MEMORANDUM 

UNITED STATES ENVIRONMENTAL PROTECTION AGENCY 

WASHINGTON, D.C. 20460 

OFFICE OF CHEMICAL SAFETY 

AND POLLUTION PREVENTION 

MAR 0 4 2016 

SUBJECT: Environmental risk assessment for the FIFRA Section 3 registration of the TGAI 

Bacillus amyloliquefaciens strain PTA-4838 (EPA File Symbol 70664-T) and two 

end-use products, Yarnimo® WSP (EPA File Symbol 70664-A) and Yarnimo®ST 

(EPA File Symbol 70664-L); Submission No. 958320, Decision No. 495952, DP 

Barcode No. 424149 

FROM: In-Soon You, Ph.D., Biologist 

Microbial Pesticide Branch 

Biopesticides and Pollution Prevention Division, 7511 P 

THROUGH: Shannon Bocges, Lead B;oJog;st \__"' ' 

Microbial Pesticide Branch ~ ~'\' Biopesticides and Pollution Prevention Division, 7511 P 

TO: Mike Mendelsohn, Regul atory Action Leader 

Microbial Pesticide Branch 

Biopesticides and Pollution Prevention Division, 7511 P 

Ecological Risk Assessment 

A. Introduction 

LidoChem, Inc. has submitted an application to register a new active ingredient, Bacillus 

amyloliquefaciens strain PTA-4838 (hereafter, B. amyloliquefaciens PTA-4838), for use as a 

suppressant against phytopathogenic fungi and nematodes and also for use as plant growth 

promoter on bushes, crops, herbs, spices, ornamentals, shrubs, trees (forest and shade), turf, 

vegetables and vines. B. amyloliquefaciens PT A-4838 is the active ingredient (a.i.) in two enduse products (EPs), Yarnimo® WSP and Varnimo®ST. Yarnimo® WSP is a water suspendable 

powder containing 0.29% a.i. at a minimum of2 x 109 cfu/g; Yarnimo®ST is a powder 

Internet Address (URL) • http //www epa gov 

Recycled/Recyclable • Pnnted w11h Vegetable Oil Based Inks on 100% Postconsumcr. Process Chlonne Free Recycled Paper 
containing 73.4% a.i. at a minimum of 5 x I 0 11 cfulg and is a biological seed treatment that 

provides early protection against selected parasitic nematodes. 

Members of the genus Bacillus are endospore forming bacteria. Endospores are formed under 

adverse growth conditions and help the Bacillus species persist in diverse environments (Logan 

and de Vos, 2009). B. amyloliquefaciens is a common bacterium present in soil, water and plant 

materials and is known to produce many secondary metabolites. EPA registered other strains of 

B. amyloliquefaciens (strains D747 and FZB24) which were reclassified from B. subtilis var. 

amyloliquefaciens (Priest et al., 1987; US EPA, 20 I 0; US EPA, 201 1 ). 

B. amyloliquefaciens PTA-4838 is a versatile bacteriun1 with multiple and unique modes of 

action. This strain produces many enzymes with special functions (e.g., phytase, chitinase, lipase 

and protease) and several secondary metabolites including antifungal lipoproteins (MRID No. 

494741 02). It 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 the ecological risk 

assessment for the registration of the new a.i. and its proposed uses. 

B. Summary of Nontarget Effects 

Table I provides the status of the Tier 1 Nontarget Organism Data Requirements for the active 

ingredient B. amyloliquefaciens PT A-4838 for ecological risk assessment. Nine original 

guideline studies with the TGAI and two sets of scientific rationale were submitted to meet data 

requirements for non-target organisms per 40 CFR Part 158.2150. Relevant information from 

the submitted and cited studies is included in the discussion of risk below (see C. Ecological 

Exposure and Rjsk Characterization) and Data Evaluation Records (DERs) for the submitted 

guideline studies are attached for this memorandum. 

Table I. Summary of data submitted to comply with Nontarget Organism Data Requirements 

per 40 CFR Part 158.1250 in support of the Section 3 registration of B. amyloliquefaciens PTA4838. 

Data Requirement OPPTS Results Summary and MRID No. 

Guideline Classification 

Avian oral 885.4050 Based on the two avian oral 49474109 

toxicity/pathogenicity studies submitted, B. 49474110 

amy/oliquefaciens strain PTA4838 is not toxic or pathogenic to 

Northern bobwhite (Colinus 

virginianus) dosed at 9.46 x l 09 

cfulml/bird/day. However, the 

birds were not tested at a 

maximum hazard dose. 

Classification: Supplemental 

2 
Data Requirement OPPTS Results Summary and MRID No. 

Guideline Classification 

Avian Oral 885.4050 Scientific rationale provided is 49474111 

sufficient to support the requested 

waiver. 

Classification: Acceptable 

Wild mammals 885.4150 Wild mammal testing was not N/A 

conducted, but studies required 

by 40 CFR Part 158.2140 are 

adequate for assessment of 

hazards to wild mammals. B. 

amyloliquefaciens PTA-4838 was 

found not toxic or pathogenic to 

laboratory rats when tested at 3.5 

x I 08 cfulrat by oral gavage 

(MRJD No. 49474105) and at 2.7 

xI 08 cfulrat by intravenous 

injection (MRID No. 494 74 1 07). 

Classification: Acceptable 

Freshwater fish 885.4200 A 30-day static renewal test with 49474112 

toxicity/pathogenicity B. amyloliquefaciens PTA-4838 

had a negative effect on growth 

of juvenile Rainbow trout 

(Oncorhynchus mykiss) dosed at 

I x I 06 cfu/mL and on survival at 

I x I 07 cfulmL. However, for 

l 00% mortality observed at 1 x 

I 0 7 cfu/mL within 15 days of 

testing, it is not possible to 

determine whether the effect was 

seen because of inherent 

toxicity/pathogenicity of the a.i. 

or due to experimental design 

(e.g., too young fish were used in 

the testing). 

Classification: Supplemental 

Freshwater 885.4240 A freshwater invertebrate study 49474113 

invertebrate with B. amyloliquefaciens PTAtoxicity/pathogenicity 4838 showed potential adverse 

effects on reproduction of 

Daphnia magna at exposure to 

I x 105 cfulmL of test medium in 

a 21-day static renewal system. 

Maximum hazard testing was not 

conducted; however, 

3 
Data Requirement OPPTS Results Summary and MRIDNo. 

Guideline Classification 

enumerations indicated exposure 

at 105 cfulmL. 

Classification: Supplemental 

Nontarget Insect 885.4340 A laboratory bioassay with adult 49474117 

Testing ladybird beetles (Hippodamia 

convergens) at the maximum 

hazard dose showed relatively 

high mortality (>20%) of B. 

amyloliquefaciens PTA-4838 to 

the test organism in all groups in 

13 days of testing. The results 

are inconclusive. 

Classification: Unacceptable 

Nontarget Insect 885.4340 A laboratory bioassay with adult 49474118 

Testing parasitic wasp (Aphidius 

coleman) at the maximum hazard 

dose showed toxic or pathogenic 

effects of B. amyloliquefaciens 

PTA-4838 to the test organism in 

all groups within 3 days of 

testing. The diet consisted of l 0% 

honey water with or without the 

test substance. The study is not of 

sufficient duration to be useful, 

and results are inconclusive. 

Classification: Unacceptable 

Nontarget Insect 885.4340 A laboratory bioassay with green 494741 16 

Testing lacewing larvae (Ch1ysoperla 

rufilabris) showed no toxic 

/pathogenic effect to the test 

organism at 1.1 X I 08 cfulcm2 

within 19 days of testing. The 

test substance and inactive test 

substance groups ate > 30% 

fewer corn earworm eggs 

(Helicoverpa zea) on Day 2 than 

the un-dosed control group, but 

these differences were not 

reported to be significantly 

different. 

Classification: Acceptable 

Honey bee testing 885.4380 A 13-day laboratory bioassay 49474 11 9 

with "young" honey bees (Apis 

mellifera) showed a relatively 

4 
Data Requirement OPPTS Results Summary and MRID No. 

Guideline Classification 

high mortality in all test groups. 

It was determined that B. 

amyloliquefaciens PTA-4838 was 

not toxic to honey bees at a rate 

of 1 O,OOOX MF AR or 1 x 1011 

cfu!mL!sq ft when administered 

orally. 

Classification: Supplemental 

given the duration ( 13 days) of 

the toxicity/pathogenicity testing, 

the stated dose rate, the rate of 

mortality(> 20%) in the control 

group and the test substance 

group, and high variability shown 

in the amount of food provided 

for consumption. 

Estuarine/marine fish 885.4280 A 30-day static renewal test with 49474114 

and invertebrate testing grass shrimp (Paleomonetes 

vulgaris) showed no chronic 

toxic/ pathogenic effect of B. 

amyloliquefaciens PTA-4838 

when tested at concentrations of 

I 05 cfu/g diet and 106 cfu/g diet. 

The ECso for Varnimo® Technical 

was determined to be > 106 cfu/g, 

based on mmtality observations. 

While there was no significant 

difference in the weight or length 

of the shrimp among the test 

groups, contamination of the 

controls makes the results less 

useful for a risk assessment. 

Classification: Supplemental 

Estuarine/marine fish 885.4280 A 30-day laboratory test with 494741 15 

and invertebrate testing inland silverside (Menidia 

beryl/ina) dosed at 106 cfu/mL 

and 107 cfu/mL showed potential 

chronic toxic and/or pathogenic 

effect at 107 cfu/mL in a 30-day 

static renewal system. Based on 

mmtality data (40% mortality at 

107 cfu/mL), the LCso for 

Varnimo® Techncal is > 1x 106 

cfu/mL. However, the submitted 

5 
Data Requirement OPPTS Results Summary and MRID No. 

Guideline Classification 

study is insufficient to detennine 

whether other sublethal effects 

may result from exposure to the 

active ingredient. 

Classification: Supplemental 

Nontarget plant 885.4300 A data waiver from nontarget 49474309 

Testing plant testing requirement for B. 

amyloliquefaciens PTA-4838 was 

submitted. Scientific rationale 

based on published literature is 

not sufficient to support no 

adverse effects to all plants. 

Classification: Unacceptable 

Additional information is needed 

to support claims of nonpersistence and lack of 

phytotoxicity. 

C. Ecological Exposure and Risk Characterization 

The EP Varnimo®WSP is a water suspendable powder that is intended for use as a suppressant 

against certain fungal pathogens and/or parasitic nematodes on bushes, crops, herbs, spices, 

ornamentals, shrubs, trees, turf, vegetables and vines, and/or as plant growth promoter. This EP 

can be applied alone or in combination and/or rotation with chemical fungicides as a tool for 

integrated disease management in agricultural crops, ornamentals, nursery plants and turfgrass. 

Varnimo® WSP may be used at the following rates: For foli ar applications, 3-32 ounces 

Varnimo®WSP mixed in a minimum of25 gallons of water can be applied per acre (1.9 x 107 

cfulmL). For drench/drip irrigation applications, 3-32 ounces Varnimo® WSP in a minimum of 

25 gallons of water can be applied. The EP Varnimo® ST is a biological seed treatment that 

provides early season protection against selected plant parasitic nematodes. For seed treatment, it 

can be applied up to 0.09 ounces per 100 lbs seed, resulting in ca.l x 106 cfu/g seed. 

The EP Varnimo®WSP is recommended to be applied in pre-emergence (e.g., 2-7 days prior to 

planting) as sprays, by chemigation (e.g., sprinkler, drip, drench, flood, in-furrows and borders), 

or by direct applications (e.g., as pre-planting, at-planting and post-planting amendments). There 

is not much drift from sprinkler irrigation, and in most chemigation applications, watering in is 

required, which would reduce runoff. All of the chemigation applications are soil directed and 

soil-directed applications with Varnimo®WSP are generally expected to have less potential for 

nontarget exposure compared to other applications (e.g., aerial sprays). Additionally, for preemergence uses, the label directs the user to irrigate immediately after treatment and to keep soil 

moist until planting. 

6 
For post-emergence applications (e.g., foliar and soil-directed applications), Varnimo®WSP is 

expected to have more potential for nontarget exposure, since nontarget organisms and/or their 

food items may be directly sprayed with the product. However, it is noted that irrigation 

immediately after application is required on the label to move the product into the root zone, so 

exposure on foliar surfaces within the treated area will be short lived. This does not eliminate 

exposure to soil-dwelling organisms. Additionally, some drift is expected from foliar sprays, and 

the label is not explicit about whether aerial applications may be made. Aerial spray applications 

would be expected to result in greater amounts of the product drifting off-site compared to 

ground spray applications. 

The EP Vamimo® ST is intended for seed treatment which is likely to result in low exposure of 

nontarget organisms to the product. Birds may be exposed by direct consumption of seeds, and 

soil-dwelling organisms may also be exposed to some degree. It is noted, however, that B. 

amyloliquefaciens occurs naturally in soil, water and plant materials, so soil-, water-, and plantborne non-target organisms in those areas currently have some exposure to the existing B. 

amyloliquefaciens strains. 

Birds and Mammals 

The guideline study submitted for the avian oral toxicity/pathogenicity testing requirement 

showed no adverse effects in birds (Northern bobwhite, Colinus virginianus) when tested at 9.46 

x I 09 cfu/mllquaillday. B. amyloliquefaciens strain PTA-4838 is not known to be pathogenic to 

birds. 

The spray concentration resulting from the highest application rate (32 oz) mixed in the 

minimum amount of water (25 gallons) based on the Varnimo WSP label is 1.9 x 107 cfu/mL 

(the product contains at least 2 x I 09 cfu/g). This concentration is below the level at which birds 

were tested and shown to have no adverse effects. Exposure by direct consumption of treated 

seed is also expected to be below this level, since a bird would need to consume> 1 OOOg of seed 

per day to achieve the same level of exposure as the birds in the study describe above. Based on 

expected field application rate and lack of adverse effects observed in birds exposed to B. 

amyloliquefaciens strain PTA-4838 at the level tested, risk to nontarget birds resulting from the 

proposed registration is not expected. Since birds were not tested at the maximum hazard dose, 

any future increases in application rate of B. amyloliquefaciens PTA-4838 would need to be 

evaluated for bird risks. 

An acute oral toxicity/pathogenicity study with laboratory rats indicated no adverse effects of B. 

amyloliquefaciens strain PTA-4838 when administered at a dose of 3.5 x 108 cfu/rat (MRlD No. 

49474105). An acute intravenous injection study (MRlD No. 49474107) was also submitted, 

which showed that B. amyloliquefaciens strain PTA-4838 was non-toxic and not infective to rats 

at a dose of2.7 x 108 cfulanimal (USEPA, 2016). 

7 
Some wild mammals may be exposed to B. amy/oliquefaciens strain PT A-4838 in treated areas; 

however, adverse effects were not observed in the mammalian studies. Therefore, risk to wild 

mammals is expected to be low from the proposed registration of the new a .i. 

Freshwater Fish and Invertebrates 

A guideline study submitted for the non-target freshwater fish testing requirement showed an 

effect of B. amyloliquefaciens PT A-4838 on Oncorhynchus mykiss (rainbow trout) when tested 

at I x I 06 cfu/mL and I x 107 cfulrnL. The a.i. was found to have an adverse effect on survival 

(e.g., 100% mortality within 15 days oftesting) ofthe test organisms at 107 cfulmL, and an 

adverse effect on growth (e.g., weight and length) of the test organisms at 106 cfulmL. 100% 

mortality seen at I 07 cfu/mL, but not at 106 cfulmL, appears to be dose-dependent, hence the a.i. 

shows a toxic effect under the conditions tested. However, for other observations (e.g., growth 

rate, abnorma l behavior/appearance, and weight), it is not possible to determine whether the 

effects were seen because of inherent toxicity/pathogenicity of the a.i. or due to experimental 

design. The study report indicates that the water in the 107 cfu/mL test substance group was 

heavily cloudy with no visibility on many days. The necropsy showed that many fish in this 

group showed discoloration, indicating stress. Many of the fish also died suddenly (e.g., 10 died 

in one replicate on day 5 due to loss of air supply, 19 died on day 15 for reasons not explained). 

No follow-up work was performed to determine if the mortality was the result of pathogenic 

effects. It is possible that the effects observed were due to toxicity and/or pathogenicity, but may 

also have been the result of the physical conditions produced by the high concentration of the test 

substance. 

A guideline study for the non-target freshwater invertebrate testing indicated no significant effect 

on mobility (indicator of mortality), but showed a potential toxic/pathogenic effect of B. 

amyloliquefaciens strain PT A-4838 on reproduction of Daphnia magna when tested at 1 x 105 

cfu/mL. The maximum hazard dose (testing at 1 x 106 cfu a.i. /mL) was not used in the study, 

and old and new test media were contaminated in some cases. However, the contamination was 

not described as the test substance, and it is possible that other microbes entered the non-sterile 

test environment. It is unclear whether there may be effects from the contamination that was 

detected; however, conclusions regarding lack of mortality are considered reliable. 

The EPA Standard Wetland, which is a wetland of l acre in size and 15 em in depth, can be used 

to determine a worst-case scenario estimate for EEC in water. The EEC for water is calculated 

to be 3 x I 03 cfu/ml ((32 ozs (product/acre) X (28.35 g/oz) X (2 X I 09 cfu/g product) X (1 

acre/40,468,564 cm2) ] ..;- 15 em). Using the EPA Standard Pond, which is a pond of 1 acre in size 

and 6ft ( 182.88 em) deep, the EEC would be 2.5 xl02 cfu/mL. To gain a more realistic EEC 

based on the potential for spray drift, the Tier I aerial agricultural application scenario in the 

AgDRIFT spray drift model can be applied to these numbers. Under that scenario assuming fine 

to medium droplet size, a maximum of 12.5% of the amount applied is expected to reach aquatic 

areas at the water's edge. Therefore, the EEC for the wetland and pond can be refined to 3.8 x 

8 
102 cfu/mL and 3.1 x 101 cfu/mL, respectively, and this exposure is expected to be even lower 

with ground spray applications. Since the potential for exposure falls three orders of magnitude 

or more below the 105 cfu/mL from the Daphnia study described above, and the I 06 and I 07 

cfu/mL in the rainbow trout study above, adverse effects to aquatic animals in freshwater 

systems are not expected. 

B. amyloliquefaciens PT A-4838 produces a biosurfactant (surfactin), chitinase and other 

secondary metabolites (MRID No. 494741 02). Surfactants are reported to be toxic to fish and 

invertebrates (Abel, 1974; Abel and Skidmore, 1975) and chitinase can be toxic to invertebrates 

containing chitin as a structural component. Some adverse sublethal effects were observed in the 

studies described above, and there is some uncertainty as the level of exposure at which such 

effects might occur. These metabolites likely were included in the test material used in the 

studies above, and they may have contributed to the effects observed. However, as described 

above, exposure to the a.i. in aquatic environments is expected to~ 1 OOOX below the levels 

tested. Therefore, any effect of these metabolites on aquatic organisms is expected to be 

minimal. Since the maximum hazard dose was not tested in the Daphnia study, any future 

increases in the application rate would need to be reevaluated for potential effects on these 

aquatic organisms. 

Marine/Estuarine Fish and Invertebrates 

The applicant submitted two guideline studies to meet the requirement for nontarget 

marine/estuarine fish and inver1ebrate testing. This testing is conditionally required if significant 

exposure of nontarget marine/estuarine fish and invertebrates to B. amyloliquefaciens strain 

PTA-4838 is expected; however, EPA did not specifically require these studies. 

A 30-day static renewal test was conducted with grass shrimp (Paleomonetes vulgaris) to 

determine the chronic toxicity/pathogenicity of B. amyloliquefaciens PTA-4938 at concentrations 

of 105 cfu/g diet or I 06 cfu/g diet. The active ingredient was shown to have no significant 

difference in mortality and in the weight/length of the shrimp among different test groups under 

the conditions tested. The EC50 was determined to be > 106 cfu/g, based on mortality 

observations. However, because of contan1ination in the un-dosed control and the sterile filtrate 

control, it is not possible to discern whether the test material had an effect on weight or length. 

A 30-day static renewal test was conducted with inland silverside (Menidia beryllina) to 

detennine the chronic toxicity/pathogenicity of B. amyloliquefaciens PTA-4938 at concentrations 

of 106 cfu/mL or 107 cfu/mL in the test water (these groups were also exposed via diet at 105 

cfu/g and 106 cfu/g diet, respectively). A mortality rate of 40% was shown in the test organisms 

when exposed at 107 cfu/mL, but no significant mortality (96.7%) was seen when exposed at 106 

cfu/mL. Based on mortality observations, the LC50 is determined to be> 106 cfu/mL. B. 

amyloliquefaciens PTA-4938 showed no statistically significant difference in the weight or 

length of the silverside among different test groups when tested at! 06 cfu/mL or l 07 cfu/mL. 

The test organisms in all groups lost 40-60% in weight and 9-13% in length. It is not clear 

9 
whether these are sublethal effects that resulted from exposure; however, since the fish were 

shown to have shrunk in length (an unusual result for studies with these animals). These 

observations suggest a husbandry issue or poor measurement technique at the testing laboratory. 

Contamination with potential test substance in the un-dosed control and sterile filtrate groups, 

introduces uncertainty in the observations of sublethal effects, so these results are not considered 

reliable 

Similar to a case with the nontarget organisms in freshwater environments, B. amyloliquefaciens 

PTA-4838 is not expected to reach marine/estuarine environments in significant quantities, and 

exposure is expected to be > 1 ,OOOX below the levels at which effects were tested in the above 

studies. Even though there is uncertainty related to the sublethal observations in these studies, 

adverse effects are expected to be very minimal, given the low level of exposure in these 

environments. 

Nontarget Insects and Honey Bees 

A guideline study submitted for the requirement for nontarget insect testing showed an effect to 

ladybird beetles (Hippodamia convergens) upon a 13-day dietary exposure to corn earworn1 

(Helicoverpa zea) eggs sprayed with B. amylofiquefaciens PTA-4838 at the maximum hazard 

dose. Adult ladybird beetles were used and their age was not specified. The use of the adult 

insects of unknown age can affect the study results. Relatively high mortality rates(> 20%) were 

shown in a ll groups, including the control group, in 14 days, but toxicity of the test substance 

could not be clearly demonstrated. The results of the study are inconclusive and the study is 

unacceptable. 

A guideline study with green lacewing (Ch1ysoperla rufllabris) larvae did not show a 

toxic/pathogenic effect to the test organism in a 19-day dietary exposure at the maximum hazard 

dose of B. amyloliquefaciens PT A-4838. There appears to be much less (30%, Table 3 of the 

MRlD No. 494 74116) amount of eggs consumed in the inactive and active test substance groups 

than in the un-dosed control group, but these differences were not reported to be significantly 

different. 

A 3-day bioassay study with parasitic wasp (Aphidius colemani) showed toxic effects of B. 

amyloliquefaciens PTA-4838 to the test organism when tested at the maximum hazard dose. The 

study ended in 3 days due to high mortality observed in the control group (77%) and >90% in 

both the test substance and inactive test substance groups. The results are inconclusive as no 

differences in mortality were found among different test groups. 

A guideline study with honey bees (Apis mellifera) was conducted for 13 days. B. 

amyloliquefaciens PTA-4838 was determined to be not toxic to honey bees when administered 

orally at a stated concentration of 1 O,OOOX the maxirmun field application (MF AR) or 1 x I 0 11 

cfulml/sq ft in 50% sugar solution. It is noted that the Agency calculation indicates the dose rate 

is much lower than the stated 1 O,OOOX MF AR, but was still well above the maximum hazard 

10 
dose. The mortality observed in the control group and the test substance group on day 13 were 

23% and 25%, respectively. It was stated in the study (MRID No. 47474119) that "young" bees 

were tested, but specific information regarding age was not provided. When young bees are 

used, the bees need be newly emerged in order to obtain the results useful for a risk assessment. 

Exposure to honey bees may occur with applications to foliage; however, based on the study, 

potential hazard to bees is not expected. 

Exposure to nontarget insects is expected in the treated areas. Nontarget insects may be directly 

sprayed within treated areas, or they may contact treated surfaces. Exposure through contact 

with treated foliage is expected to be short-lived within areas targeted for treatment, since the 

label requires immediate irrigation of treated plants to move the product off of foliar surfaces and 

into the root zone. Therefore, insect exposure in the treated area is primarily expected to occur 

through direct contact with spray during application or through contact with soil in treated areas. 

Within soil, exposure is expected to be somewhat spatially limited, since B. amyloliquefaciens 

PTA-4838 is expected to inhabit plant roots once applied. B. amyloliquefaciens PTA-4838 may 

persist with formation of endospores; however, it is noted that B. amyloliquefaciens is a common 

inhabitant of soil and nontarget insects likely are already exposed at some level to this bacterium. 

Foliar sprays are likely to result in exposure to nearby areas through drift of the applied product. 

In these areas, exposure will be lower, since a fraction of the sprayed amount is expected to drift. 

However, unlike treated areas that will be immediately irrigated after treatment, B. 

amyloliquefaciens PTA-4838 is expected to remain on fol iar surfaces for a longer period of time 

after appli cation. Therefore, exposure at some level cannot be avoided in these areas. 

While exposure is expected to be somewhat limited in treated areas, the potential for exposure to 

nontarget insects cannot be ruled out. Data available for other nontarget insects are incomplete, 

since two of the submitted studies were inconclusive. One study indicates that applications of B. 

amyloliquefaciens PTA-4838 may be relatively safe for certain nontarget insects. However, as 

described above, B. amyloliquefaciens is known to produce secondary metabolites and/or 

enzymes that may adversely affect insects, and several recent reports in the literature indicate the 

potential for use of this bacterium or its biosurfactants in insect control (Gadhave and Gange, 

2016; Khedher et al., 2015; Li et al., 2015; Yun et al. 2013). On the other hand, Bacillus 

amyloliquefaciens has been isolated from the gut of certain insects (see US EPA, 201 0). Concerns 

for nontarget insects had previously been raised for a currently registered strain of B. 

amyloliquefaciens, but EPA determined that the weight of the evidence indicated that adverse 

effects were not expected (US EPA, 201 0). However, given the varying information regarding the 

potential for adverse effects on nontarget insects, including more recent reports, the lack of 

acceptable data on this specific strain, and the potential for exposure particularly associated with 

fo liar sprays, adverse effects to nontarget insects cannot be ruled out with high certainty. 

Removal of foliar sprays from the proposed label would significantly reduce exposure to 

nontarget insects both within the treated areas and in adjacent areas. This application method 

may be better supported in the future with additional acceptable testing on insects. 

11 
Nontarget Plants 

A guideline study for Nontarget Plant Testing, Tier I was not submitted, but a data waiver was 

requested for the new a.i. B. amyloliquefaciens grows in association with plants (Alvarez et al., 

20 II) and is a plant growth-promoting bacterium (Chen et al., 2007), but is not a known plant 

pathogen. Few adverse effects of B. amyloliquefaciens on plants have been reported by the 

applicant from the published literature on existing B. amyloliquefaciens strains on greenhouse, 

growth chamber or field studies (MRJD No. 49474309). Scientific rationale submitted for a 

waiver of the requirement for Nontarget Plant Testing should be strengthened as some references 

were either too general or outdated to be useful. The rationale provided was insufficient to 

conclude that the proposed uses of B. amyloliquefaciens PTA-4838 are not expected to result in 

increased exposure to, or adverse effects in nontarget plants of economic importance. 

Bacillus amyloliquefaciens produces secondary metabolites that may have adverse effects on 

certain nontarget organisms (e.g., fish, aquatic invertebrates, insects - see discussion above). Its 

reported biology (e.g., an endospore former) and the wide range of use sites and application 

methods may argue against the applicant's claim of no adverse effects and nonpersistence of the 

active ingredient in all environments. Additionally, Talboys et al. (2014) reported reductions in 

inorganic phosphorus (Pi) uptake by Triticum aestivium seeds treated with B. amyloliquefaciens 

FZB42, resulting from the action of auxin produced by this strain in low Pi environments. These 

effects were not observed in high Pi environments, where the effect was observed to be 

beneficial. While this paper may not signal potential for adverse effects in all conditions, the 

auU1ors conclude the importance of understanding bacteria-plant interactions when deciding 

among pesticide alternatives. More information like this would better inform users of 

biopesticide products and also future pesticide risk assessments. 

Exposure to nontarget plants is expected with the proposed registration of B. amyloliquefaciens 

PTA-4838, particularly with foliar sprays that may cause drift or to nontarget plants on the field 

that may contact B. amyloliquefaciens in the soil. B. amylolique.faciens is not a known plant 

pathogen and is a common soil bacterium that has a close association with many plant species. 

Adverse effects to nontarget plants also have not been documented. Therefore, adverse effects 

are not anticipated from the proposed registration of the products containing this a.i. 

D. Environmental Persistence 

Bacillus amyloliquefaciens is a common bacterium found in soil, water and plant materials. Like 

other Bacillus species, it forms endospores under adverse growth conditions. Endospores can 

survive extreme environmental conditions (e.g., heat, desiccation and chemicals) that would 

normally kill the bacterium, which makes the bacterium persistent in diverse environments. It is 

also known to produce special enzymes and secondary metabolites, including antibiotics and 

antifungal lipoproteins (Alvarez et al., 2011; Chen et al., 2007). 

12 
Bacillus amyloliquefaciens PT A-4838 is a nutritionally and biochemically versatile bacterium 

with multiple and unique modes of action. This strain produces many enzymes with special 

functions (e.g., phytase, chitinase, lipase and protease) and several secondary metabolites such as 

fengycin, iturin, and surfactin (MRID No. 494741 02). The EPs likely contain both cells with 

endospores and vegetative cells. Therefore, the active ingredient is expected to persist in the 

environment for a prolonged period once introduced. Whether it persists at levels that are likely 

to have pesticidal benefits or adverse effects is uncertain. 

E. Federally Listed Threatened and Endangered Species 

Based on available information, EPA has determined that no adverse effects are anticipated for 

birds, mammals, fish, aquatic invertebrates, and plants exposed to B. amylofiquefaciens PTA4838 as a result of the proposed labeled applications. Therefore, a "No Effect" determination is 

made for direct and indirect effects to listed species of these taxa and their designated critical 

habitats resulting from the proposed registration of B. amyfoliquefaciens PTA-4838. Concerns 

were raised for nontarget insects for the proposed registration of B. amyloliquefaciens PT A-4838 

as labeled, due to uncertainties related to lack of acceptable data and reports of potential effects 

in the literature. Therefore, direct effects to listed insects cannot be precluded, nor can indirect 

effects to listed obligate insectivores whose food supply may be affected. However, adverse 

effects are not expected to nontarget insects if foliar applications are removed from the proposed 

Yarnimo® WSP label , as this is expected to significantly reduce exposure to nontarget insects. In 

such case, EPA could make a "No Effect" detennination for direct effects to listed insects and 

for indirect effects to listed obligate insectivores. 

References 

Abel, P.O. 1974. Toxicity of synthetic detergents to fish and aquatic invertebrates. Journal of 

Fish Biology, 6: 279-298. 

Abel, P.D. and J.F. Skidmore. 1975. Toxic effects of an anionic detergent on the gills of rainbow 

trout. Water Research, 9: 59-65. 

Alvarez, F., M. Castro, A. Principe, G. Borioli, S. Fisher, G. Fischer, G. Mori and E. Jofre. 2011. 

The plant-associated Bacillus amyloliquefaciens strains MEP218 and ARP23 capable of 

producing the cyclic lipopipetides iturin or surfactin and fengycin are effective in biocontrol of 

sclerotinia stem rot disease. Journal of Applied Microbiology, 112: 159-174. 

Chen, X.H., A. Koumoutsi, R. Scholz, A. Eisenreich, K. Schneider, I. Heinemeyer, B. 

Morgenstern, B. Voss, W.R. Hess, 0 . Reva, H. Junge, B. Voigt, P. R. Jungblut, J. Yater, R. 

Sussmuth, H. Liesegang, A. Strittmatter, G. Gottschalk and R. Borriss. 2007. Comparative 

13 
analysis of the complete genome sequence of the plant growth-promoting bacterium Bacillus 

amyloliquefaciens FZB42. Nature Biotechnology, Advance Online Publication pages 7-14. 

Gandhave, K.R. and A.C. Gange. 2016. Plant-associated Bacillus spp. Alter life-history traits of 

the specialist insect Brevicoryne brassicae L. Agricultural and Forest Entomology. 18:35-42. 

Khedher, S.B., H. Boukedi, 0. Kilani-Feki, I. Chaib, A. Laarif, L. Abdelkefi-Mesrati and S. 

Tounsi. 2015. Bacillus amyloliquefaciens AG 1 biosurfactant: Putative receptor diversity and 

histopathological effects on Tufa absoluta midgut. Journal oflnvertebrate Pathology, 132: 42-

47. 

Li, H., M.A. Soares, m. S. Torres, M. Bergen and J.F. White. 2015. Endophytic bacterium, 

Bacillus amyloliquefaciens, enhances ornamental hosta resistance to disease and insect pests. 

Journal of Plant Interactions, 10: 224-229. 

Logan, N.A. and P. de Vos. 2009. Geneus I. Bacillus. Pp. 2l-128ln: P.de Vos, G.M. Garrity, D. 

Jones, N.R. Krieg, W. Ludwig, F.A. Rainey, K.H. Schleifer, and W. Whitman (Eds.) Bergey's 

Manual of Systematic Bacteriology, Volume 3, 2nd Ed. Springer, New York. 

Priest, F.G., M. Goodfellow, L.A. Shute, and R.C.W. Berleley. 1987. Bacillus 

amyloliquefaciens sp. nov., nom. Rev. International Journal of Systematic Bacteriology, 37: 69-

71. 

Talboys P.J., Owen D.W., Healey J. R., Withers P.J., and D.L. Jones. 20 14. Auxin secretion by 

Bacillus amyloliquefaciens FZB42 both stimulates root exudation and limits phosphorus uptake 

in Triticum aeslivum. BMC plant biology, 14: 51 

US EPA. 2010. Bacillus subtilis Final Registration Review Decision Case 6012. Docket No. 

EPA-.HQ-OPP-2007-1 026. Washington, D.C. 

US EPA. 201 1. Memorandum from S. Borges (OPP/BPPD) to S. Cerrelli, Re: Environmental 

risk assessment for the FIFRA Section 3 registration of B. amyloliquefaciens strain D474 (EPA 

File Symbols 70051-RNI and -RNT); Submission No. 879171, Decision No. 437753, DP 

Barcode No. 382915. Dated October 3, 2011. 

US EPA. 2016. Memorandum from M. Djurickovic (OPP/BPPD) toM. Mendelsohn, Re: 

Review of 5 Batch Enumeration, Product Chemistry, Manufacturing Process, Storage Stability, 

Acute Toxicity Studies, and Tolerance Exemption for Varnimo® Technical/ST (EPA Reg. No. 

760644-T) submitted by Lidochem Inc. Draft dated March 4, 2016. 

Yun, D.C., S.I. Yang, Y.C. Kim, I.S. Kim and Y.H. Kim. 2013. Identification of surfactin as an 

aphicida l metabolite produced by Bacillus amyloliquefaciens G 1. Journal of Korean Society for 

Applied Biological Chemistry, 56: 751-753. 

14