bacillus thuringiensis subspecies galleriae, with no known pathogenesis to fish species [8] and a limited host range to certain insect orders. A study with Daphnia magnawas also submitted, inwhichDaphnia were exposed to water containing a range ofnominal concentrations ofthe TGAI for 21 days. Based onthe percentage ofthe a.i. in the EP, a direct application to a body ofwater equivalent to the EPA Standard Wetland wouldbe approximately 10.0 mg a.i./1, which is less than the NOEC values for reproduction and growth (i.e., 50 mg!L) and for survival (100 mg/L). Although high daphnid mortality and low neonateproductionwere noted in the filtered treatment, the adverse effects did not affectthe overall conclusions ofthe study and were attributed to low dissolved oxygen content, and anomalous values for conductivity and water hardness. Since exposure resulting from the proposed applications is well belowthe defined toxicity endpoints, adverse effects to freshwater invertebrates are not expected. Marine/Estuarine Fish andInvertebrates A scientific rationale was submitted in lieu ofa study for freshwater fish. Resubmitted information and data confirmedthe taxonomic classification as Bacillus thuringiensis subspecies galleriae, with no knownpathogenesis to fish species and a limited host range to certain insect orders. AquaticPlants As discussed above, Btg SDS-502 is not related to knownplant pathogens. Therefore, adverse effects to aquatic plants are not anticipated. 

### D. Environmental Persistence and Fate Microbial pesticides containing B. thuringiensis readily breaks down in the environment, degrades within 48 hrs under UV light exposures [6], and has a half-life of3.8 hrs under normal sunlit conditions [7]. The microorganism can persist for several months in soil [4], and certain Cryproteins may degrade within 32 days. Half-life values (DT5o) ranged from 8.3 to 31.3 days [5]. A spray-dried powder ofBtg SDS-502 provided nearly 100% control ofA

. cuprealarvae as long as 56 days after aninitial application of0.12 g/m2 [1].

7E. Threatened and Endangered Species Assessment EPA has determined that no effects are anticipated for most nontarget species exposedto Btg SDS-502 as a result ofthe proposed labeled applications, including mammals, birds, terrestrial and aquatic plants, and freshwater and marine/estuarine animals. Therefore, a "No Effect" determination is made for direct and indirect effects to federally listed endangered and threatened ("listed") mammals, birds, terrestrial and aquatic plants, and freshwater and marine/estuarine animals and for their designated critical habitat. Sustainedpopulation-level effects to nontarget insects are not anticipated, however it is known thatBt subsp. galleriae can affect certain species ofinsects (e.g., Coleopterans and Lepidopterans). Direct effects to individuals oflisted insect species [9] cannotbe precluded, nor can indirect effects to listed species that depend specifically on a listed insect. Additional analysis ofthe potential effects to these is necessary to make a determination for these species. References Cited 1. Asano, S., C.Yamashita, T.lizuka, K.Tak:euchi, S.Yamanak:a, D.Cerf, and T.Yamamoto.2003. A strain ofBacillus thringiensis subsp. galleriae containing a novel cry8 gene highly toxic to Anomala cuprea (Coleoptera:Scarabaeidae). Also in MRID 48553227. 2. Chestukhina, G., L.Kostina, I.Zalunin, L.Revina, A.Mikhailova, and V.Stepanov.1994. Production ofmultiple o-endotoxins byBacillus thuringiensis: o-endotoxins producedby strains ofthe subspecies galleriae and wuhanensis. 3. Masson, L., M.Erlandson, M.Puzstai-Carey, R.Brousseau, V.Juarez-Perez, and R.Frutos.1998. A Holistic Approach for Determining the Entomopathogenic Potential ofBacillus thuringiensis Strains. Appl.Environ.Microbiol.(64):4782-4788. 4. Melin, B.E. and E.M. Cozzi. 1990. Safety to nontarget invertebrates ofLepidopteran strains of Bacillus thuringiensis and their P-exotoxins. Pp. 149-167 In Laird, M., Lacey, L.A. & Davidson, E.W.D., Eds., Safety of Microbial Insecticides. CRC Press, Boca Raton. 5. Mendelsohn, M., J.Kough, Z.Vaituzis and K. Matthews. 2003. Are Bt crops safe? Nature Biotechnology (21):1003-1009). 6. Myasnik, M., R.Manasherob, E.Ben-Dov, A.Zartsky, Y.Margalith, Z.Barak:. 2001. Comparative Sensitivityto UV-B Radiation oftwo Bacillus thuringiensis Subspecies and Other Bacillus sp. Current Microbiol.(43):140-143. 7. U.S.D.A. Extension Service, National Agricultural Pesticide Impact Assessment Program. 1994. As cited in EXTOXNET. Pesticide Information Profile- Bacillus Thuringiensis. 8. U.S. Environmental Protection Agency (USEPA). 1998. Reregistration Eligibility Decision (RED): Bacillus thuringiensis. USEPA Office ofPrevention, Pesticides, and Toxic Substances, Washington, DC. EPA 738-R-98-004.



### 89. U.S. FishandWildlife Services, DOL 2013,

### May 10. (http: www.fws.gov /endangered).



### 9