BIOPESTICIDES REGISTRATION ACTION DOCUMENT

Bacillus subtilis strain QST 713 variant Soil 

Pesticide Chemical (PC) Code: 006579

U.S. Environmental Protection Agency

Office of Pesticide Programs

Biopesticides and Pollution Prevention Division

DRAFT – October 3, 2012
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TABLE OF CONTENTS

I. EXECUTIVE SUMMARY...........................................................................................................................4

II. ACTIVE INGREDIENT OVERVIEW .......................................................................................................7

III. REGULATORY BACKGROUND ..............................................................................................................7

A. Applications for Pesticide Product Registration ............................................................................7

B. Food Tolerance Exemption ..............................................................................................................7

IV. RISK ASSESSMENT....................................................................................................................................7

A. Product Analysis Assessment (40 CFR § 158.2120).......................................................................8

B. Human Health Assessment (40 CFR § 158.2140)...........................................................................8

C. Environmental Assessment (40 CFR § 158.2150).........................................................................14

V. ENVIRONMENTAL JUSTICE.................................................................................................................16

VI. RISK MANAGEMENT DECISION .........................................................................................................16

VII. ACTIONS REQUIRED OF THE REGISTRANT...................................................................................17

VIII. GLOSSARY OF ACRONYMS AND ABBREVIATIONS ......................................................................18

IX. BIBLIOGRAPHY .......................................................................................................................................19

A. Studies Submitted to Support the Active Ingredient, Bacillus subtilis strain QST 713 variant 

Soil, and Pesticide Product, Serenade Soil DPZ, Registration...............................................................................19

B. Environmental Protection Agency Risk Assessment Memoranda .............................................22

C. Other References..........................................................................................................................223

APPENDIX A. MICROBIAL PESTICIDES DATA REQUIREMENTS ...........................................................24

APPENDIX B. PESTICIDE PRODUCT................................................................................................................28
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BIOPESTICIDES REGISTRATION ACTION DOCUMENT TEAM

Office of Pesticide Programs (OPP)

Biopesticides and Pollution Prevention Division

Microbial Pesticides Branch

Science Reviews

Ibrahim S. Barsoum, Ph.D. Product Analysis, Human Health

John L. Kough, Ph.D. Product Analysis, Human Health

Shannon Borges Environmental Effects

Regulations

Kimberly Nesci Acting Chief, Microbial Pesticides Branch

Michael Glikes Regulatory Action Leader
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I. EXECUTIVE SUMMARY

Background

In July 2011, Agraquest, Inc., submitted an application for a new end-use pesticide product, 

Serenade Soil DPZ (EPA File Symbol 69592-EI), containing a new active ingredient, Bacillus 

subtilis strain QST 713 variant Soil, to the United States Environmental Protection Agency

(EPA) under section 3 of the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA). 

Concurrently with this application, Agraquest, Inc., filed a petition for an amendment to an 

existing exemption from the requirement of a tolerance for Bacillus subtilis strain QST to include 

residues of products containing the active ingredient Bacillus subtilis strain QST variant Soil.

Bacillus subtilis strain QST 713 variant Soil is a naturally occurring variant of Bacillus subtilis 

strain QST 713. In 2000, EPA first registered the latter as a pesticide active ingredient for use on 

several minor crops to treat a variety of plant diseases and fungal pathogens including gray mold, 

powdery mildew, early and late blight, fire blight, scab, sour rot, bacterial spot, and walnut 

blight. Bacillus subtilis is commonly found in various ecological niches including soil, water and 

air, and it does not have a history of pathogenicity from contact in the environment. Trade names 

for the registered end-use products include: Serenade®, Rhapsody®, Rhapsody® ASO, Serenade® 

ASO, Serenade® MAXTM , Serenade Biofungicide Wettable Powder, Serenade® AS, Serenade® 

Garden Disease Control Concentrate, and Serenade® Garden Disease Control Ready To Use. In 

addition, there are other strains of Bacillus subtilis registered as microbial pesticides.

The bacterium, Bacillus subtilis, is prevalent in soils and has been found in a variety of habitats 

worldwide. The QST 713 strain of Bacillus subtilis is known to be antagonistic toward many fungal 

plant pathogens. This antagonism may be achieved in several ways including nutrient competition, 

site exclusion, colonization, and attachment of the bacteria to the fungal pathogen. In addition, 

Agraquest reports that the QST 713 strain of Bacillus subtilis has been shown to induce plants' 

natural systemic resistance or systemic acquired resistance (SAR) of certain plants against bacterial 

pathogens. These bacteria can stop plant pathogen spores from germinating, disrupt germ tube 

growth, and inhibit attachment of the plant pathogen to the leaf. 

The applicant submitted data to the EPA demonstrating that Bacillus subtilis strain QST 713 

variant Soil is stable and does not revert back to the parent strain, Bacillus subtilis strain QST 

713, over multiple generations. When compared to the parent strain, the variant can be identified 

by its distinct rough (sandpaper), adherent phenotypic colony morphology. This phenotype is 

associated with enhanced biofilm formation, which enhances growth promotion and disease 

protection of the parental strain. This gene has been identified as an essential gene for Bacillus to 

move over solid substrates. After researchers transferred the test substance in flasks 15 times, no 

Bacillus subtilis strain QST 713 wild type colonies were found when they plated Bacillus subtilis 

strain QST 713 variant Soil. These results demonstrate that the particular genetic sequence 

mutant is stable in Bacillus subtilis strain QST 713 variant Soil and, hence, the strain breeds trueto-form. The EPA determined that the data are acceptable.

The proposed end-use product (EP) containing this active ingredient, Bacillus subtilis strain QST 

713 variant Soil, is Serenade Soil DPZ, a biofungicide to be primarily applied as a soil drench or 

in-furrow spray. This EP is intended for use in field applications, greenhouses, glasshouses, 

nurseries, shade houses, landscapes, interiorscapes, seedling production sites, and forest seedling 

production sites, as well as turf and home and garden sites.
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EPA scientists reviewed product analysis, toxicology, and nontarget organism data and 

information (40 CFR §§ 158.2120, 158.2140, and 158.2150, respectively) submitted to support 

the registration of the proposed Bacillus subtilis strain QST 713 variant Soil pesticide product. 

They found that, overall, such data and information are adequate for risk assessment purposes, 

fulfill the current microbial pesticide data requirements, and support registration of the proposed 

product under FIFRA section 3(c)(5).

Product Analysis

For the purposes of FIFRA section 3(c)(5) registration, the product analysis data requirements 

for Bacillus subtilis strain QST 713 variant Soil, including product chemistry and composition, 

analysis of samples, physical and chemical characteristics, and additional data concerning 

genetic stability, were fulfilled by acceptable guideline studies.

Toxicology

The applicant submitted adequate mammalian toxicology data and information to support the 

pesticide products. Acute oral, pulmonary, and injection toxicity/pathogenicity studies showed 

that, at a single high dose, Bacillus subtilis strain QST 713 variant Soil, is not toxic and/or 

pathogenic via these routes of exposure. Moreover, the applicant reported that no 

hypersensitivity incidents occurred during research, development, or testing of Bacillus subtilis 

strain QST 713 variant Soil. In light of the results of the acute toxicity/pathogenicity data and the 

absence of hypersensitivity incidents, testing at higher tiers (i.e., Tiers II and III) was not 

required. EPA concluded that Bacillus subtilis strain QST 713 and Bacillus subtilis strain QST 

713 variant Soil are substantially similar for the purposes of assessing toxicity, pathogenicity, 

and infectivity (Review of Product Chemistry and Acute Toxcity Studies, Memorandum from 

Barsoum to Glikes, July 11, 2012). 

Tolerance Exemption

The EPA is amending the existing exemption for the requirement of a tolerance for Bacillus 

subtilis strain QST at 40 CFR 180.1209 to include residues of the active ingredient Bacillus 

subtilis strain QST variant Soil in or on all agricultural commodities. The EPA concluded that 

there is reasonable certainty that no harm will result to the U.S. population, including infants and 

children, from aggregate exposure to residues of Bacillus subtilis strain QST 713 variant Soil. 

Data and information submitted by the petitioner support the conclusion that no dietary risks are 

expected to occur as a result of the use of Bacillus subtilis strain QST 713 variant Soil as an 

active ingredient in pesticide products. The EPA concludes that there will not be significant 

exposure to Bacillus subtilis strain QST 713 variant Soil through food and drinking water. This 

conclusion is based on: (1) the proposed application methods of the end-use pesticide products 

(soil directed, soil incorporated, and/or seed directed; no aquatic applications); (2) the filtering 

effect of many particulate soil types; and (3) the conditions (e.g., filtration and pH adjustments) 

water is subjected to in wastewater treatment systems and drinking water facilities. Based on 

their review of the acute oral toxicity and pathogenicity data, the EPA determined that, even if 

Bacillus subtilis strain QST 713 variant Soil were present on food or in drinking water, human 

exposure to this bacterium would not result in unreasonable adverse effects.
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Occupational Exposure

Despite the low toxicological profile of Bacillus subtilis strain QST 713 variant Soil, the EPA is 

requiring baseline personal protective equipment (PPE) for handlers that, due to their occupation,

may be subject to prolonged or repeated exposure to the active ingredient. The product label 

directs handlers working with Bacillus subtilis strain QST 713 variant Soil in agricultural 

settings to wear a long-sleeved shirt, long pants, socks, and shoes. For future products, EPA may

require additional PPE (e.g., protective eyewear and/or a dust/mist filtering respirator meeting 

National Institute for Occupational Safety and Health (NIOSH) standards of at least N-95, R-95, 

or P-95) on a product-specific basis. 

Nontarget Organisms

The applicant submitted rationale bridging studies previously submitted for the registration of 

Bacillus subtilis strain QST 713 to support the Agency’s waiving nontarget organism testing for 

the new active ingredient. Because the two variants of the bacterium are closely related, the EPA 

accepted the bridging rationale and determined that the data are sufficient to fulfill the relevant 

microbial pesticide data requirements and for risk assessment purposes. Further testing of 

nontarget organisms at higher tier levels (i.e., Tiers II, III, and IV) is not required. The EPA

performed an environmental risk assessment, based on data and other information provided by

the applicant, and determined that adverse effects to nontarget organisms are not anticipated 

from the proposed pesticidal uses of Bacillus subtilis strain QST 713 variant Soil. Moreover, the 

EPA made a “No Effect” determination for direct and indirect effects to listed species and their 

designated critical habitats resulting from these same proposed pesticidal uses. 

Public Participation

On October 1, 2009, the EPA announced a new policy to provide a more meaningful opportunity

for the public to participate in major registration decisions before they occur. According to this 

policy, the EPA intends to provide a public comment period prior to making a registration 

decision for, at minimum, the following types of applications: new active ingredients; first food 

uses; first outdoor uses; first residential uses; or any other registration actions for which the 

Agency believes there may be significant public interest.

 

Consistent with the policy of making registration actions more transparent, the proposed 

pesticide product containing Bacillus subtilis strain QST 713 variant Soil, a new active 

ingredient, is subject to a 15-day comment period. In addition to containing a new active 

ingredient, the registration of the pesticide product would result in the first outdoor and food uses 

for Bacillus subtilis strain QST 713 variant Soil. The docket identification number associated 

with these registration actions, and accessed through either http://www.regulations.gov or 

http://www.epa.gov/pesticides/regulating/registration-status.html, is EPA-HQ-OPP-2011-0774. 

The following documents are available for comment in EPA-HQ-OPP-2011-0774: (1) draft 

Bacillus subtilis strain QST 713 variant Soil Biopesticide Registration Action Document 

(BRAD); (2) environmental risk assessment for Bacillus subtilis strain QST 713 variant Soil; and 

(3) draft label for the end-use pesticide product, Serenade Soil DPZ (EPA File Symbol 69592-

EI). While a final decision on registration is contingent upon review and consideration of public 

comments, EPA believes that, based upon the data and information submitted in support of 

Serenade Soil DPZ, it is appropriate to issue the registration. The basis for this preliminary

decision can be found in the risk assessment for Bacillus subtilis strain QST 713 variant Soil, 
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which is characterized throughout this BRAD. 

II. ACTIVE INGREDIENT OVERVIEW

Biological Name: Bacillus subtilis strain QST 713 variant soil

Culture Deposit: USDA Agricultural Research Service Patent Culture 

Collection: NRLL B-50421 

OPP Chemical Code: 006579

Type of Pesticide: Microbial Pesticide – Biofungicide

See Appendix B for specific information (e.g.,

use sites, application rates, methods of application, 

formulation types, and target pests) regarding

the proposed pesticide products containing this 

active ingredient.

III. REGULATORY BACKGROUND

A. Applications for Pesticide Product Registration

In July 2011, Agraquest, Inc. (address: 1540 Drew Avenue; Davis, California 95618) submitted 

an application to register an end-use pesticide product, Serenade Soil DPZ (EPA File Symbol 

69592-EI), under FIFRA section 3. On October 12, 2011 (76 FR 63298), EPA announced receipt 

of the application to register a pesticide product containing a new active ingredient for use in 

field applications, greenhouses, glasshouses, nurseries, shade houses, landscapes, interiorscapes, 

seedling production sites, and forest seedling production sites, as well as turf and home and 

garden sites. EPA opened a 30-day public comment period pursuant to the provisions of FIFRA 

section 3(c)(4). No comments were received following this publication. 

B. Food Tolerance Exemption

Concurrent with its registration applications and under Federal Food, Drug, and Cosmetic Act 

(FFDCA) section 408(d), Agraquest, Inc., submitted a petition to amend the exemption from the 

requirement of a tolerance for Bacillus subtilis strain QST 713 to include residues of Bacillus 

subtilis strain QST 713 variant Soil (Pesticide Petition (PP) IF7896). In the Federal Register of 

September 7, 2011 (76 FR 55329), the EPA announced that Agraquest, Inc., proposed to amend 

the exemption from the requirement of a tolerance for residues of the microbial pesticide, 

Bacillus subtilis strain QST 713, in or on all raw agricultural crops, to include residues of 

Bacillus subtilis strain QST 713 variant Soil and the Agency opened a 30-day comment period. 

No comments were received following this publication. 

IV. RISK ASSESSMENT

In the Federal Register of October 26, 2007 (72 FR 61002), the EPA issued a Final Rule on the 

data requirements to support registration of microbial pesticides and updated the definition for 

microbial pesticides The rule became effective on December 26, 2007. The data and information 

evaluated for this BRAD were considered in light of these requirements.
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The EPA classifies each data submission with an indication of the usefulness of the information 

contained in the documents for risk assessment. A rating of “acceptable” indicates the study is 

scientifically sound and is useful for risk assessment. A “supplemental” rating indicates the data 

provide some information that can be useful for risk assessment. The studies may have certain 

aspects determined not to be scientifically acceptable (“supplemental: upgradeable”). If a study

is rated as “supplemental: upgradeable,” EPA always provides an indication of what is lacking or 

what can be provided to change the rating to “acceptable.” If there is simply a “supplemental” 

rating, the reviewer will often state that the study is not required by 40 CFR Part 158. Both 

“acceptable” and “supplemental” studies may be used in the risk assessment process as 

appropriate. An “unacceptable” rating indicates that new data must be submitted.

For the acute toxicity data requirements, Toxicity Categories are assigned based on the 

hazard(s) identified from studies and/or other information submitted to the EPA in support of a 

pesticide registration. The EPA classifies the active ingredient or particular product into Toxicity

Category I, II, III, or IV, where Toxicity Category I indicates the highest toxicity and Toxicity

Category IV indicates the lowest toxicity (see 40 CFR § 156.62). 

A. Product Analysis Assessment (40 CFR § 158.2120)

For purposes of registration under FIFRA section 3(c)(5), the EPA determined that all product 

analysis data requirements for Bacillus subtilis strain QST 713 have been fulfilled. Refer to 

Tables 1, 2, 3, and 4 in Appendix A for a summary of the data requirements, including both 

generic and product-specific information.

B. Human Health Assessment (40 CFR § 158.2140)

1. Toxicity

All toxicology data requirements for Bacillus subtilis strain QST 713 variant Soil have been 

fulfilled. Acceptable Tier I mammalian toxicology data and information support registration of 

the pesticide product, Serenade Soil DPZ. EPA concluded that Bacillus subtilis strain QST 713 

and Bacillus subtilis strain QST 713 variant Soil are substantially similar for the purposes of 

assessing toxicity, pathogenicity, and infectivity (Review of Product Chemistry and Acute 

Toxcity Studies, Memorandum from Barsoum to Glikes, July 11, 2012). Furthermore, Tier II and 

Tier III studies were not required for Bacillus subtilis strain QST 713 variant Soil because of the 

lack of acute toxicity/pathogenicity in the Tier I studies.

For a summary of the toxicology data described in sections IV(B)(1)(a) and IV(B)(1)(b) (see 

pages 8–10), refer to Table 5 in Appendix A. For additional information on the generic 

toxicology data on Bacillus subtilis strain QST 713, see the March 24, 2010, Final Registration 

Review Decision for Bacillus subtilis available in EPA’s docket at www.regulations.gov, docket 

number EPA-HQ-OPP-2007-1026.

a. Acute Toxicity/Pathogenicity – Tier I

Acute Oral Toxicity/Pathogenicity – Rat (Harmonized Guideline 885.3050; Master Record 

Identification Number (MRID No. 446519-06): QST 713 Technical was administered orally to 

male and female rats at a dose of approximately 1.13 x 108colony-forming units (cfu)/animal in 
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a l millileter (mL) volume. No adverse clinical signs were observed, and gross necropsy did not 

reveal any abnormalities. The organism cleared from all tissues by Day 14. This study was rated 

“ACCEPTABLE for risk assessment purposes (U.S. EPA 2006; Watson et al. 1999). 

Acute Pulmonary Toxicity/Pathogenicity – Rat (Harmonized Guideline 885.3150; MRID 

No.446519-09): Male and female rats were dosed intratracheally with QST 713 technical powder 

as a suspension in purified water at a concentration of 1.2 x 108cfu/animal. There was no 

mortality, and other than a mottle lung parenchyma on Day 0, no other adverse effects were 

observed via gross necropsy. The test microbe (pre- and post-heat treatment) was detectable in 

lungs of rats through Day 35, although at significantly less levels when compared to Day 0. The 

organism (post-heat treatment) was cleared from the spleen, liver, and kidneys of all animals by

Day 21. This study was rated “ACCEPTABLE” for risk assessment purposes (U.S. EPA 2006; 

Watson et al. 1999). 

Acute Injection Toxicity/Pathogenicity (Intravenous) – Rat (Harmonized Guideline 885.3200; 

MRID No 485309-09 ): Researchers exposed thirty 49- to 84-day-old Sprague-Dawley, albino 

rats (15 per sex) by intravenous route to Bacillus subtilis strain QST 713 variant Soil at doses of 

6.6 x 108cfu/rat. They then observed the rats for up to 21 days. No animals were killed from 

dosing, and all animals appeared normal for the duration of the study. The researchers observed 

no abnormalities, except for 2 enlarged and/or discoloured spleens, at the end of the study. The 

test organism had cleared completely from treatment group blood and brains by day 21 and 

showed a pattern of clearance from other organs. They determined the test substance to be nontoxic, non-infective and non-pathogenic to rats when injected intravenously at a dose of 6.6 x 108

cfu/rat. The EPA classified this intravenous toxicity/pathogenicity study as acceptable.

Hypersensitivity Incidents (Harmonized Guideline 885.3400; MRID 485309-02): The applicant 

reported that no hypersensitivity incidents, including immediate-type or delayed-type reactions 

of humans and domestic animals, occurred during research, development, or testing of Bacillus 

subtilis strain QST713 variant Soil. Any future hypersensitivity incidents must be reported to 

EPA (refer to test note #3 of 40 CFR § 158.2140(d)).

Cell Culture (Harmonized Guideline 885.3500): This study is not required because Bacillus 

subtilis strain QST713 variant Soil is not a virus (refer to test note #4 of 40 CFR § 158.2140(d)).

Acute Oral Toxicity– Rat (Harmonized Guideline 870.1100; Master Record Identification 

Number (MRID No.) 485309-10): Researchers fed three 56- to 84-day-old female SpragueDawley, albino rats a single dose of Serenade Soil DPZ (1.72 %, undiluted test article, Bacillus 

subtilis strain QST 713 variant Soil) at approximately 2.87 x 109cfu/g at doses of 4.89 mL/kg, or 

5000 mg/kg body weight (bw). After 14 days, they observed no deaths or abnormalities. Based 

on the results of this study, Bacillus subtilis strain QST 713 variant Soil does not appear to be 

toxic and/or pathogenic in rats when dosed at approximately 5000 mg/kg bw. The EPA classified 

the acute oral toxicity/pathogenicity as Toxicity Category IV, the lowest toxicity category, and 

the Agency rated the study as acceptable. 

Acute Inhalation Toxicity– Rat (Harmonized Guideline 870-1300; MRID No.485309-12): 

Researchers exposed groups of 56- to 84-day-old Sprague-Dawley, albino rats (5 per sex) nose 

only via the inhalation route to Serenade Soil DPZ at a dose of 2.87 x 109cfu/g (1.72 % a.i.) in 

undiluted test articles for 4 hours at concentrations of 5.15 mg/L. They observed a few symptoms

that did not continue after the fourth day. None of the rats died after 14 days. The EPA classified 
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the acute inhalation toxicity as Toxicity Category IV, the lowest toxicity category, and the 

Agency rated the study as acceptable

Acute Dermal Toxicity - Rat (Harmonized Guideline 870.1200; MRID No 485309-11): 

Researchers treated ten 56- to 84-day-old Sprague-Dawley, albino rats (5 per sex) to Serenade 

Soil DPZ, Bacillus subtilis strain QST 713 variant Soil at doses of 4.94 mL/kg, or 5,050 mg/kg 

body weight (bw), for 24 hours. They observed the animals one hour after exposure and at 

periodic intervals for 14 days. The researchers did not find signs of dermal irritation during any 

of these observations, and they did not observe any abnormalities after the rats were sacrificed 

and dissected. The EPA classified the dermal toxicity as Toxicity Category IV, and the Agency

rated the study as acceptable.

Acute Eye Irritation Study - Rabbit (Harmonized Guideline 870.2400; MRID No 485309-13):

Researchers instilled 0.1ml of Serenade Soil DPZ (Bacillus subtilis strain QST 713 variant Soil, 

2.87 x 109cfu/g or 1.72 % a.i. into the conjunctival sac of the right eye of 3 female, 84-day-old, 

albino rabbits for 24 hours. They observed the animals for 3 days and rated the effects with a 

maximum average irritation score of 2.0, meaning that the formulation is practically nonirritating to the eye. Since there were no positive effects and minimal effects cleared in less than 

24 hours, the EPA classified the eye irritation as Toxicity Category IV, and the Agency rated the 

study as acceptable.

Acute Dermal Irritation Study - Rabbit (Harmonized Guideline 870.2500; MRID No 485309-

14):Researchers applied 0.5 mL of Serenade Soil DPZ (Bacillus subtilis strain QST713 variant 

Soil, 2.87 x 109cfu/g, 1.72 % a.i.) to the trunks of 2 male and 1 female albino rabbits and then 

covered the exposed skin for 4 hours. After observing the rabbits for 3 days (72 hours), the 

researchers determined that there was no dermal irritation to the skin, based on a maximum 

irritation score of 0 at 24, 48 and 72 hour observations. The EPA classified the primary dermal 

irritation as Toxicity Category IV and rated the study as acceptable.

b. Acute Toxicology and Subchronic Toxicity/Pathogenicity – Tier II; 

ReproductiveFertility Effects, Carcinogenicity, Immunotoxicity, and 

Infectivity/Pathogenicity Analysis – Tier III

Tier II and Tier III studies were not required for Bacillus subtilis strain QST 713 variant Soil,

based on the lack of acute toxicity/pathogenicity in the Tier I studies for both Bacillus subtilis 

strain QST 713 and Bacillus subtilis strain QST 713 variant Soil.

c. Endocrine Disruptors

As required by the Administrator under the Federal Food, Drug, and Cosmetic Act (FFDCA), the 

EPA has developed the Endocrine Disruptor Screening Program (EDSP) and has begun to 

implement the screening program that is to be used to test all pesticides to determine whether 

certain substances (including pesticide active and other ingredients) may have an effect in 

humans or wildlife similar to an effect produced by a “naturally occurring estrogen, or other such 

endocrine effects as the Administrator may designate.” FFDCA section 408(p)(4) authorizes the 

Administrator, by order, to exempt from the requirements of the Endocrine Disruptor Screening 

Program a biologic substance or other substance if a determination is made that the substance is 

anticipated to not produce any effect in humans similar to an effect produced by a naturally 

occurring estrogenic substance. Between October 2009 and February 2010, EPA issued test 
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orders/data call-ins for the first group of 67 chemicals, which contains 58 pesticide active 

ingredients and 9 inert ingredients. Bacillus subtilis is not among the group of 58 pesticide active 

ingredients on the initial list to be screened under the EDSP. 

The EPA believes that Bacillus subtilis is a substance that would not produce any effect in 

humans similar to an effect produced by a naturally occurring estrogenic substance. As such, and 

pursuant to Section 408(p)(4), the EPA will determine in the future whether it can exempt 

Bacillus subtilis from the requirements of the Section 408(p)(4) EDSP. In the event the EPA 

does determine to exempt this substance from the EDSP, an order will be issued. For further 

information on the status of the EDSP, the policies and procedures, the list of 67 chemicals, the 

test guidelines and the Tier 1 screening battery, please visit our website: 

http://www.epa.gov/endo/.

2. Federal Food, Drug, and Cosmetic Act (FFDCA) Considerations

Section 408(c)(2)(A)(i) of FFDCA allows the EPA to establish an exemption from the 

requirement for a tolerance (the legal limit for a pesticide chemical residue in or on a food) only 

if the EPA determines that the exemption is “safe.” Section 408(c)(2)(A)(ii) of FFDCA defines 

“safe” to mean that “there is a reasonable certainty that no harm will result from aggregate 

exposure to the pesticide chemical residue, including all anticipated dietary exposures and all 

other exposures for which there is reliable information.” This includes exposure through drinking 

water and in residential settings, but does not include occupational exposure. Pursuant to section 

408(c)(2)(B) of FFDCA, in establishing or maintaining in effect an exemption from the 

requirement of a tolerance, the EPA must take into account the factors set forth in section 

408(b)(2)(C) of FFDCA, which require the EPA to give special consideration to exposure of 

infants and children to the pesticide chemical residue in establishing a tolerance exemption, and 

to “ensure that there is a reasonable certainty that no harm will result to infants and children from 

aggregate exposure to the pesticide chemical residue....” Additionally, section 408(b)(2)(D) of 

FFDCA requires that the EPA consider “available information concerning the cumulative effects 

of [a particular pesticide's] . . . residues and other substances that have a common mechanism of 

toxicity.”

The EPA performs a number of analyses to determine the risks from aggregate exposure to 

pesticide residues. First, the Agency determines the toxicity of a pesticide. Second, the EPA 

examines exposure to the pesticide through food, drinking water, and through other exposures 

that occur as a result of pesticide use in residential settings.

Consistent with section 408(b)(2)(D) of FFDCA, the EPA has reviewed the available scientific 

data and other relevant information, and considered its validity, completeness, and reliability and 

the relationship of this information to human risk. The Agency also considered available 

information concerning the variability of the sensitivities of major identifiable subgroups of 

consumers, including infants and children.

Based on the acute toxicity/pathogenicity data and information discussed previously and 

presented in Table 5 in Appendix A, the data required for a FFDCA risk assessment for Bacillus 

subtilis strain QST 713 variant Soil have been fulfilled. Hence, the EPA amended the existing

exemption for the requirement of a tolerance for Bacillus subtilis strain QST to include residues 

of Bacillus subtilis strain QST variant soil.
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a. Aggregate Exposure

In examining aggregate exposure, section 408 of FFDCA directs EPA to consider 

available information concerning exposures from the pesticide residue in food and all other 

nonoccupational exposures, including drinking water from ground water or surface water and 

exposure through pesticide use in gardens, lawns, or buildings (residential and other indoor 

uses).

Food Exposure and Risk Characterization: The EPA found that dietary exposure to Bacillus 

subtilis strain QST 713 variant Soil, a naturally occurring soil bacterium, is anticipated to be 

negligible. Due to the ubiquitous nature of the organism, and the concentrations of Bacillus 

subtilis and other closely related Bacillus species that already exist in the environment, the 

likelihood of increased risk to humans or animals from the use of Serenade Soil DPZ is low. The 

EPA in its registration review decision concluded “the risk posed to adults, infants, and children 

is likely to be minimal because of the low acute oral toxicity/pathogenicity potential of Bacillus 

subtilis strain QST 713.” The EPA concluded, based on the similarity in product composition 

and production, measured physical/chemical, and pathogenicity/infectivity toxicity data, that the 

risk from any potential exposure to Bacillus subtilis strain QST 713 variant Soil in Serenade Soil 

DPZ also would be minimal.

Drinking Water Exposure and Risk Characterization: Exposure to residues of Bacillus subtilis

strain QST 713 variant Soil in consumed drinking water is unlikely. The proposed use patterns 

for Serenade Soil DPZ are soil directed and/or soil incorporated, thereby limiting contact with 

surface water by drift and runoff. Furthermore, ground water is not expected to have significant 

exposure to Bacillus subtilis strain QST 713 variant Soil since, like other microorganisms, this 

microbial pesticide would likely be filtered out by the particulate nature of many soil types. If it 

were to be transferred to surface or ground waters that are intended for eventual human 

consumption (e.g., through spray drift or runoff) and directed to wastewater treatment systems or

drinking water facilities, it likely would not survive the conditions water is subjected to in such 

systems or facilities, including chlorination, pH adjustments, filtration, and/or occasionally high 

temperatures (Centers for Disease Control and Prevention 2009; U.S. EPA 2004). In the remote 

likelihood that Bacillus subtilis strain QST 713 variant Soil is present in drinking water (e.g., 

water not subject to treatment systems or facilities), its target pest specificity and available data

indicate no toxicity and/or pathogenicity is likely to occur with any drinking water exposure to 

Bacillus subtilis strain QST 713 variant Soil that results from pesticide applications made in 

accordance with good agricultural practices (see section IV(B)(1)(a) on pages 8–9 and Table 5 in 

Appendix A).

Non-occupational, Residential Risk Characterization: Given Bacillus subtilis strain QST 713 

variant Soil’s natural occurrence in soil, EPA determined that non-occupational exposure to the 

bacterium likely is already occurring. Additional exposure to the microorganism, due to 

pesticidal applications, is not expected because such exposures do not exceed EPA’s level of 

concern, particularly in light of available data that demonstrate Bacillus subtilis strain QST 713 

and Bacillus subtilis strain QST 713 variant Soil are not toxic (acute dermal toxicity and acute 

pulmonary toxicity/pathogenicity), non-irritating (primary dermal irritation), and not pathogenic 

(acute pulmonary toxicity/pathogenicity) (see section IV(B)(1)(a) on pages 8–9 and Table 5 in 

Appendix A).
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b. Cumulative Effects from Substances with a Common Mechanism of Toxicity

Section 408(b)(2)(D)(v) of FFDCA requires that, when considering whether to establish, modify, 

or revoke a tolerance exemption, the EPA consider “available information concerning the 

cumulative effects of [a particular pesticide’s] . . . residues and other substances that have a 

common mechanism of toxicity.”

The EPA has not found Bacillus subtilis strain QST 713 variant Soil to share a common 

mechanism of toxicity with other substances, and Bacillus subtilis strain QST 713 variant Soil

does not appear to produce a toxic metabolite against target pests. For the purposes of the 

tolerance action, therefore, the EPA has assumed that Bacillus subtilis strain QST 713 variant 

Soil does not have a common mechanism of toxicity with other substances. Thus, section 

408(b)(2)(D)(v) of the FFDCA does not apply. For information regarding the EPA’s efforts to 

determine which chemicals have a common mechanism of toxicity and to evaluate the 

cumulative effects of such chemicals, see the EPA’s website at 

http://www.epa.gov/pesticides/cumulative.

c. Determination of Safety for the United States Population, Infants and Children

In considering the establishment of a tolerance or tolerance exemption for a pesticide chemical 

residue, FFDCA section 408(b)(2)(C) provides that the EPA shall assess the available 

information about consumption patterns among infants and children, special susceptibility of 

infants and children to pesticide chemical residues, and the cumulative effects on infants and 

children of the residues and other substances with a common mechanism of toxicity. In addition, 

FFDCA section 408(b)(2)(C) provides that the EPA shall apply an additional tenfold (10X) 

margin of exposure (safety) for infants and children in the case of threshold effects to account for 

prenatal and postnatal toxicity and the completeness of the database on toxicity and exposure 

unless the EPA determines that a different margin of exposure (safety) will be safe for infants 

and children. This additional margin of exposure (safety) is commonly referred to as the Food 

Quality Protection Act Safety Factor. In applying this provision, the EPA either retains the 

default value of 10X or uses a different additional safety factor when reliable data available to 

the EPA support the choice of a different factor.

Based on the acute toxicity and pathogenicity data/information discussed in section IV(B)(1)(a) 

(see pages 8–9) and Table 5 in Appendix A, the EPA concludes that there are no threshold 

effects of concern to infants, children, or adults when Bacillus subtilis strain QST 713 variant 

Soil is used as labeled in accordance with good agricultural practices. As a result, the EPA 

concludes that no additional margin of exposure (safety) is necessary to protect infants and 

children and that not adding any additional margin of exposure (safety) will be safe for infants 

and children.

Moreover, based on the same data/information and EPA analysis as presented directly above, the 

Agency is able to conclude that there is a reasonable certainty that no harm will result to the 

United States population, including infants and children, from aggregate exposure to the residues 

of Bacillus subtilis strain QST 713 variant Soil when it is used—as labeled and in accordance 

with good agricultural practices—as a biofungicide. Such exposure includes all anticipated 

dietary exposures and all other exposures for which there is reliable information. The EPA has 

arrived at this conclusion because, considered collectively, the data and information available on 

Bacillus subtilis strain QST 713 and Bacillus subtilis strain QST 713 variant Soil do not 
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demonstrate toxic and/ pathogenic potential to mammals, including infants and children. 

3. Occupational Exposure and Risk Characterization

The EPA does not expect handler exposure to Bacillus subtilis strain QST 713 variant Soil to 

pose any undue risk. Regardless, appropriate personal protective equipment (PPE) and 

precautionary statements are required on pesticide product labels to mitigate any potential risks 

to pesticide handlers due to prolonged or numerous exposures. Handlers applying Serenade Soil 

DPZ in agricultural settings must wear a long-sleeved shirt, long pants, socks, and shoes. For 

future products containing this microorganism as an active ingredient, the EPA may require 

additional PPE (e.g., protective eyewear and/or dust/mist filtering respirators) on a productspecific basis.

4. Human Health Risk Characterization

The EPA considered human exposure to Bacillus subtilis strain QST 713 variant Soil in light of 

the standard for registration in FIFRA and the relevant safety factors in FFDCA. The Agency

determined that no unreasonable adverse effects to the United States population in general, and 

to infants and children in particular, will result when Bacillus subtilis strain QST 713 variant Soil

is used in accordance with EPA-approved labeling.

C. Environmental Assessment (40 CFR § 158.2150)

Data and other information (e.g., scientific literature) submitted by the applicant to support 

requests to waive nontarget organism testing for Bacillus subtilis strain QST 713 variant Soil are 

sufficient to fulfill the relevant microbial pesticide data requirements and for risk assessment 

purposes. Further testing of nontarget organisms at higher tier levels (i.e., Tiers II, III, and IV) is 

not required. The EPA performed an environmental risk assessment based on data and other

information provided by the applicant, and the Agency determined that adverse effects to 

nontarget organisms are not anticipated from the proposed pesticidal uses of Bacillus subtilis

strain QST 713 variant Soil. Moreover, the EPA has made a “No Effect” determination for direct 

and indirect effects to listed species and their designated critical habitats resulting from these 

same proposed pesticidal uses. 

For a summary of the generic data requirements described in sections IV(C)(1) (see pages 14–

17), refer to Table 6 in Appendix A.

1. Ecological Exposure and Risk Characterization 

a. Terrestrial Animals and Plants

Birds (Harmonized Guideline 885.4050), Nontarget Insects (Harmonized Guideline 885.4340),

and Honey Bees (Harmonized Guideline 885.4380) (MRID No. 4853909-15): 

While applications of the proposed end use product containing Bacillus subtilis strain QST 713 

variant Soil are soil directed, birds and wild mammals could be exposed. Such exposure is 

expected to occur through ingestion of food items that contact the active ingredient, or it may

occur through incidental ingestion of soil or drinking water (e.g., puddles) that contain Bacillus

subtilis strain QST 713 variant Soil. The percentage of the a.i. in the proposed end use product is 
Bacillus subtilis strain QST 713 variant Soil Page 15 of 28

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similar to that of Serenade ASO, a currently-registered end use product containing Bacillus

subtilis strain QST 713. Since the potency and maximum application rates are the same for both 

products, the proposed uses of the end-use product, Serenade Soil DPZ, are expected to result in 

similar field exposures and the maximum hazard doses used in previously submitted studies are 

sufficient for determining risk. Birds and wild mammals were not adversely affected when 

exposed at high dosage rates, as demonstrated in bird studies with B. subtilis strain QST 713

(MRID 44651910), and adverse effects were not observed in rats (MRID 44651906). Therefore, 

adverse effects to birds and wild mammals are not expected if they are exposed to Bacillus

subtilis strain QST 713 variant Soil as a result of the proposed labeled uses. 

Nontarget insects and honey bees may be exposed if they contact surfaces, including soil, treated 

with Bacillus subtilis strain QST 713 variant Soil, or if this a.i. is present on food items. Studies 

submitted for the registration of Bacillus subtilis strain QST 713 show that adverse effects to 

several species of nontarget insects and honey bees do not occur when the bees are exposed at 

high levels. Researchers observed mortalities in studies with green lacewing (Chrysoperla 

carnea) and ladybird beetle (Hippodamia convergens), but the dead insects were not caused by

treatment with the a.i. Several supplemental field studies demonstrated that B. subtilis strain QST 

713 applied at field rates is not hazardous to honey bees. Based on the results of these studies 

and expected field exposure levels, EPA scientists do not expect adverse effects to nontarget 

insects and honey bees from the proposed applications of B. subtilis strain QST 713 variant Soil.

The EPA determined that, since Bacillus subtilis strain QST 713 variant Soil is not 

taxonomically related to known plant pathogens, adverse effects to nontarget plants are not 

expected. 

b. Aquatic Animals and Plants

Freshwater Fish (Harmonized Guideline 885.4200, Freshwater Invertebrates (Harmonized 

Guideline 885.4240), Estuarine/Marine Fish and Invertebrates (Harmonized Guideline 

885.4280) (MRID No. 4853909-15):

Freshwater fish and invertebrates could be exposed to the Bacillus subtilis strain QST 713 

variant Soil from runoff and drift into aquatic areas. While most of the proposed applications are 

soil directed, some applications are allowed to occur via spray equipment (e.g., for turf uses). 

These applications could result in some runoff and drift of the applied Bacillus subtilis strain 

QST 713 variant Soil into aquatic areas, but the concentrations resulting from drift and runoff are 

expected to be a small fraction of the amount applied. A study conducted with rainbow trout 

(MRID 45445202) demonstrated that fish died when exposed to high levels of the a.i.; however,

the reviewers determined that these deaths resulted from water fouling by high concentrations of 

the test material and not from toxicity or pathogenicity. The reviewers observed similar problems 

in a study on Daphnia magna, and they determined that mortalities at high exposure levels were 

also due to fouling of the water (MRID 48530905). Therefore, adverse effects to freshwater fish 

and invertebrates are not expected from proposed applications of Bacillus subtilis strain QST 713 

variant Soil as labeled.

The EPA determined, based on its analysis of freshwater animals, that high concentrations of 

Bacillus subtilis strain QST 713 variant Soil are not expected to be deposited and persist in 

estuarine or marine areas. Therefore, estuarine and marine fish and invertebrates are not likely to 

be exposed, and the risk to these species as a result of the proposed applications of Bacillus
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subtilis strain QST 713 variant Soil is minimal.

2. Environmental Fate Data 

As the data and information provided are sufficient to fulfill the Tier I nontarget organism data 

requirements and allow for nontarget organism risk assessment for Bacillus subtilis strain QST 

713 variant Soil, further testing at higher tier levels (i.e., Tiers II, III, and IV) is not required. 

3. Threatened and Endangered Species Assessment

Since the EPA has determined that no effects are anticipated for any nontarget species exposed 

to Bacillus subtilis strain QST 713 variant Soil as a result of the proposed applications, effects to 

federally listed threatened and endangered species and their designated critical habitats also are 

not expected. Therefore, a “No Effect” determination is made for direct and indirect effects to 

listed species and their designated critical habitats resulting from the proposed pesticidal uses of

Bacillus subtilis strain QST 713 variant Soil, as labeled.

V. ENVIRONMENTAL JUSTICE

The EPA seeks to achieve environmental justice—the fair treatment and meaningful involvement 

of all people regardless of race, color, national origin, or income—with respect to the 

development, implementation, and enforcement of environmental laws, regulations, and policies. 

Fair treatment means that no group of people, including racial, ethnic, or socioeconomic groups, 

should bear a disproportionate share of the negative environmental consequences resulting from 

industrial, municipal, and commercial operations or the execution of federal, state, local, and 

tribal environmental programs and policies. Meaningful involvement means that (1) potentially

affected community residents have an appropriate opportunity to participate in decisions about a 

proposed activity that will affect their environment and/or health; (2) the public’s contribution 

can influence the regulatory agency’s decision; (3) the concerns of all participants involved will 

be considered in the decision-making process; and (4) the decision-makers seek out and facilitate 

the involvement of those potentially affected. The EPA has this goal for all communities and 

persons across the United States. 

To help address potential environmental justice issues, the EPA seeks information on any groups 

or segments of the population who, as a result of their location, cultural practices, or other 

factors, may have atypical, unusually high exposure to Bacillus subtilis strain QST 713 variant 

Soil, compared to the general population. Please comment if you are aware of any

subpopulations that may have atypical, unusually high exposure compared to the general 

population.

For additional information regarding environmental justice issues, please visit the EPA’s web 

site at http://www.epa.gov/compliance/environmentaljustice/index.html.

VI. RISK MANAGEMENT DECISION

Section 3(c)(5) of FIFRA permits for the registration of a pesticide provided that all the 

following determinations are made:

(1) Its composition is such as to warrant the proposed claims for it;
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(2) Its labeling and other material required to be submitted comply with the

 requirements of FIFRA;

(3) It will perform its intended function without unreasonable adverse effects on 

 the environment; AND

(4) When used in accordance with widespread and commonly recognized 

 practice, it will not generally cause unreasonable adverse effects on the 

 environment.

To satisfy criterion 1, the Bacillus subtilis strain QST 713 variant Soil product has well-known 

properties. The EPA has no knowledge that would contradict the claims made on the Serenade 

Soil DPZ label, and this pesticide product is not expected to cause unreasonable adverse effects

on the environment when used according to label instructions. Criterion 2 is satisfied by the 

current product label, as well as the data and information presented in this document. The 

Agency believes that the Bacillus subtilis strain QST 713 variant Soil pesticide product will not 

cause any unreasonable adverse effects on the environment. In fact, Serenade Soil DPZ is likely 

to provide protection against many plant diseases, satisfying criterion 3. Criterion 4 is satisfied in 

that the Bacillus subtilis strain QST 713 variant Soil pesticide product is not expected to cause 

unreasonable adverse effects when used according to label instructions. Therefore, Serenade Soil 

DPZ, containing Bacillus subtilis strain QST 713 variant Soil as a new active ingredient, is

eligible for registration under FIFRA section 3(c)(5) for the labeled uses. 

VII. ACTIONS REQUIRED OF THE REGISTRANT

A. Final Printed Labeling

Before Serenade Soil DPZ— the pesticide product containing Bacillus subtilis strain QST 713 

variant Soil –may be released for shipment, the registrant will be required to provide appropriate 

final printed labeling to the EPA.

B. Terms of Registration

As a term of a Serenade Soil DPZ registration, Agraquest, Inc. will be required to submit 

additional information (confirmatory) on the product within six months of registration.

Additionally, as terms of a Serenade Soil DPZ registration, Agraquest, Inc. will be required to 

submit within one year of the date of registration a study on storage stability (OCSPP 830.6317)

and a study demonstrating that Bacillus subtilis strain QST 713 variant Soil does not revert to 

Bacillus subtilis strain QST 713.

C. Reporting of Adverse Effects and Hypersensitivity Incidents

Notwithstanding the information stated in the previous sections, it should be clearly understood 

that certain specific data must be reported to the EPA as a requirement for maintaining the 

federal registration for a pesticide product. Reports of all incidents of adverse effects to the 

environment must be submitted to the EPA under the provisions stated in FIFRA section 6(a)(2). 

Additionally, all incidents of hypersensitivity (including both suspected and confirmed incidents) 

must be reported to EPA under the provisions of 40 CFR § 158.2140(d).
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VIII. GLOSSARY OF ACRONYMS AND ABBREVIATIONS

BRAD Biopesticides Registration Action Document

CFR Code of Federal Regulations

cSt centistokes

EDSP Endocrine Disruptor Screening Program

EP end-use pesticide product

EPA United States Environmental Protection Agency (the “Agency”)

FFDCA Federal Food, Drug, and Cosmetic Act

FIFRA Federal Insecticide, Fungicide, and Rodenticide Act

FR Federal Register

g/mL grams per milliliter

lb/gal pounds per gallon

LD50 median lethal dose. A statistically derived single dose that can be expected 

to cause death in 50% of the test animals when administered by the route 

indicated (oral, dermal, or inhalation). It is expressed as a weight of 

substance per unit weight of animal (e.g., mg/kg).

mg/kg milligrams per kilogram

MP manufacturing-use pesticide product

MRID No. Master Record Identification Number

NIOSH National Institute for Occupational Safety and Health

OPP Office of Pesticide Programs

PC Code Pesticide Chemical Code

PP Pesticide Petition

PPE personal protective equipment

TGAI technical grade of the active ingredient

U.S. United States
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IX. BIBLIOGRAPHY

A. Studies Submitted to Support the Active Ingredient, Bacillus subtilis strain QST 713 

variant Soil, and Pesticide Product Registration, Serenade Soil DPZ.

MRID No. Study Information

44651901 Bellet, E. (1998) Product Chemistry for QST 713 Technical. Unpublished study

prepared by Chemical Consultants International, Inc. 4 p.

44659104 Bellet, E. (1998) Manufacturing and Analytical Data for Technical QST 713.

Unpublished study prepared by Chemical Consultants International, Inc. 48 p.

45206101 Hilbig, L. (2000) Manufacturing Process Amendment for QST 713 Technical

Powder. Unpublished study prepared by AgraQuest, Inc. 23 p.

44651905 Gingras, B. (1998) Sensitivity of Detection of Bacillus subtilis Strain QST 713 for 

Toxicity/Pathogenicity Testing in Rats: Lab Project Number: L08726SN3. 

Unpublished study prepared by IIT Research Institute. 30 p

44651906 Harrington, K. (1998) Toxicity/Pathogenicity Testing of QST 713 Following

Acute Oral Challenge in Rats: Lab Project Number: L08726SN4. Unpublished

study prepared by IIT Research Institute. 126 p.

44651907 Findlay, J. (1998) Acute Dermal Toxicity/Pathogenicity Study of QST 713 in 

Rabbits: Lab Project Number: L08726SN7. Unpublished study prepared by IIT

Research Institute. 27 p.

44651909 Harrington, K. (1998) Toxicity/Pathogenicity Testing of QST 713 Following 

Acute Intratracheal Challenge in Rats: Lab Project Number: L08726SN6. 

Unpublished study prepared by IIT Research Institute. 137 p.

44651908 Harrington, K. (1998) Toxicity/Pathogenicity Testing of QST 713 Following 

Acute Intravenous Challenge in Rats: Lab Project Number: L08726SN5. 

Unpublished study prepared by IIT Research Institute. 119 p.

44651910 Foster, J.; Grimes, J.; Beavers, J. (1998) An Avian Oral Pathogenicity and 

Toxicity Study in the Northern Bobwhite: (QST 713): Lab Project Number: 489-

101. Unpublished study prepared by Wildlife International Ltd. 28 p.

45455202 Drottar, K.; Flaggs, R.; Krueger, H. (2001) QST 713 Technical: A FiveConcentration Toxicity and Pathogenicity Test with the Rainbow Trout

(Oncorhynchus mykiss): Final Report: Lab Project Number: 489A-108. 

Unpublished study prepared by Wildlife International, Ltd. 39 p.

44651912 Drottar, K.; Krueger, H. (1998) A 48-Hour Static Acute Toxicity Test with the 

Cladoceran (Daphnia Magna): (QST 713): Final Report: Lab Project Number: 

489A-103. Unpublished study prepared by Wildlife International Ltd. 22 p.
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MRID No. Study Information

45455203 Drottar, K.; Flaggs, R.; Krueger, H. (2001) QST 713 Technical: A 21-Day LifeCycle Toxicity and Pathogenicity Test with the Cladoceran (Daphnia magna): 

Final Report: Lab Project Number: 489-107A. Unpublished study prepared by

Wildlife International, Ltd. 39 p.

45455205 Bryan, R.; Porch, J.; Krueger, H. (2001) Bacillus Subtilis Strain QST 713: A 

Dietary Pathogenicity and Toxicity Study with the Parasitic Hymenopteran 

(Nasonia vitripennis): Final Report: Lab Project Number: 489-107B. Unpublished 

study prepared by Wildlife International, Inc. 25 p.

44651915 Hoxter, K.; Krueger, H.; Palmer, S. (1998) A Dietary Pathogenicity Toxicity 

Study with the Ladybird Beetle (Hippodamia convergens): (QST 713): Lab 

Project Number: 489-103B. Unpublished study prepared by Wildlife International 

Ltd. 24 p.

44651916 Hoxter, K.; Krueger, H.; Palmer, S. (1998) A Dietary Pathogenicity Toxicity 

Study with the Green Lacewing Larvae (Chrysoperia carnea): (QST 713): Lab 

Project Number: 489-104. Unpublished study prepared by Wildlife International 

Ltd. 17 p.

46172902 Jimenez, D.; Richards, K. (2004) Evaluation of the Dietary Effect(s) of QST 713 

Technical Powder on Larval Honeybee Development (Apis mellifera L.). Project 

Number: CAR/158/03. Unpublished study prepared by California Agricultural 

Research Inc. 70 p.

46172901 Jimenez, D. (2004) Discussion of the Results of Honeybee Studies Conducted 

with QST 713 Technical and Serenade Products. Unpublished study prepared by

Agraquest Inc and California State Polytechnic College and Montana State

University. 237 p.

45385301 Van Koppenhagen, J.; Hilbig, L. (2001) Product Chemistry of Serenade AS: Lab 

Project Number: QC-012301. Unpublished study prepared by AgraQuest, Inc. 95 

p.

45385309 Freeman, D. (2000) Dermal Sensitization Study (Closed-Patch Repeated Insult) in 

Guinea Pigs with Serenade AS: Lab Project Number: 012774-1. Unpublished 

study prepared by Ricerca, LLC. 36 p.

46012201 Lin, J.; Martinez, T.; Czochor, L. (2003) Product Chemistry of Serenade ASO. 

Project Number: AC/F/007/1. Unpublished study prepared by AgraQuest Inc. 79 

p.

45662301 Czochor, L. (2002) Amendment to MRID No. 45385301, Manufacturing Process: 

Submission of Replacement Page 17. Unpublished study prepared by AgraQuest, 

Inc. 8 p.
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MRID No. Study Information

47720601 Heins, S. (2009) Product Chemistry of QRD 146. Project Number: 090225/1. 

Unpublished study prepared by AgraQuest, Inc. 67 p.

48530900 AgraQuest, Inc. (2011) Submission of Product Chemistry and Toxicity Data in 

Support of the Application for Registration of Serenade Soil DPZ. Transmittal of 

15 Studies.

48530901 Heins, S.; Joo, D. (2011) Supplemental Information on Bacillus Identification as 

Discussed in Amended Final Report 14676-10 and Final Report 15246-11. Project 

Number: REG110624/1. Unpublished study prepared by AgraQuest, Inc. 126 p.

48530902 Heins, S.; Thurston, J.; Guilhabert-Goya, M.; et al. (2011) Product Chemistry of 

Bacillus subtilis Strain QST 713 Variant Soil (TGAI). Project Number: 110509/2. 

Unpublished study prepared by AgraQuest, Inc. 107 p.

48530903 Kaminsky, M. (2011) QST 713 Strain of Bacillus subtilis Variant Soil (TGAI): 

Product Chemistry: Amended Final Report. Project Number: 15001/11. 

Unpublished study prepared by Stillmeadow, Inc. 19 p.

48530904 Monds, K. (2011) QST 713 Strain of Bacillus subtilis Variant Soil: Identification 

and Enumeration of a Microbial Pest Control Agent (MPCA): Final Report. 

Project Number: 15246/11. Unpublished study prepared by Stillmeadow, Inc. 10 

p.

48530905 Heins, S.; Thurston, J.; Guilhabert-Goya, M.; et al. (2011) Product Chemistry of 

Serenade Soil DPZ (EP). Project Number: 110509/1. Unpublished study prepared 

by AgraQuest, Inc. 70 p.

48530906 Kaminsky, M. (2011) Serenade Soil DPZ (EP): Product Chemistry: Final Report. 

Project Number: 15000/11. Unpublished study prepared by Stillmeadow, Inc. 9 p.

48530907 Sehn, A.; Heins, S. (2007) Serenade ASP Storage Stability at 25 Degrees Celsius. 

Project Number: AQ/040514/2, AQ/061121/1. Unpublished study prepared by

AgraQuest, Inc. 42 p.

48530908 Heins, S. (2011) Bacillus subtilis Strain QST 713 Variant Soil: Request for Acute 

Mammalian Toxicity / Pathogenicity Study Waivers. Project Number: 

REG110602/1. Unpublished study prepared by AgraQuest, Inc. 11 p.

48350909 Monds, K. (2011) QST 713 Strain of Bacillus subtilis Variant Soil: Intravenous 

Toxicity / Pathogenicity Study in Rats: Amended Final Report. Project Number: 

14676/10. Unpublished study prepared by Stillmeadow, Inc. 42 p.

48530910 Monds, K. (2011) QST 713 Strain of Bacillus subtilis Variant Soil: Intravenous 

Toxicity / Pathogenicity Study in Rats: Amended Final Report. Project Number: 

14676/10. Unpublished study prepared by Stillmeadow, Inc. 42 p.
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MRID No. Study Information

48530911 Kuhn, J. (2011) Serenade Soil DPZ: Acute Dermal Toxicity Study in Rats: Final 

Report. Project Number: 14932/11. Unpublished study prepared by Stillmeadow, 

Inc. 13 p.

48530912 Doig, A. (2011) Serenade Soil DPZ: Acute Inhalation Toxicity Study in Rats: 

Final Report. Project Number: 14933/11. Unpublished study prepared by

Stillmeadow, Inc. 18 p.

48530913 Kuhn, J. (2011) Serenade Soil DPZ: Acute Eye Irritation Study in Rabbits: Final 

Report. Project Number: 14934/11. Unpublished study prepared by Stillmeadow, 

Inc. 17 p.

48530914 Kuhn, J. (2011) Serenade Soil DPZ: Acute Dermal Irritation Study in Rabbits: 

Final Report. Project Number: 14935/11. Unpublished study prepared by

Stillmeadow, Inc. 11 p.

48530915 Heins, S. (2011) Bacillus subtilis Strain QST 713 Variant Soil: Nontarget 

Organism Toxicity/Pathogenicity Study Waiver Requests. Project Number: 

REG110603/2. Unpublished study prepared by AgraQuest, Inc. 15 p.

48906300 AgraQuest, Inc. (2012) Submission of Product Chemistry Data in Support of the 

Application for Registration of Serenade Soil DPZ. Transmittal of 1 Study.

48906301 Curtis, D. (2012) Bacillus subtilis Strain QST 713 Variant Soil (TGAI): Product 

Identity: Further Information on the Characterization of the MPCA. Project 

Number: 120810/AQ/REG/01. Unpublished study prepared by AgraQuest, Inc. 

6p.

B. Environmental Protection Agency Risk Assessment Memoranda

U.S. EPA. 2012a. Review of Product Chemistry and Acute Toxicity Studies for section 3 

registration of a new TGAI Bacillus subtilis strain QST 713 variant Soil and a new EP 

Serenade Soil DPZ (EPA Reg. no. 69592-EI). Memorandum from Ibrahim S. Barsoum, 

Ph.D. and John L. Kough, Ph.D. to Michael Glikes, dated July 11, 2012.

U.S. EPA. 2012b. Environmental Risk Assessment for a FIFRA Section 3 Registration of 

Bacillus subtilis strain QST 713 variant Soil (EPA File Symbol 69592-EI). Memorandum 

from Shannon Borges to Michael Glikes, dated August 2, 2012.

U.S. EPA. 2012c. Review of additional data concerning the genetic stability of Bacillus subtilis

strain QST 713 variant Soil for section 3 registration of the TGAI and a new EP Serenade 

Soil DPZ (EPA Reg. no. 69592-EI). Memorandum from Ibrahim Barsoum, Ph.D. and 

John L. Kough, Ph.D. to Michael Glikes, dated August 23, 2012.

U.S. EPA. 2010. Bacillus subtilis Case 6012 Final Registration Review Decision, March 2010.
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C. Other References

Centers for Disease Control and Prevention. 2009. Drinking Water - Water Treatment. Available 

from http://www.cdc.gov/healthywater/drinking/public/water_treatment.html.

DeFelice K, Wollenhaupt N, Buchholz D. 1993. Aquifers and Soil Filter Effect. Available from 

http://extension.missouri.edu/p/WQ24.

U.S. EPA. 2004. Primer for Municipal Wastewater Treatment Systems. EPA 832-R-04-001. 

Available from http://www.epa.gov/npdes/pubs/primer.pdf.
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APPENDIX A. MICROBIAL PESTICIDES DATA REQUIREMENTS 

(40 CFR PART 158 – SUBPART V)

TABLE 1. Product Analysis Data Requirements for the Active Ingredient (TGAI), Bacillus subtilis strain QST 713 variant Soil

(40 CFR § 158.2120)

Harmonized 

Guideline 

Number

Data Requirement Results MRID No.

885.1100 Product Identity

Submitted data fulfill the requirement for product identity. Bacillus subtilis

strain QST 713 variant Soil is a naturally occurring variant of Bacillus subtilis

strain QST 713.

446519-01

885.1200 Manufacturing 

Process Submitted data fulfill the requirement for manufacturing process.

446519-01,

446419-04

N/A

Deposition of a 

Sample in a 

Nationally 

Recognized Culture 

Collection

Bacillus subtilis strain QST 713 variant Soil is on deposit with the USDA 

Agricultural Research Service Patent Culture Collection: NRLL B-50421. 446519-04

885.1300

Discussion of 

Formation of 

Unintentional 

Ingredients

As a term of registration, EPA is requiring additional information 

(confirmatory) on this data requirement. 446519-04

885.1400 Analysis of Samples Submitted data fulfill the requirement for analysis of samples. 446519-02

885.1500

Certification of 

Limits Submitted data fulfill the requirement for manufacturing process. 450845-01

TABLE 2. Product Analysis Data Requirements for the End-Use Pesticide Product (EP), Serenade Soil DPZ

(40 CFR § 158.2120)

Harmonized

Guideline 

Number

Data Requirement Results MRID No.

885.1100 Product Identity

Submitted data fulfill the requirement for product identity. 

Serenade Soil DPZ contains 1.57% by weight Bacillus subtilis

strain QST 713 variant Soil (contains at least 1 x 109cfu per 

gram).

485309-05

885.1200 Manufacturing Process Submitted data fulfill the requirement for manufacturing 

process.

485309-05

N/A

Deposition of a Sample in a 

Nationally Recognized 

Culture Collection

N/A N/A

885.1300 Discussion of Formation of 

Unintentional Ingredients

As a term of registration, EPA is requiring additional 

information (confirmatory) on this data requirement. 485309-05

885.1400 Analysis of Samples Submitted data fulfill the requirement for analysis of samples 

for purposes of FIFRA section 3(c)(5) registration. 

485309-05

885.1500 Certification of Limits Limits listed on the confidential statement of formula are 

adequate/acceptable.

485309-05
Bacillus subtilis strain QST 713 variant Soil Page 25 of 28

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TABLE 3. Physical and Chemical Characteristics for the Technical Grade of the Active Ingredient (TGAI), Bacillus subtilis strain 

QST 713 variant Soil (40 CFR § 158.2120)

Harmonized 

Guideline Number Data Requirement Results MRID No.

830.6302 Color Brown or light brown

830.6303 Physical State Liquid 485309-03

830.6304 Odor Like yeast

830.6313

Stability to Normal and 

Elevated

Temperatures, Metals,

and Metal Ions

Unstable to elevated temperatures and to metals and metal 

ions. 485309-03

830.6317 Storage Stability As a term of registration, EPA is requiring additional 

information (confirmatory) on this data requirement. 485309-03

830.6319 Miscibility Non emulsifiable liquid. N/A

830.6320 Corrosion 

Characteristics N/A N/A

830.7000 pH 7.34 (1% solution in distilled water) 485309-03

830.7100 Viscosity N/A (not a homogenous solution) 485309-03

830.7300

Density/Relative 

Density/Bulk Density 

(Specific Gravity)

1.02 485309-03

TABLE 4. Physical and Chemical Characteristics for the End-Use Pesticide Product (EP), Serenade Soil DPZ

(40 CFR § 158.2120)

Harmonized

Guideline Number Data Requirement Results MRID No.

830.63021 Color Medium brown

N/A 485309-06

830.63031Physical State Liquid 485309-06

830.63041 Odor Yeast like odor 481460-06

830.63131

Stability to Normal and 

Elevated Temperatures, 

Metals, and Metal Ions

Unstable to elevated temperatures and to metals and 

metal ions. 460122-01

830.6317 Storage Stability As a term of registration, EPA is requiring additional 

information (confirmatory) on this data requirement. 485309-08

830.6319 Miscibility

Not required because the end-use pesticide product, 

Serenade Soil DPZ, is not an emulsifiable liquid form of 

microbial pesticides (refer to test note #2 of 40 CFR § 

158.2120(d)).

N/A

830.6320 Corrosion Characteristics N/A 460122-01

830.7000 pH 5.2 - 5.4 485309-06

830.7100 Viscosity 10 – 100 cPs 485309-06

830.7300

Density/Relative 

Density/Bulk Density 

(Specific Gravity)

1.000 – 1.075 g/cc 485309-03

1 According to 40 CFR § 158.2120, these data are only required for the technical grade of the active ingredient. Since Agraquest, 

Inc. included this information with its application for Serenade Soil DPZ, it is summarized appropriately in this table.
Bacillus subtilis strain QST 713 variant Soil Page 26 of 28

Biopesticides Registration Action Document

TABLE 5. Toxicology Data Requirements for the Active Ingredient Bacillus subtilis strain QST 713 variant Soil

and the End Use Pesticide Product Serenade Soil DPZ (40 CFR § 158.2140)

Harmonized 

Guideline 

Number

Data Requirement Results MRID No.

Tier I

885.3050 Acute Oral 

Toxicity/Pathogenicity

Waived based on the results of MRID No. 446519-06, conducted on

Bacillus subtilis strain QST 713

Classification: Acceptable

TOXICITY CATEGORY IV

446519-06

489309-08

885.3150 Acute Pulmonary 

Toxicity/Pathogenicity

Waived based on the results of MRID No. 446519-09, conducted on

Bacillus subtilis strain QST 713

No mortality and no adverse effects. The test microbe was detectable (both 

pre- and post- heat treatments) in the lung through day 35, but at 

significantly reduced levels compared to day 0. 

Classification: Acceptable

446519-09

489309-08

885.3200

Acute Injection 

Toxicity/

Pathogenicity

No mortality and no adverse clinical signs in rats. The test substance 

(Bacillus subtilis strain QST 713 variant Soil) cleared from all organs by 

day 21.

Classification: Acceptable

483509-09

885.3200

Acute Injection 

Toxicity/Pathogenicity 

(Intravenous)

No mortality and no adverse clinical signs in rats dosed with QST 713 

Technical powder. The test microbe cleared from most organs by day 35; 

however, low levels were detected in the spleen and liver after 35 days. 

Classification: Acceptable

446519-08

885.3400 Hypersensitivity

Incidents

The applicant reported that no hypersensitivity incidents, including 

immediate-type or delayed-type reactions of humans and domestic animals, 

occurred during research, development, or testing of the TGAI. Any future 

hypersensitivity incidents must be reported to EPA (refer to test note #3 of 

40 CFR § 158.2140(d)). 

48530902

885.3500 Cell Culture Not required because Bacillus subtilis strain QST 713 variant Soil is not a 

virus (refer to test note #4 of 40 CFR § 158.2140(d)). N/A

870.1100 Acute Oral Toxicity

Oral LD50 female rats > 5,000 mg/kg body weight (bw)

Classification: Acceptable

TOXICITY CATEGORY IV

485309-10

870.1200 Acute Dermal Toxicity

Dermal LD50 combined (male and female rats) > 5,050 mg/kg bw

Classification: Acceptable

TOXICITY CATEGORY IV

485309-11

870.1300 Acute Inhalation 

Toxicity

LC50 combined (male and female rats) >5.15 mg/L

Classification: Acceptable

TOXICITY CATEGORY III

483509-12

870.2400 Acute Eye Irritation

Serenade Soil DPZ was essentially non-irritating to the eyes of rabbits.

Classification: Acceptable

TOXICITY CATEGORY IV

485309-13

870.2500 Primary Dermal 

Irritation

The EP test substances were essentially non-irritating to the skin of rats.

Classification: Acceptable

TOXICITY CATEGORY IV

485309-14

Tiers II and III

Not required for Bacillus subtilis strain QST 713 variant Soil, based on the lack of acute toxicity/pathogenicity in the Tier I studies.
Bacillus subtilis strain QST 713 variant Soil Page 27 of 28

Biopesticides Registration Action Document

TABLE 6. Nontarget Organism Toxicity and Environmental Fate Data Requirements for the Technical Grade of the Active 

Ingredient (TGAI), Bacillus subtilis strain QST 713 variant Soil (40 CFR § 158.2150)

Harmonized Guideline 

Number

Data Requirement Results MRID No.

Tier I

885.4050 Avian Oral Toxicity

Data and other information provide sufficient 

information to determine that 

toxicity/pathogenicity to avian wildlife is not 

expected as a result of the proposed pesticidal

uses.

Classification: Acceptable

485309-15

885.4100 Avian Inhalation 

Toxicity/Pathogenicity

Not required as the nature of the microbial 

pesticide does not indicate potential pathogenicity 

to birds or relatedness to any known bird 

pathogens (refer to test note #3 of 40 CFR § 

158.2150(e)).

N/A

885.4150 Wild Mammal 

Toxicity/Pathogenicity

Tests required by 40 CFR § 158.2140 are 

adequate and appropriate for assessment of 

hazards to wild mammals. Studies submitted with 

laboratory rats indicate no adverse effects due to 

oral exposure.

Classification: Acceptable for wild mammal 

risk assessment

N/A

885.4200 Freshwater Fish 

Toxicity/Pathogenicity

Data and other information provide sufficient 

information to determine that 

toxicity/pathogenicity to freshwater fish and 

invertebrates is not expected as a result of the 

proposed pesticidal uses.

Classification: Acceptable

485309-15

885.4240 Freshwater Invertebrate 

Toxicity/Pathogenicity

885.4280 Estuarine/Marine Fish and 

Invertebrate Testing

Not required as the microbial pesticide will not be 

applied directly to water and is not expected to 

enter marine/estuarine environments in amounts 

that would result in significant concentrations 

(refer to test note #6 of 40 CFR § 158.2150(e)).

N/A

885.4300 Nontarget Plant Testing

Not required as the microbial pesticide is not 

taxonomically related to any known plant 

pathogens (refer to test note #7 of 40 CFR § 

158.2150(e)).

N/A

885.4340 Nontarget Insect Testing

Data and other information provide sufficient 

information to determine that 

toxicity/pathogenicity to honey bees and

nontarget insects is not expected as a result of the 

proposed pesticidal uses.

Classification: Acceptable

485309-15

885.4380 Honey Bee Testing

Tiers II, III, and IV

Not required for Bacillus subtilis strain QST 713 variant Soil based on the acceptability of the data and other information provided for

Tier I.
Bacillus subtilis strain QST 713 variant Soil Page 28 of 28

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APPENDIX B. PESTICIDE PRODUCT

EPA File 

Symbol

Registration 

Name

Percentage 

Active 

Ingredient

Formulation 

Type

Use Site(s) Method(s) of 

Application

Application Rate(s) Target Pest

69592-EI Serenade 

Soil DPZ 1.57%

End-use 

product

Agricultural

Seed treatment

Residential

Ground application

Aerial

Chemicagation

Tank mix

Seed treatment

Hose-end sprayer

1.5 – 14.7 fluid ounces/ 

1000 row-ft.

Soil drench: 1 -6 

qt./acre

Seed treatment: 1-10 

oz./100 lbs. of seed

Home and Garden:

2-8 fl. oz. per 1,000 

square feet

Soil diseases