UNITED STATES ENVIRONMENTAL PROTECTION AGENCY 

WASHINGTON, D.C. 20460 

MAY 1 4 2013 

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); Decision Nos. 452525,453161, and 

472696; Submission Nos. 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 

Shannon Borges, Lead Biologist\\ ' 

Microbial 

Biopesticides 

Pesticides 

and Pollution 

Branch 

Prevention 

~ Division, 7511P ,...,~ 

Jeannine Kausch, Regulatory Action Leader 

Microbial Pesticides Branch 

Biopesticides and Pollution Prevention Division, 7511P 

I. Ecological Risk Assessment 

A. Introduction 

Phyllom, LLC has applied for FIFRA Section 3 registration of Bacillus thuringiensis subsp. 

galleriae strain SDS-502 (hereafter "Btg SDS-502") for control of Coleopteran 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 

include turf and ornamental plant sites in recreational (e.g., parks and golf courses), residential 

(e.g., ornamental gardens) and commercial areas (e.g. plantscapes and turfscapes in an office 

park). In addition, there is a proposed poultry premise use to control darkling and hide beetles 

by treatments to outdoor perimeters and indoor structural areas (e.g., floor, cracks, crevices and 

support beams). 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 on limited forest acreage. 
Btg SDS-502 is the active ingredient (a.i.) in the 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 Phyllom boreGONE! under the EUP) is the product intended for 

use in the EUP cited above. Phyllom SDS-502 (EPA File Symbol 88347-R), is a manufacturing 

product that is used to make the end-use products. The EP 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 1010 cfu/g 

of Btg 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 Phyllom beetleGONE! 

contains a minimum of 0.85 x 1010 cfu/g. 

Btg SDS-502 is a new a.i., and thus requires a review to 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 of Nontarget Effects Data 

Table 1 provides the status of the data requirements for the ecological risk assessment of the 

active ingredient Btg SDS-502. Guideline studies and scientific rationales were submitted to 

satisfy data requirements for nontarget organisms as published in 40 CFR § 158.2150 and are 

summarized in Table 1. Information from the studies submitted and cited and the rationales is 

included in the discussion of risk 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 of data submitted to comply with nontarget organism data requirements 

published in 40 CFR § 158.2150 for support of the registration of Bacillus thuringiensis subsp. 

ga ll erzae . (B tg) , s t . ram SDS -502 

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 sufficient to 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 of hazards 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 

2 
Data Requirement OPPTS Results Summary and Classification MRID 

Guideline No. a 

885.4200 Scientific rationale was sufficient to satisfy the data 48553222 

Freshwater fish requirement. Btg SDS-502 is not considered toxic 

toxicity/pathogenicity or pathogenic to fish. 

Classification: Acceptable 

Freshwater 885.4240 A 21-day study shows that the EC50 for survival, 48553223 

invertebrate reproduction, and growth of Daphnia magna is 48940107 

toxicity/pathogenicity > 100 mg/L. The NOEC for survival is 100 mg!L 49094000 

(2.5 x 107 cfu/ml); and the NOEC 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 sufficient to satisfy the data 48553224 

and invertebrate requirement. Btg SDS-502 is not considered toxic 

testing or pathogenic to estuarine or marine organisms. 

Classification: Acceptable 

Nontarget plant 885.4300 Scientific rationale was sufficient to determine that 48553225 

testing adverse effects are not expected in plants as a result 

of exposures 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 of Colorado 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 (Tetrastichus planipennisi, 48940107 

Oobius agrili, Spathius agrili, Atanycolus 49094000 

cappaerti, Order Hymenoptera), whereas 52% 

mortality was noted for the adult Emerald Ash 

Borers (Agrilus planipenis) when exposed to 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, Plutella xylostella, Spodoptera litura) are 48940107 

useful for demonstrating potential spectra of 49094000 

insecticidal activity. The test duration (2 to 5 

days) may have been of insufficient length for 

insect species that showed no mortality (B. 

mori, S. litura ). Maximum hazard doses 

could not be confirmed. 

Classification: Supplemental. 

3 
Data 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 

undecimpunctata howardi) were exposed to 49094000 

reported concentrations of purified Cry8Da protein 

in artificial diets. There were no quantitative data 

or information to confirm the concentrations of the 

test material and reported results. Maximum hazard 

doses could not be confirmed. 

Classification: Unacceptable 

Honey bee testing 885.4380 The 20-day NOEC for Btg SDS-502 was 500 ng 48553230 

Cry8 Da protein/honeybee larval cell. There is 48940107 

uncertainty in the amount of Cry8Da protein that 49094000 

might remain in the environment after applications 

of the 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. 

Subsequent MRIDs were reviewed for confirmation of test 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 application to many types of grain, vegetable and 

small fruit crops, including fruit and nut trees, greenhouse/shadehouse and ornamental plants and 

trees, and poultry premises. Proposed application methods include ground and aerial sprays, and 

chemigation. The EP, Phyllom grubGONE!G, is proposed for use on turf, turfgrass, ornamentals 

plants and trees, and is intended to be applied by ground application equipment. The turfgrass 

sites include golf course 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 in contact of the 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 of active ingredient1 in 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. 

1 Terrestrial 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 and Mammals 

Birds and mammals may be exposed to Btg SDS-502 primarily through ingestion or contact with 

treated surfaces. Information presented to BPPD indicates low risk to birds and wild mammals 

as a result of the proposed applications. An acute oral toxicity study with birds at the maximum 

hazard dose (MRID 48553219) showed that the acute oral LD50 is 3.0 x lOll cfu/kg of body 

weight per day. Studies with laboratory rats exposed orally (MRID 48553208) showed that Btg 

SDS-502 is not toxic, infective, or pathogenic when administered at maximum hazard doses. 

Other acute toxicity studies (MRIDs 48553211, 48940108) provide additional information to 

conclude that toxicity to mammals is not expected. 

Nontarget Insects and Honey Bees 

The Agency reviewed submitted studies and information from the open literature. Most of the 

submitted insect studies ("bioassays") provided limited information regarding potential dietary 

toxicity of the Cry8Da protein, extracted and purified from Btg SDS-502 (Table 1). Notably, 

these studies may have been of insufficient duration to note potential activity by an insecticide 

which must be ingested by susceptible insect larvae for toxic effects. Insect bioassays may be 

influenced by relative proportions of the different proteins within the crystal, thereby obscuring 

the role an individual toxin may play in insect pathogenesis. Variability in the composition and 

potency of crystals 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]. 

It is not possible to determine whether these exposures were conducted at maximum hazard 

concentrations, based on submitted information about the protein levels in the test materials for 

some of the studies (MRID 48553204). A couple of submitted studies used different protein 

extraction, purification and quantification and did not report the percentage of the Cry8Da 

protein. 

Itemized studies (b-f) were determined supplemental, however all studies are useful for the risk 

assessment of Btg SDS-502 because they provide a spectrum of the tested protein's (Cry8Da) 

activity. The conclusions of these insect bioassays/studies were: 

a. No sustained adverse effects are anticipated for populations of H convergens (Order 

Coleoptera). 

b. Potential Cry8Da toxicities were demonstrated for Plutella xylostella, (Order 

Lepidoptera); defined as "weakly toxic" (cited by [1], in MRID 48553227). 

c. No Cry8Da toxicity was demonstrated for Bombyx mori (Oriental silkworm moth), 

Spodoptera litura (Orientalleafworm moth) 

d. No Cry8Da toxicity was demonstrated for Leptinotarsa decemlineata (Colorado Potato 

beetle) 

e. No Cry8Da toxicity to 4 species of adult parasitic Hymenoptera 

5 
f. High toxicity of purified Cry8Da from Btg SDS 502 to the Emerald Ash Borer (Agrilus 

planipennis or Agrilus 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 of the Scarabadiae (e.g., cupreous chafer, Anomola 

cuprea; Japanese beetle, Popilliajaponica Newman) are affected by other strains or subspecies 

of B. thuringiensis (e.g., Bt galleriae NRRL B-4027 and Btjaponensis) and other cry8 genes 

were active against Cotinis sp. (June beetle). 

Although the Cry8Da protein was reported as weakly toxic to a lepidopteran pest, Plutella 

xylostella, another formulation with B. t. subspecies galleriae was developed to control the wax 

moth (Galleriae sp) in honeybee hives [4]. Additional text is presented below, Honeybees. 

Based on these studies and the information presented above, adverse effects to insect species in 

the Coleopteran and Lepidopteran Orders are anticipated as a result of Btg SDS-502 applications. 

However, refined estimates of the potential insecticidal activity to other (untested) species in 

these orders is not possible because: (1) the majority of the studies were not conducted at 

maximum hazard concentrations, and (2) other uncertainties associated with bioassays and 

activity levels associated with different protein preparations of individual studies. Notably these 

bioassays (non-GLP) were conducted primarily to determine the potential insecticidal activity of 

Cry8Da protein. 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 

Cry8Da protein. Based on this study, the NOEC was 500 ng SDS-502 Cry8Da protein/honeybee 

larval cell; however, the study was supplemental because of the uncertainty in the amount of 

Cry8Da protein that might remain in the environment after applications of products containing 

Btg SDS-502. 

A commercial formulation containing Bt subsp. galleriae (strain not identified) with 

Lepidopteran activity was discussed in a review paper by Melin 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 on the surface of the honeycomb or impregnated 

into the wax. Hives treated with B. 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, brood production, 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 satisfy the data requirement for nontarget plants. Bacillus 

thuringiensis is well characterized with respect to effects on plants, and is not taxonomically 

6 
related to known plant pathogens. Therefore, adverse effects on nontarget plants are not expected 

to result from the proposed applications Btg SDS-502. 

2. Aquatic Animals and Plants 

Freshwater Fish and Invertebrates 

A scientific rationale was submitted in lieu of a study for freshwater fish. Resubmitted 

information and data confirmed the 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 magna was also submitted, in which Daphnia were exposed to water 

containing a range of nominal concentrations of the TGAI for 21 days. Based on the percentage 

of the a.i. in the EP, a direct application to a body of water equivalent to the EPA Standard 

Wetland would be 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 neonate production were noted in the filtered treatment, the adverse effects did 

not affect the overall conclusions of the 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 below the defined toxicity endpoints, adverse effects to 

freshwater invertebrates are not expected. 

Marine/Estuarine Fish and Invertebrates 

A scientific rationale was submitted in lieu of a study for freshwater fish. Resubmitted 

information and data confirmed the taxonomic classification as Bacillus thuringiensis subspecies 

galleriae, with no known pathogenesis to fish species and a limited host range to certain insect 

orders. 

Aquatic Plants 

As discussed above, Btg SDS-502 is not related to known plant 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 of 3.8 hrs under normal 

sunlit conditions [7]. The microorganism can persist for several months in soil [4], and certain 

Cry proteins may degrade within 32 days. Half-life values (DT5o) ranged from 8.3 to 31.3 days 

[5]. A spray-dried powder of Btg SDS-502 provided nearly 100% control of A. cuprea larvae as 

long as 56 days after an initial application of0.12 g/m2 [1]. 

7 
E. Threatened and Endangered Species Assessment 

EPA has determined that no effects are anticipated for most nontarget species exposed to Btg 

SDS-502 as a result of the 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. 

Sustained population-level effects to nontarget insects are not anticipated, however it is known 

that Bt subsp. galleriae can affect certain species of insects (e.g., Coleopterans and 

Lepidopterans). Direct effects to individuals oflisted insect species [9] cannot be precluded, nor 

can indirect effects to listed species that depend specifically on a listed insect. Additional 

analysis of the 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 of Bacillus 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 of multiple o-endotoxins by Bacillus thuringiensis: o-endotoxins produced by 

strains of the 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 of Bacillus 

thuringiensis Strains. Appl.Environ.Microbiol.(64):4 782-4 788. 

4. Melin, B. E. and E.M. Cozzi. 1990. Safety to nontarget invertebrates of Lepidopteran 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 Sensitivity to UV -B Radiation of two 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 of Prevention, Pesticides, and Toxic 

Substances, Washington, DC. EPA 738-R-98-004. 

8 
9. U.S. Fish and Wildlife Services, DOL 2013, May 10. (http: www.fws.gov/endangered). 

9